Neuromedin U receptor 2 does not play a role in the development of neuropathic pain following nerve injury in mice.

BACKGROUND: Previous studies have identified neuromedin U receptor 2 (NMUR2) as the subtype mediating the effects of neuromedin U on acute chemo-nociception induced by capsaicin or formalin injection. The aims of this study were to determine whether NMUR2 is required for the development of mechanical hypersensitivity after nerve injury or heat hypersensitivity after inflammation and whether there is a gender difference in the contribution of NMUR2 to nociception. METHODS: Mechanical sensitivity was assessed with von Frey filaments in wild type (WT) and NMUR2-null mice at baseline and following spared tibial nerve (STN) injury. Heat sensitivity was also assessed at baseline and after induction of inflammation with Freund's complete adjuvant (FCA). RESULTS: The response to von Frey filaments at baseline was similar for WT and NMUR2-null mice and for males and females. The response of male NMUR2-null mice was slightly but significantly decreased when exposed to 52 °C but not 58 °C heat stimuli. There was no difference between the stimulus-response curve for WT and NMUR2-null mice 7, 13 and 16 days after nerve injury. Similarly, after FCA-induced inflammation, there was no significant difference in heat hyperalgesia between WT and NMUR2-null mice or male or female mice in responses to temperatures ranging from 44 to 48 °C. CONCLUSIONS: The present data do not support a significant contribution of NMUR2 to the development of hypersensitivity after nerve injury or tissue inflammation, suggesting that pharmacological intervention aimed at the NMUR2 receptor might not be a valuable approach for the treatment of chronic pain.

Widely expressed transcripts for chemokine receptor CXCR1 in identified glutamatergic, gamma-aminobutyric acidergic, and cholinergic neurons and astrocytes of the rat brain: a single-cell reverse transcription-multiplex polymerase chain reaction study.

Increasing evidence suggests that the chemokine interleukin (IL)-8/CXCL8 plays important roles in CNS development, neuronal survival, modulation of excitability, and neuroimmune response. Recently, we have shown that CXCL8 can acutely modulate ion channel activity in septal neurons expressing receptors CXCR1 and/or CXCR2. This was a surprising finding, insofar as CXCR1 expression had not been described for the mammalian brain. Here we investigated whether CXCR1 transcripts are present in other brain regions, whether they are expressed at the single-cell level in molecularly identified neurons and astrocytes, and how they are regulated during early postnatal development. In addition, possible cellular colocalization of CXCR1 and CXCR2 transcripts was examined. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that CXCR1 mRNAs were expressed in the septum, striatum, hippocampus, cerebellum, and cortex (temporoparietal and entorhinal) at different levels and appeared to be regulated independently from CXCR2 during development. By using RT multiplex PCR on acutely dissociated cells from these brain regions, we show that CXCR1 transcripts were expressed in 83% of 84 sampled neurons displaying cholinergic (choline acetyltransferase mRNAs), gamma-aminobutyric acidergic (glutamic acid decarboxylases 65 and 67 mRNAs), or glutamatergic (vesicular glutamate transporters 1 and 2 mRNAs) phenotypes. CXCR1 and CXCR2 transcripts were colocalized in 45% of neurons sampled and also were present in some glial fibrillary acidic protein mRNA-expressing astrocytes. This is the first study to demonstrate the widespread expression of CXCR1 transcripts in the brain and suggests that CXCR1 may have hitherto unsuspected roles in neuromodulation and inflammation.

Pro-nociceptive effects of neuromedin U in rat.

The neuropeptide neuromedin U (NMU) has been shown to have significant effects on cardiovascular, gastrointestinal and CNS functions. The peptide was first isolated from the porcine spinal cord and later shown to be present in spinal cords of other species. Little is known about the distribution of neuromedin U receptors (NMURs) in the spinal cord and the spinal action of the peptide. Here we report on the expression of NMURs and a potential role in nociception in the rat spinal cord using a combination of behavioral and electrophysiological studies. Receptor autoradiography showed that NMU-23 binding was restricted to the superficial layers of spinal cord, a region known to be involved in the control of nociception. In situ hybridization analysis indicated the mRNA of NMUR2 was located in the same region (laminae I and IIo) as NMU-23 binding, while the mRNA for NMU receptor 1 was observed in a subpopulation of small diameter neurons of dorsal root ganglia. Intrathecal (i.t.) administration of neuromedin U-23 (0.4-4.0 nmol/10 microl) dose-dependently decreased both the mechanical threshold to von Frey hair stimulation and the withdrawal latency to a noxious thermal stimulus. Mechanical allodynia was observed between 10 and 120 min, peaking at 30 min and heat hyperalgesia was observed 10-30 min after i.t. administration of NMU-23. A similar mechanical allodynia was also observed following i.t. administration of NMU-8 (0.4-4 nmol/10 microl). A significant enhancement of the excitability of flexor reflex was induced by intrathecal administration of NMU-23 (4 nmol/10 microl). Evoked responses to touch and pinch stimuli were increased by 439+/-94% and 188+/-36% (P<0.01, n=6) respectively. The behavioral and electrophysiological data demonstrate, for the first time, a pro-nociceptive action of NMU. The restricted distribution of NMU receptors to a region of the spinal cord involved in nociception suggests that this peptide receptor system may play a role in nociception.

The chemokine interleukin-8 acutely reduces Ca(2+) currents in identified cholinergic septal neurons expressing CXCR1 and CXCR2 receptor mRNAs.

The chemokine IL-8 is known to be synthesized by glial cells in the brain. It has traditionally been shown to have an important role in neuroinflammation but recent evidence indicates that it may also be involved in rapid signaling in neurons. We investigated how IL-8 participates in rapid neuronal signaling by using a combination of whole-cell recording and single-cell RT-PCR on dissociated rat septal neurons. We show that IL-8 can acutely reduce Ca(2+) currents in septal neurons, an effect that was concentration-dependent, involved the closure of L- and N-type Ca(2+) channels, and the activation of G(ialpha1) and/or G(ialpha2) subtype(s) of G-proteins. Analysis of the mRNAs from the recorded neurons revealed that the latter were all cholinergic in nature. Moreover, we found that all cholinergic neurons that responded to IL-8, expressed mRNAs for either one or both IL-8 receptors CXCR1 and CXCR2. This is the first report of a chemokine that modulates ion channels in neurons via G-proteins, and the first demonstration that mRNAs for CXCR1 are expressed in the brain. Our results suggest that IL-8 release by glial cells in vivo may activate CXCR1 and CXCR2 receptors on cholinergic septal neurons and acutely modulate their excitability by closing calcium channels.

Hippocampal theta rhythm in anesthetized rats: role of AMPA glutamate receptors.

The present study describes the effects of intraseptal infusions of 1 nmol AMPA and 12 nmol NBQX on both frequency and amplitude of physostigmine-induced theta rhythm in urethane-anesthetized rats. Infusion of AMPA increased the theta frequency. This effect was blocked by a prior infusion of NBQX. Infusion of NBQX decreased the theta amplitude, and this effect was not altered by AMPA. These results suggest that the septal AMPA/glutamate receptors exert subtle modulatory influences on septohippocampal cells involved in theta rhythm generation.

Nicotine improves memory in an object recognition task in rats.

Nicotine was investigated for its mnemonic effect in a two trials object recognition task. In the first trial, two copies of the same object were presented. In the second trial (24 h after), one of the familiar object and a new object were presented. The time spent exploring the new object by control rats was not significantly different from the exploration time of the familiar object, indicating that they did not remember the familiar object. Rats injected with nicotine before the first trial, after the first trial or before the second trial spent more time in exploring the new object than the familiar one at the second trial. These results suggest that, in normal rats, acute nicotine enhances acquisition, consolidation and restitution of the information in an object recognition task.

Intraseptal infusions of a low dose of AP5, a NMDA receptor antagonist, improves memory in an object recognition task in rats.

The present study describes the effects of intraseptal microinjections of 2 nmol of AP5 upon memory of rats subjected to a two trial object recognition task. This task allows us to detect either a disruption or an improvement of memory according to the duration of the interval between the sample trial (T1) and the choice trial (T2). AP5 injected before T1 did not disrupt memory in a schedule able to detect an amnesia. In a schedule able to detect an improvement of memory, AP5 injected either 10 min before or just after T1, or 10 min before T2, improved retention. These results suggest that microinjection of a low dose of AP5 in the septum improves the acquisition, the consolidation and the restitution of the information in a working memory task.

Effects of intraseptal infusions of N-methyl-D-aspartate receptor ligands on memory in an object recognition task in rats.

The present study describes the effects of intraseptal microinjections of N-methyl-D-aspartate (NMDA) or AP5, an agonist and an antagonist of the NMDA receptors, respectively, upon memory of rats. Animals were injected with the drug or vehicle immediately after the first exposure to two identical objects, and the duration of exploration of the familiar and a new object were evaluated 45 min or 24 h later. Vehicle-treated rats explored the new object longer than the familiar object when the intertrial time was 45 min, indicating that they remembered the familiar object, but not when the intertrial time was 24 h. The difference of exploration time between the objects was increased by NMDA, but not by AP5, when the intertrial time was 24 h, and decreased by AP5 when the intertrial interval was 45 min. These results suggest that NMDA and AP5 improves and disrupts, respectively, the consolidation in a working memory task.

Relations between acetylcholine release and electrophysiological characteristics of theta rhythm: a microdialysis study in the urethane-anesthetized rat hippocampus.

In urethane-anesthetized rats, recording electrodes were implanted in the left dorsal hippocampus and a dialysis probe was placed in the contralateral dorsal or ventral hippocampus. Samples of extracellular acetylcholine (ACh) levels were assessed at 10-min intervals over a period of 30 min using microdialysis with high-performance liquid chromatography with electrochemical detection. EEG was recorded during the same period and amplitude, frequency, and duration of theta rhythm were calculated for each of the three 10-min intervals. Data were analyzed using the two-tailed Spearman rank-order correlation test. A positive and high rank correlation coefficient (rho = 0.90, p < 0.01, n = 8) was seen between the average ACh outflow in the dorsal hippocampus and the average theta amplitude, both being calculated for the entire collection period. A lower but statistically significant positive correlation (rho = 0.59, p < 0.01) between dorsal hippocampus ACh output and theta amplitude was also found when the couples of values collected for the 30-min period were pooled (n = 20). In contrast, frequency and duration of theta were not significantly correlated with dorsal hippocampus ACh release. Also, no statistically significant correlation (p > 0.05) was found between ACh output in the ventral hippocampus and theta parameters. Because changes in hippocampal ACh outflow are believed to be the reflection of changes in number and/or level of activity of cholinergic afferents to the dorsal hippocampus, our present findings support the view that, at least in the dorsal hippocampus of the urethane-anesthetized rat, the septohippocampal cholinergic projection regulates the theta amplitude but not frequency. Finally, the possibility that ACh outflow increase and tonic release in the hippocampus is not a sufficient condition to induce and maintain theta in the urethane-anesthetized rat is discussed.

Intraseptal infusion of selective and competitive glutamate receptor agonist NMDA and antagonist D-2-amino-5-phosphonopentanoic acid spectral implications for the physostigmine-induced hippocampal theta rhythm in urethane-anesthetized rats.

Theta (theta) rhythm may be mediated, at least in part, by a glutamate neurotransmitter. Thus, in the present study, it was hypothesized that the septum glutamatergic NMDA receptor subtype may be involved in the modulation of physostigmine-induced theta rhythm. To test this hypothesis, we analyzed, in the urethane-anesthetized rat, the effects of septum application of NMDA and D-2-amino-5-phosphonopentanoic acid (AP5), selective and competitive NMDA agonist and antagonist, respectively, on the spectral characteristics of hippocampal theta rhythm elicited by intravenous injection of a anticholinesterase agent, physostigmine. A low dose (16 nmol) of AP5 did not significantly affect EEG recordings, whereas a high dose (50.75 nmol) resulted in significant decreases in phase (-61.8%) at theta frequency, peak theta power (-64.2%), and absolute power of the low-frequency theta band (-67%). These electroencephalographic alterations, which appeared at 50.75 nmol AP5, were amplified following application of massive doses of the drug (121.8 nmol, n = 1, and 162 nmol, n = 1). Amplification, however, was slight and the theta waves remained clearly detectable. On the other hand, the infusion of NMDA resulted in a significant increase in frequency (+25%) of this rhythm, but this effect was completely antagonized by prior local administration of 16 nmol AP5. Our data suggest that the septal NMDA receptors exert subtle modulatory influences on the septohippocampal cells involved in physostigmine-induced theta wave production, which has not been reported elsewhere: tonic with respect to both low-frequency theta band power and theta phase, and phasic with respect to theta frequency. Our data also indicate that the septum may be a sensitive action site for exogenously administered glutamatergic drugs.

Involvement of M2 and non-M2 muscarinic receptors in hippocampal theta rhythm induced by carbachol infusion into the septum of the rat.

Binding and autoradiographic studies have shown the presence of a rather high density of M2 muscarinic subtype receptors and the apparent absence or low density of the M1 subtype in the septum. We tested the hypothesis that, in the urethane-anesthetized rat, septal M2 receptors are involved in the generation of the hippocampal theta (theta) rhythm induced by intraseptal administration of carbachol, a potent cholinomimetic agent. Carbachol-induced theta was blocked by local infusion of the unspecific muscarinic antagonist agent, atropine (20 micrograms (29.55 nM)), given 10 min prior to carbachol. The intraseptal administration of low to high doses of gallamine (range: 20-180 micrograms (22.43-201.90 nM)), a specific M2 antagonist which displays high affinity for the septal region, resulted in significant changes in the electrophysiological characteristics of carbachol-induced theta but failed to abolish this rhythm. It is suggested that the latter may have resulted from a combined activation of both M2 and non-M2 receptors at septal level.