Effects of prostaglandins and other putative chemical intermediaries on the activity of canine testicular polymodal receptors studied in vitro

(1) An in vitro testis-superior spermatic nerve preparation was used to evaluate the effects of chemical agents applied in the bathing solution. Both directly evoked discharges and responses to algesic solutions [bradykinin (BK) 9×10^–8 M, hypertonic saline 616 mM and high K⁺ solution 60 mM] of polymodal receptors were studied. (2) Prostaglandin (PG)-E₂ (1.4×10^–6–1.4×10^–5 M) and serotonin (5-HT) (1.1×10^–6 to 1.4×10^–4 M) had only a weak excitatory effect. However, test responses to algesic substances were regularly greatly increased by PG-E₂,-I₂ and 5-HT. Concentrations of PG-E₂ of 1.4×10^–8 M or grealer augmented BK responses; higher concentrations and/or longer applications were needed to enhance responses to algesic salt solutions. Effective concentrations for the PGs and 5-HT were near those reported for inflamed tissues and exudate. (3) Aspirin (ASA) (5.5×10^–4 M or greater, for more than 4 min) suppressed the responses to BK but not those evoked by hypertonic saline. The ASA effect on the BK response was largely restored by an addition of PG-E₂. (4) Substance P also had a weak excitatory effect on some polymodal receptors, but no significant enhancement of the response to BK was noted. (5) These results further support a role of polymodal receptors in transmitting nociceptive information, of inflammatory origin.     

Involvement of prostaglandin E(2) derived from enteric glial cells in the action of bradykinin in cultured rat myenteric neurons

We characterized bradykinin (BK)-induced changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) and membrane potential in cultured rat myenteric neurons using ratiometric Ca(2+) imaging with fura-2 and the whole-cell patch-clamp technique, respectively. BK evoked a dose-dependent increase of [Ca(2+)]i that was abolished by HOE 140, a B2 receptor antagonist but not by [Lys-des-Arg(9)]-BK, a B1 receptor antagonist. [Lys-des-Arg(9)]-HOE140, a B1 receptor agonist, failed to cause a [Ca(2+)]i response. Double staining with antibodies against the B2 receptor together with PGP9.5 or S100 indicated that B2 receptors were expressed in neurons and glial cells. The BK-evoked [Ca(2+)]i increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E(2) (PGE(2)). The release of PGE(2) from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca(2+)]i in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca(2+)]i in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin. These results indicated that BK caused a [Ca(2+)]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE(2) released from glial cells in response to BK. It is suggested that a neuron-glial interaction plays an important role in the effect of BK in the rat myenteric plexus.     

Strong heat stimulation sensitizes the heat response as well as the bradykinin response of visceral polymodal receptors.

1. Hyperalgesia after thermal injury is a common phenomenon, but its mechanism is poorly understood. As a step toward understanding the mechanism for heat sensitization, we searched conditions that would induce sensitization consistently and studied changes in polymodal receptors in their responses to heat (45 and 48 degrees C) and to bradykinin. Experiments were conducted in vitro, with the use of testis-superior spermatic nerve preparations excised from anesthetized dogs. 2. Stimulation at 55 degrees C for 30 s induced clear augmentation of the responses to stimulations at 45 and 48 degrees C when tested within 10 min in 22 out of 24 testicular polymodal units. In the remaining two units, weak or delayed augmenting tendencies were observed. The response to stimulation at 45 degrees C increased from 0.28 +/- 0.08 to 2.99 +/- 0.46 (SE) imp/s, whereas at 48 degrees C it increased from 1.08 +/- 0.16 to 4.60 +/- 0.63 imp/s (P < 0.001, n = 24). A low-level ongoing discharge appeared in about one-half of the units studied. 3. Despite frequent rinsing of the receptive field, augmentation of the heat response observed after stimulation at 55 degrees C continued for up to approximately 3 h. 4. The response to bradykinin (94 nM) was also potentiated: the net mean discharge rate (NMDR) during a 1-min application period of bradykinin as well as the total number of impulses induced by one bradykinin application significantly increased from 0.91 +/- 0.27 to 1.97 +/- 0.32 imp/s and from 213.9 +/- 42.3 to 433.5 +/- 58.7 impulses, respectively (P < 0.001, n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)     

Response properties of polymodal receptors studied using in vitro testis superior spermatic nerve preparations of dogs

Characteristics of the polymodal receptor were studied using in vitro testis superior spermatic nerve preparations excised from anesthetized dogs. They were in most aspects similar to those reported previously using in vivo preparations. The majority (90%) of the tested polymodal units had small myelinated nerve fibers; the rest had nonmyelinated fibers. The mean mechanical threshold as determined by von Frey hairs was 17.5 g/mm2 (n = 476). There was a tendency for a unit with a higher conduction velocity to have a lower mechanical threshold. Bradykinin and hypertonic saline consistently caused a dose-dependent increase in discharge rate of these units; high K+ solution was also found to be a consistent stimulant. The responses of C-fiber receptors were not significantly different from those of A-delta-fibers. Heat stimulation up to 50 degrees C evoked discharges in 99 out of the total 103 units tested. The mean threshold temperature was 44.4 degrees C for the first trial. In 19 units in which the same heat stimulation was tested after an interval of 10 min, 10 units showed sensitization; 3 units were deactivated; and no clear difference was observed in the rest. No unit responded with a substantial increase in discharge rate to cold stimuli of 20 degrees C or less. A small temperature rise of 2 degrees C from the normal surface temperature of the testis (34 degrees C) significantly increased the response to hypertonic saline (616 mM) (2.41 +/- 0.22 impulses/s at 34 degrees C to 3.23 +/- 0.44 impulses/s at 36 degrees C) and to bradykinin (9 X 10(-8) M) (1.95 +/- 0.35 impulses/s at 34 degrees C to 2.85 +/- 0.19 impulses/s at 36 degrees C). The majority of the units recorded from the superior spermatic nerve in this experiment were most probably of polymodal receptor type, although the heat response was tested in a limited number of units. A very small number of a different type of receptor was discovered: rapidly adapting mechano-receptors, which responded almost exclusively to mechanical stimulation and were especially sensitive to a light mechanical stimulus moving across the receptive fields. The response properties of receptors studied in vitro remained practically unchanged during the experiments of several hours. The present experiments have shown that this preparation is suitable for systematic investigations, especially of the effects of chemical agents, on the polymodal receptor, which plays important roles in nociceptive functions.     

Effects of ionic calcium on the responses of canine testicular polymodal receptors to algesic substances

1. To explore possible mechanisms of the responses to algesic substances (bradykinin, hypertonic saline, and high K+ solution) of polymodal receptors in the canine testis, the Ca2+ concentration was varied in vitro. 2. After 1 min in Ca2+-free media, the responses to both high K+ solution (60 mM K+) and hypertonic saline (0.6 M Na+) were significantly augmented and tended to increase further with time; return to normal Ca2+ concentration quickly reversed these changes. These augmenting effects were blocked by the substitution of Mg2+ for Ca2+. The excitation produced by 60 mM K+ was decreased by increasing Ca2+ in a concentration-dependent fashion. 3. Reducing the bath concentration of K+ decreased responses evoked by 9 X 10(-8) M bradykinin (BK), whereas increased K+ concentration had the opposite effect. 4. The excitatory effects of BK were significantly suppressed in extracellular Ca2+-free condition. The suppression was not affected by the addition of Mg2+. Prostaglandin E2, which has been known to be released by BK and to augment the BK response, failed to restore the suppressed response by either preapplication (2.8 X 10(-7) M) or simultaneous application in high concentration (1.4 X 10(-5) M). 5. On the basis of these observations, it was postulated that Ca2+ concentration-dependent changes of the responses to 60 mM K+ and 0.6 M Na+ results from Ca2+-dependent "membrane surface potential" changes. The suppressed response to BK by Ca2+ depletion may be explained by the intervention of Ca2+-dependent processes other than PG production.     

P2Y1 receptor modulation of Ca2+-activated K+ currents in medium-sized neurons from neonatal rat striatal slices

ATP signaling to neurons and glia in the nervous system occurs via activation of both P2Y and P2X receptors. Here, we investigated the effects of P2Y(1) receptor stimulation in developing striatal medium-sized neurons using patch-clamp recordings from acute brain slices of 7- and 28-day-old rats. Application of the selective P2Y(1) receptor agonist 2-(Methylthio) ADP trisodium salt (2-MeSADP; 250 nM) increased outward K(+) currents evoked by a ramp depolarization protocol in voltage-clamp recordings. This effect was observed in 59 out of 82 cells (72%) and was blocked completely by the P2Y(1) antagonist, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate. The averaged 2-MeSADP-sensitive conductance was fitted by the sum of a linear conductance and a Boltzmann relation, giving one-half activation voltage of -14.2 mV and an equivalent charge of 2.91. The 2MeSADP-mediated effect was sensitive to submillimolar concentrations of tetraethylammonium (TEA; 200 μM), to 200 nM iberiotoxin and to 100 nM apamin, suggesting the involvement of both big and small potassium (BK and SK, respectively) calcium-activated channels. In current-clamp experiments, 2-MeSADP decreased depolarization-evoked action potential (AP) firing in all 26 cells investigated, and this effect was reversed by TEA and by apamin but not by iberiotoxin. We conclude that the stimulation of P2Y(1) receptors in developing striatal neurons leads to activation of calcium-activated potassium channels [I(K(Ca))] of both BK and SK subtypes, the latter responsible for decreasing the frequency of AP firing in response to current injection. Therefore, P2Y(1) signaling leading to activation of I(K(Ca)) may be important in regulating the activity of medium-sized neurons in the striatum.     

Chemosensitivity of fine afferents from rat skin in vitro

1. Properties of sensory receptors with slowly conducting nerve fibers (less than 10 m/s) were studied using a rat skin-saphenous nerve in vitro preparation where receptive fields of identified single units can be isolated and superfused at the corium side with defined chemical solutions. 2. With mechanical search stimuli, 150 slowly adapting units were identified, 88% C-fibers, and the remainder, A delta-fibers. The majority of these units (65%) were categorized as mechano-heat sensitive ("polymodal") with controlled radiant heat stimulation. The remaining units were classified as low- or high-threshold mechanoreceptors according to their von Frey thresholds. 3. Bradykinin (BK), in concentrations of 10(-8) to 10(-4) M, was repeatedly applied for 1 min at 10-min intervals. Fifty-six percent of the polymodal C-fibers responded to BK (up to 10(-5) M), in contrast to 17% of the heat-insensitive units (P less than 0.01). No correlation between BK sensitivity and conduction velocity or von Frey threshold was found. 4. The BK "threshold concentrations" to excite C- and A delta-fibers were about equally distributed over a range from 10(-8) to 10(-5) M. 5. There was a large interindividual variability in pattern and magnitude of the response to BK. Intraindividually, a marked tachyphylaxis upon repeated BK stimulation was observed. 6. In fibers with a slow development of tachyphylaxis, the effects of conditioning application of different chemicals on BK responsiveness were studied. Norepinephrine in 10(-7) M concentration did not produce a significant effect, whereas 10(-5) M and 10(-4) M seemed to increase the BK responses. 7. Prostaglandin E2 (10(-6) M) caused a weak sensitization to BK on average (n.s.), but serotonin (10(-6) M) was clearly effective (P less than 0.05). 8. The strongest sensitization to BK (P = 0.01) resulted from conditioning heat stimulation, which also uncovered a responsiveness in some units initially insensitive to BK. 9. In some experiments the calcium concentration in the superfusate of receptive fields was lowered to 0.3 mM, which induced ongoing activity in C-fibers and markedly increased the BK responses in two polymodal units tested. Increasing the calcium concentration to 3.0 mM reversed these effects. 10. After completing the BK test protocol, polymodal C-fibers were exposed to other chemicals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alterations in NMDA receptors in a rat model of cortical dysplasia

Recent studies have demonstrated an important role for the N-methyl-D-aspartate receptor (NMDAR) in epilepsy. NMDARs have also been shown to play a critical role in hyperexcitability associated with several animal models of human epilepsy. Using whole-cell voltage clamp recordings in brain slices, we studied evoked paroxysmal discharges in the freeze-lesion model of neocortical microgyria. The voltage dependence of epileptiform discharges indicated that these paroxysmal events were produced by a complex pattern of excitatory and inhibitory inputs. We examined the effect of the NMDAR antagonist D-2-amino-5-phosphopentanoic acid (APV) and the NMDA receptor subunit type 2B (NR2B)-selective antagonist ifenprodil on the threshold, peak amplitude, and area of evoked epileptiform discharges in brain slices from lesioned animals. Both compounds consistently raised the threshold for evoking the discharge but had modest effects on the discharge peak and amplitude. For comparison with nonlesioned cortex, we examined the effects of ifenprodil on the epileptiform discharge evoked in the presence of 2 microM bicuculline (partial disinhibition). In slices from nonlesioned cortex, 10 microM ifenprodil had little effect on the threshold whereas 71% of the recordings in bicuculline-treated lesioned cortex showed a >25% increase in threshold. These results suggest that NR2B-containing receptors are functionally enhanced in freeze-lesioned cortex and may contribute to the abnormal hyperexcitability observed in this model of neocortical microgyria.     

A combined blockade of glycine and calcium-dependent potassium channels abolishes the respiratory rhythm

In order to test whether glycinergic inhibition is essential for the in vivo respiratory rhythm, we analysed the discharge properties of neurones in the medullary respiratory network after blockade of glycine receptors in the in situ perfused brainstem preparation of mature wild type and oscillator mice with a deficient glycine receptor. In wild type mice, selective blockade of glycine receptors with low concentrations of strychnine (0.03-0.3 microM) provoked considerable changes in neuronal discharge characteristics: The cycle phase relationship of inspiratory, post-inspiratory and expiratory specific patterns of membrane potential changes was altered profoundly. Inspiratory, post-inspiratory and expiratory neurones developed a propensity for fast voltage oscillations that were accompanied by multiple burst discharges. These burst discharges were followed by "after-burst" hyperpolarisations that were capable of triggering secondary burst discharges. Blockade of glycine receptors and the "big" Ca2+-dependent K+-conductance by charybdotoxin (3.3 nM) resulted in loss of the respiratory rhythm, whilst only tonic discharge activity remained. In contrast, rhythmic activity was only weakened, but preserved after the "small" Ca2+-dependent activated K+ conductance was blocked with apamin (8 nM). Also low concentrations of pentobarbital sodium (6 mg/kg) abolished rhythmic respiratory activity after blockade of glycine receptors in the wild type mice and in glycine receptor deficient oscillator mice. The data imply that failure of glycine receptors provokes enhanced bursting behaviour of respiratory neurones, whilst the additional blockade of BKCa channels by charybdotoxin or with pentobarbital abolishes the respiratory rhythm.     

B2 receptor-mediated enhanced bradykinin sensitivity of rat cutaneous C-fiber nociceptors during persistent inflammation.

Bradykinin (BK), which has potent algesic and sensitizing effect on nociceptors, is of current interest in understanding the mechanisms of chronic pain. BK response is mediated by B2 receptor in normal conditions; however, findings that B1 receptor blockade alleviated hyperalgesia in inflammation have been highlighting the role of B1 receptor in pathological conditions. It has not yet been clear whether nociceptor activities are modified by B1 receptor agonists or antagonists during inflammation. In addition, previous studies reported the change in BK sensitivity of nociceptors during short-lasting inflammation, and data in persistent inflammation are lacking. Therefore we investigated whether an experimentally induced persistent inflammatory state modulates the BK sensitivity of nociceptors and which receptor subtype plays a more important role in this condition. Complete Freund's adjuvant was injected into the rat-tail and after 2-3 wk, persistent inflammation developed, which was prominent in the ankle joint. Using an in vitro skin-saphenous nerve preparation, single-fiber recordings were made from mechano-heat sensitive C-fiber nociceptors innervating rat hairy hindpaw skin, and their responses were compared with those obtained from C-fibers tested similarly in normal animals. BK at 10(-8) M excited none of the 10 C-fibers in normal animals while it excited 5 of 11 (45%) C-fibers of inflamed animals, and at 10(-6) M BK excited all of the 11 inflamed C-fibers (or 94% of 36 tested C-fibers) but only 4 of 10 (or 45% of 58 tested C-fibers) in normal animals. Thus the concentration-response curves based on the incidence of BK induced excitation, and the total number of impulses evoked in response to BK were significantly shifted to the left. Moreover, an increased percentage of the inflamed C-fibers responded to 10(-6) M BK with bursting or high-frequency discharges. Thirty-percent of inflamed C-fibers had spontaneous activity, and these fibers showed comparatively less tachyphylaxis to consecutive second and third 10(-6) M BK stimulation. A B2 receptor antagonist (D-Arg-[Hyp3, Thi5,8,D-phe7]-BK) completely eliminated BK responses in inflamed rats, while B1 receptor antagonists (B 9958 and Des-Arg9-[Leu8]-BK) had no effect. Selective B1 receptor agonist (Des-Arg10-Kallidin) excited 46% (n = 13) of inflamed C-fibers at 10(-5) M concentration, which is 1,000 times higher than that of BK needed to excite the same percentage of inflamed C-fibers. We conclude that in chronically inflamed tissue, sensitivity of C-fiber nociceptors to BK, which is B2 receptor mediated, is strongly increased and that B1 receptor may not be important to a persistent inflammatory state, at least at the primary afferent level.     

N-methyl-D-aspartate receptors and large conductance calcium-sensitive potassium channels inhibit the release of opioid peptides that induce mu-opioid receptor internalization in the rat spinal cord

Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn mediate analgesia, inhibition of spinal opioid release could contribute to the hyperalgesic actions of spinal N-methyl-D-aspartate receptors.     

Influence of histamine on the bradykinin response of canine testicular polymodal receptors in vitro

Effects of histamine on the testicular polymodal receptors were studied in vitro using canine testisspermatic nerve preparations. Histamine induced distinct increase in the discharge rate in 6 out of 17 units tested (high responders), while it only weakly excited the remaining 11 units (low responders). The bradykinin response of low responders tended to be slightly facilitated by pretreatment with histamine, while that of high responders tended to be suppressed. Significant correlation was observed between the magnitude of histamine-induced discharges and the magnitude of change in the bradykinin responses.     

Mechano-insensitive nociceptors encode pain evoked by tonic pressure to human skin

Single unmyelinated axons in the superficial branch of the peroneal nerve of human subjects were recorded (microneurography) and the response patterns during tonic pressure stimulation (14N at 30 mm(2); 120 s) were assessed using the previously described "marking technique". It was found that tonic pressure stimuli induced augmenting pain responses which were matched by the discharges of initially mechano-insensitive ("silent") C-units, whereas mechano- and heat-responsive "polymodal" C-nociceptors showed a response pattern incompatible with the stimulus-induced perceptions, namely strong initial excitation, followed by adaptation. Eighteen mechano- and heat-responsive "polymodal" C-fibers and 11 mechano-insensitive units were studied. The former had von Frey thresholds <160 mN, the latter were not excited by a von Frey filament of 750 mN (six of them responded to radiant heat stimulation). However, in the course of pressure stimulation, nine of the 11 mechano-insensitive units were activated after more than 20s. A second, identical pressure stimulus induced a stronger response in mechano-insensitive and a weaker response in mechano-responsive units. The stronger response, indicating sensitization, matched the more intense pain perception during the second pressure stimulus.It is concluded that mechano-insensitive C-nociceptors encode pressure-induced pain in human hairy skin and that they play an important role in static mechanical hyperalgesia.     

Possible contribution of protein kinase C in the effects of histamine on the visceral nociceptor activities in vitro

To clarify the possible contribution of protein kinase C activation in histamine-induced excitation and sensitization of the heat response of testicular polymodal receptors, the effects of staurosporine, a protein kinase C inhibitor, and phorbol 12,13-dibutyrate, a protein kinase C activating phorbol ester, were studied in visceral polymodal receptors. Single polymodal receptor activities were recorded in vitro from testis-spermatic nerve preparations obtained from deeply anesthetized dogs (pentobarbital sodium, 30 mg/kg, i.v.). Histamine (10 microM)-induced excitation and facilitation of the heat response of polymodal receptors were both suppressed by staurosporine (1 microM), suggesting that activation of protein kinase C is involved in both these effects of histamine. Application of phorbol 12,13-dibutyrate (0.1 microM) mixed with histamine increased the histamine-induced excitation, whereas a 5 min application of phorbol 12,13-dibutyrate before histamine suppressed it. These results suggest that phorbol 12,13-dibutyrate-activated protein kinase C has inactivation as well as activation effects on the intracellular cascade connected to histamine receptors, and that the former has a slower time course.     

Sensitization to mechanical stimulation by inflammatory mediators and by mild burn in canine visceral nociceptors in vitro

Hyperalgesia to mechanical stimulation and heat is commonly observed in inflamed conditions. Although sensitization to heat is well documented and its mechanism has also been well studied, it remains unclear whether and how nociceptors are sensitized to mechanical stimulation. Therefore we conducted in vitro investigation of which inflammatory mediators (bradykinin, histamine, prostaglandin E2, and protons) sensitize nociceptors to suprathreshold mechanical stimulation and at what concentrations. In addition, we studied the effects of possible second messengers for these mediators downstream of the receptors and also the effects of mild burn. Single polymodal receptor activities were recorded in canine testis-spermatic nerve preparations excised from deeply anesthetized dogs. Mechanical stimulation was applied to the identified receptive field for 10 s with a servo-controlled mechanical stimulator. Bradykinin at 0.001 microM induced neither excitation nor facilitation of the mechanical response; however, it facilitated the mechanical response at 0.01 microM and higher, levels at which significant excitation was also induced by bradykinin alone. Histamine excited the nociceptor and sensitized it to mechanical stimulation at 10 microM and higher. PG E(2) also sensitized the mechanical response, but starting at 1 microM, without inducing excitation by itself. The effects of two possible intracellular messengers for these mediators were studied using forskolin (10 microM), which increases intracellular cAMP, and a protein-kinase-C-stimulating phorbol ester, phorbol 12,13-dibutyrate (0.1 microM). Both substances reversibly facilitated the mechanical response of testicular polymodal receptors. In contrast, low-pH solution (pH: 6.6-4.5) seldom induced excitation and failed to facilitate the mechanical response. After 55 degrees C, 30-s heat stimulation, testicular polymodal receptors were sensitized to mechanical stimulation. These results demonstrated that inflammatory mediators and burn sensitized nociceptor responses to mechanical stimulation and provide support for the idea that peripheral nociceptor sensitization is a mechanism involved in hyperalgesia to mechanical stimulation in inflamed tissues.     

Nicotine modulates evoked GABAergic transmission in the brain

The effects of nicotine on evoked GABAergic synaptic transmission were examined using whole cell recordings from neurons of the lateral spiriform nucleus in embryonic chick brain slices. All synaptic activities were abolished by the GABA(A) receptor antagonist, bicuculline (20 microM). Under voltage-clamp with KCl-filled pipettes (holding potential -70 mV), nicotine (0.1-1.0 microM) increased the frequency of spontaneous GABAergic currents in a dose-dependent manner. Nicotine enhanced electrically evoked GABAergic transmission only at relatively low concentrations of 50-100 nM (but not 25 nM), which approximate the concentrations of nicotine in the blood produced by cigarette smoking. At higher concentrations nicotine had either no effect (0.25 microM) or diminished (0.5-1.0 microM) evoked GABAergic neurotransmission. Nicotine had no significant effect on the postsynaptic current induced by exogenous GABA (30-50 microM). These data imply that nicotine levels attained in smokers are sufficient to enhance evoked GABAergic transmission in the brain, and that this effect is most likely mediated through activation of presynaptic nicotinic receptors.     

NMDA receptor-dependent plasticity of granule cell spiking in the dentate gyrus of normal and epileptic rats

Because granule cells in the dentate gyrus provide a major synaptic input to pyramidal neurons in the CA3 region of the hippocampus, spike generation by granule cells is likely to have a significant role in hippocampal information processing. Granule cells normally fire in a single-spike mode even when inhibition is blocked and provide single-spike output to CA3 when afferent activity converging into the entorhinal cortex from neocortex, brainstem, and other limbic regions increases. The effects of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in gamma-aminobutyric acid-A (GABA(A)) receptor-dependent inhibition on spike generation were examined in granule cells of the dentate gyrus. In contrast to the single-spike mode observed in normal bathing conditions, perforant path stimulation in Mg(2+)-free bathing conditions evoked graded burst discharges in granule cells which increased in duration, amplitude, and number of spikes as a function of stimulus intensity. After burst discharges were evoked during transient exposure to bathing conditions that relieve the Mg(2+) block of the NMDA receptor, there was a marked increase in the NMDA receptor-dependent component of the EPSP, but no significant increase in the non-NMDA receptor-dependent component of the EPSP in normal bathing medium. Supramaximal perforant path stimulation still evoked only a single spike, but granule cell spike generation was immediately converted from a single-spike firing mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg(2+)-free conditions and the expression of burst discharges evoked in normal bathing medium with subsequent disinhibition were both blocked by DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in the perforant path, which is induced by NMDA receptors and is also expressed by alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of evoked synaptic response, and in the dentate gyrus reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. NMDA receptor-dependent plasticity of granule cell spike generation, which can be distinguished from LTP and induces long-term susceptibility to epileptic burst discharge under conditions of reduced inhibition, could modify information processing in the hippocampus and promote epileptic synchronization by increasing excitatory input into CA3.     

B1 and B2 antagonists and bradykinin-induced blood flow in rat skin inflammation

OBJECTIVE AND DESIGN: The aim of the present study was to examine the contribution of the two kinin receptors B1 and B2 to the increased blood flow observed in response to bradykinin (BK) in a blister model under different injury conditions. MATERIAL: Young male Sprague-Dawley rats weighing 250-350 g were used. METHODS: A vacuum-induced blister was raised in the rat hind paw and blood flow measured in the superfused blister base under four different conditions including, early phase acute injury; late phase acute injury; recurrent injury and early phase acute injury in the setting of chronic nerve damage. BK (10 microM) was superfused alone, or in the presence of the B1 antagonist DesArg9Leu8BK (DALBK), (10 nM) and/or the B2 antagonist [D-Arg,Hyp3,Thi5 D-Tic7,Oic8] Bradykinin (HOE 140) (10 nM). RESULTS: HOE 140 significantly inhibited the BK response in all models. Significant inhibition of BK-induced vasodilatation by DALBK was only observed in the late phase acute and recurrent injury models. CONCLUSIONS: The results suggest that the involvement of the inducible B1 receptor in skin inflammation site is related to the site, duration and recurrence of the injury condition.     

Thin-fibre receptors responding to mechanical, chemical, and thermal stimulation in the skeletal muscle of the dog

1. Unitary activities of muscular thin fibre afferents, which were not sensitive to muscle stretching, were recorded from the nerve of the medial gastrocnemius muscle of the dog. Responses to mechanical stimulation, intra-arterial injection and local application of chemical solutions, and thermal stimulation of the surface of the muscle were studied. It was observed that polymodal receptors which responded to all types of stimulation existed in the thin fibre afferents of the muscle.2. The receptive area of these units tested by mechanical stimulation was spot-like and appeared to be located not only on the surface but in the midst of the muscle.3. The mechanical response varied among these units with respect to the threshold and the pattern of discharges.4. In these units, NaCl, KCl, and bradykinin consistently evoked responses, with differences in the latencies and discharge patterns, while solutions of histamine, acetylcholine and sodium citrate caused responses less consistently and less effectively. In the stretch receptors, chemical stimulation applied in the same way as tested in the thin fibre afferents produced quite different features in their responses.5. Heating the receptive area of the muscle surface caused responses in twenty-five out of thirty-six units, which were sensitive both to mechanical and to chemical stimulations. The threshold varied from 38.0 to 48.3 degrees C, with a mean of 43.1 degrees C for C fibre units and 41 degrees C for A-delta fibre units.6. The responses to heating were consistently obtained in the units responding to the surface application of chemical solutions. However, the above response was never obtained in the units which did not respond to surface chemical stimulation but responded to intra-arterial injection. These results suggest a large population of polymodal receptors in the muscular thin fibre afferents.     

Inhibitory control of the GABAergic transmission in the rat neostriatum by D2 dopamine receptors

The aim of the study was to determine the role of dopamine on the GABAergic input to striatal projection neurons. Accordingly, the effect of the activation of dopamine D2-like receptors on GABA-mediated depolarizing postsynaptic potentials evoked in striatal slices by local stimulation was studied. Conventional intracellular recording techniques were used to record the synaptic responses. The experiments were done in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM) and (+)-2-amino-5-phosphonovaleric acid (40 microM) to block the participation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate and N-methyl-D-aspartate receptors in the synaptic response. The GABAergic nature of the response was assessed by its potentiation by pentobarbital (50 microM) and by its elimination by bicuculline or picrotoxin. At 100 nM, a concentration already maximal, dopamine inhibited by 55% the GABAergic synaptic response. The inhibitory effect was totally blocked by the selective antagonist of D2-like receptors, sulpiride (100 nM). The dopamine inhibition was observed only in one-third of the studied neurons and was concentration dependent (IC50 = 14 nM). The inhibition was not associated with changes in the input resistance or any other membrane property. In addition, dopamine (50 nM) reduced the frequency but not the amplitude of spontaneous, bicuculline-sensitive depolarizing postsynaptic potentials. The D2-like receptor agonist quinpirole also dose-dependently (IC50 = 10 nM) inhibited the GABAergic synaptic response. As with dopamine, the inhibition did not change the membrane properties of the studied neurons. In addition, the quinpirole induced inhibition of the GABA response was accompanied by increased paired-pulse facilitation. The results indicate that D2-like receptors located on intrinsic GABAergic terminals in the rat striatum exert an inhibitory control of the GABAergic input to striatal projection neurons. The dopaminergic effect would be translated in facilitation of the firing of the neurons upon the arrival of the cortical input.