Sex-dependent roles of prolactin and prolactin receptor in postoperative pain and hyperalgesia in mice.

Although surgical trauma activates the anterior pituitary gland and elicits an increase in prolactin (PRL) serum levels that can modulate nociceptive responses, the role of PRL and the PRL-receptor (PRL-R) in thermal and mechanical hyperalgesia in postoperative pain is unknown. Acute postoperative pain condition was generated with the use of the hindpaw plantar incision model. Results showed endogenous PRL levels were significantly increased in serum, operated hindpaw and spinal cords of male and female rats 24h after incision. These alterations were especially pronounced in females. We then examined the role of the PRL system in thermal and mechanical hyperalgesia in male and female mice 3-168 h after plantar incision with the use of knock-out (KO) mice with PRL or PRL-R gene ablations and in wild-type (WT) mice. WT mice showed postoperative cold hyperalgesia in a sex-dependent manner (only in females), but with no effect on heat hyperalgesia or mechanical allodynia in either sex. Studies in KO mice showed no effect of PRL and PRL-R gene ablation on heat and cold hyperalgesia in male mice, while heat hyperlgesia were reduced 3-72 h post-surgery in female PRL and PRL-R KO mice. In contrast, PRL and PRL-R ablations significantly attenuated mechanical allodynia 3-72 h post-surgery in both male and female mice. Overall, we found elevated PRL levels in serum, hindpaws and spinal cords after incision, and identify a contributory role for the PRL system in postoperative pain responses to thermal stimuli in females and to mechanical stimuli in both males and females.

LPS sensitizes TRPV1 via activation of TLR4 in trigeminal sensory neurons.

Recent studies have demonstrated that the lipopolysaccharide (LPS) receptor (TLR4) is expressed in TRPV1 containing trigeminal sensory neurons. In this study, we evaluated whether LPS activates trigeminal neurons, and sensitizes TRPV1 responses via TLR4. To test this novel hypothesis, we first demonstrated that LPS binds to receptors in trigeminal neurons using competitive binding. Second, we demonstrated that LPS evoked a concentration-dependent increase in intracellular calcium accumulation (Ca(2+))(i) and inward currents. Third, LPS significantly sensitized TRPV1 to capsaicin measured by (Ca(2+))(i), release of calcitonin gene-related peptide, and inward currents. Importantly, a selective TLR4 antagonist blocked these effects. Analysis of these data, collectively, demonstrates that LPS is capable of directly activating trigeminal neurons, and sensitizing TRPV1 via a TLR4-mediated mechanism. These findings are consistent with the hypothesis that trigeminal neurons are capable of detecting pathogenic bacterial components leading to sensitization of TRPV1, possibly contributing to the inflammatory pain often observed in bacterial infections.

Transient receptor potential V1 regulates activation and modulation of transient receptor potential A1 by Ca2+.

The transient receptor potential A1 (TRPA1) channel contributes to nociceptive signaling in certain pain models. It has been suggested that Ca(2+), which activates and modulates TRPA1, could play a critical regulatory role in this process. Since TRPA1 and transient receptor potential V1 (TRPV1) channels are co-expressed and interact in neurons, we investigated whether activation and modulation of TRPA1 by Ca(2+) is regulated by TRPV1. Cell-attached recordings showed that TRPA1 is activated by extracellular Ca(2+) ([Ca(2+)](e)) in concentration-response fashion. This activation, especially by 2 mM [Ca(2+)](e) was substantially suppressed by co-expression with TRPV1. Inside-out recordings demonstrated that intracellular Ca(2+) ([Ca(2+)](i))-triggered activation of TRPA1 was attenuated by the presence of TRPV1 only at 2 mM [Ca(2+)](e), but not in Ca(2+)-free conditions. Further, depletion of internal Ca(2+) stores by thapsigargin generated TRPA1-mediated currents, which is affected by TRPV1 in both Chinese hamster ovary cells and sensory neurons. Since mustard oil current (I(MO)) is modulated by [Ca(2+)](e), we next examined whether alterations in the Ca(2+)-permeability of TRPV1 by mutating Y671 effect I(MO) properties. First it was demonstrated that the mutations in TRPV1 did not affect association of the TRPA1 and TRPV1 channels. However, these TRPV1 mutations, particularly Y671K, altered the following characteristics of TRPA1: magnitude of I(MO) in presence and absence of [Ca(2+)](e); the influence of [Ca(2+)](e) on the voltage-dependency of I(MO), and open probability of single-channel I(MO). In summary, activation of TRPA1 by [Ca(2+)](e) and [Ca(2+)](i) is controlled by the TRPV1 channel, and characteristics of I(MO) depend on Ca(2+) permeability of the TRPV1 channel.

NGF up-regulates TRPA1: implications for orofacial pain.

The transient receptor potential ankyrin repeat 1 (TRPA1) channel is believed to be involved in many forms of acute and chronic hyperalgesia. Nerve Growth Factor (NGF) regulates chronic inflammatory hyperalgesia by controlling gene expression in sensory neurons, including genes involved in inflammatory hyperalgesia in the dental pulp. We hypothesized that NGF increases functional activities of the TRPA1 channel in trigeminal ganglion neurons. Here, we show that NGF induced a concentration- and time-dependent up-regulation of TRPA1 mRNA in trigeminal ganglia neurons, as detected by real-time RT-PCR and in situ hybridization. In addition, NGF evoked a time-dependent increase of mustard oil (MO)-evoked TRPA1 activation in trigeminal ganglia neurons. Collectively, these findings demonstrate that NGF participates in the functional up-regulation of TRPA1 in trigeminal ganglia neurons. These enhanced activities of TRPA1 could play an important role in the development of hyperalgesia following nerve injury and inflammation in the orofacial region.

[Pathological types of breathing during acute hypoxia]. / Patologicheskie tipy dykhaniia pri vozdeistvii ostroi gipoksii.

Different breathing pathologies (apneusis, gasping, Cheyne-Stokes breathing and Kussmaul breathing) were observed in anaesthetized rabbits and rats exposed to hypoxia at the altitude of 7500-8000 meters. Dominance of the high-voltage slow EKG activity (the delta-wave type) suggested deterioration of the brain functioning. Registration of impulse activities of the inspiration and expiration breathing neurons in medulla oblongata revealed a higher tolerance of the inspiration neurons to severe hypoxia which is attributed to the metabolic specifics and functional heterogeneity of these neurons. Character of the neuron impulsation is indicative of the reflectory effects of hemo- and mechanoreceptors, and the direct effects of different humoral substances resulting in impairment of the central control of pacemakers and breathing pathologies. It appears that various breathing pathologies in altitude chambers and mountains are triggered by the hypoxic factor. However, no dependence was established either between the initial breathing rhythm (before "ascent") and various types of breathing at the "altitude" or these types of breathing and magnitude of hypoxia.

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors.

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain. The ATP receptor P2X3 is selectively expressed by nociceptors and is one of seven ATP-gated, cation-selective ion channels. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X(2/3) heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious 'warming' stimuli.

A new member of the acid-sensing ion channel family.

Acid-sensing ion channels (ASICs) are members of the epithelial sodium channel (ENaC)-degenerin family of two-pass transmembrane segment protein subunits which form multimeric cation channels. Members of the ENaC-degenerin family are gated by stimuli as diverse as protons, peptides and mechanical distension. Here we describe a new member of the family, SPASIC or ASIC 4 (spinal cord ASIC) which is expressed throughout the central nervous system in an overlapping population of neurons that also express the ASIC subunit MDEG2. ASIC-4 which shows 44% identify with ASIC is developmentally regulated and expressed in a subset of sensory neurons as well as in the CNS. However, despite the strong homology with ASIC, the ASIC-4 transcript does not encode a proton gated cation channel.

A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons.

TTX-resistant (TTX-R) sodium currents are preferentially expressed in small C-type dorsal root ganglion (DRG) neurons, which include nociceptive neurons. Two mRNAs that are predicted to encode TTX-R sodium channels, SNS and NaN, are preferentially expressed in C-type DRG cells. To determine whether there are multiple TTX-R currents in these cells, we used patch-clamp recordings to study sodium currents in SNS-null mice and found a novel persistent voltage-dependent sodium current in small DRG neurons of both SNS-null and wild-type mice. Like SNS currents, this current is highly resistant to TTX (Ki = 39+/-9 microM). In contrast to SNS currents, the threshold for activation of this current is near 70 mV, the midpoint of steady-state inactivation is -44 +/- 1 mV, and the time constant for inactivation is 43+/-4 msec at 20 mV. The presence of this current in SNS-null and wild-type mice demonstrates that a distinct sodium channel isoform, which we suggest to be NaN, underlies this persistent TTX-R current. Importantly, the hyperpolarized voltage-dependence of this current, the substantial overlap of its activation and steady-state inactivation curves and its persistent nature suggest that this current is active near resting potential, where it may play an important role in regulating excitability of primary sensory neurons.

The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel alpha subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

Trans-splicing of a voltage-gated sodium channel is regulated by nerve growth factor.

Mammalian sensory neurons express a voltage-gated sodium channel named SNS. Here we report the identification of an SNS transcript (SNS-A) that contains an exact repeat of exons 12, 13 and 14 encoding a partial repeat of domain II. Because the exons 12-14 are present in single copies in genomic DNA, the SNS-A transcript must arise by trans-splicing. Nerve growth factor, which regulates pain thresholds, and the functional expression of voltage-gated sodium channels increases the levels of the SNS-A transcript several-fold both in vivo and in vitro as measured by RNase protection methods, as well as RT-PCR. These data demonstrate a novel regulatory role for the nerve growth factor and are the first example of trans-splicing in the vertebrate nervous system.

A sensory neuron-specific, proton-gated ion channel.

Proton-gated channels expressed by sensory neurons are of particular interest because low pH causes pain. Two proton-gated channels, acid-sensing ionic channel (ASIC) and dorsal root ASIC (DRASIC), that are members of the amiloride-sensitive ENaC/Degenerin family are known to be expressed by sensory neurons. Here, we describe the cloning and characterization of an ASIC splice variant, ASIC-beta, which contains a unique N-terminal 172 aa, as well as unique 5' and 3' untranslated sequences. ASIC-beta, unlike ASIC and DRASIC, is found only in a subset of small and large diameter sensory neurons and is absent from sympathetic neurons or the central nervous system. The patterns of expression of ASIC and ASIC-beta transcripts in rat dorsal root ganglion neurons are distinct. When expressed in COS-7 cells, ASIC-beta forms a functional channel with electrophysiological properties distinct from ASIC and DRASIC. The pH dependency and sensitivity to amiloride of ASIC-beta is similar to that described for ASIC, but unlike ASIC, the channel is not permeable to calcium, nor are ASIC-beta-mediated currents inhibited by extracellular calcium. The unique distribution of ASIC-beta suggests that it may play a specialized role in sensory neuron function.

Structure and chromosomal mapping of the mouse P2X3 gene.

P2X3 is one of seven cloned ATP-gated non-selective cation channels. We have isolated a full-length mouse P2X3 gene from a phage lambda-129/Sv genomic library. The gene consists of 12 exons spanning a locus of approximately 40 kb. No significant similarities have been found between the genomic organisation of the mouse P2X3 gene and genes encoding other ion channels. The encoded mouse P2X3 protein consists of 397 amino acids and shows 99% identity with rat P2X3. Using RNase protection and primer extension assays, multiple transcription initiation sites have been mapped in the mouse P2X3 promoter to a region 162-168 bp upstream of the translation initiation codon. The P2X3 gene has been mapped to mouse chromosome 2p by fluorescence in situ hybridisation. The RAG locus-associated gene T160 is located 1.8 kb upstream of the transcription start site of mouse P2X3 gene. The promoter region of the mouse P2X3 gene lacks a conventional TATA and CCAAT consensus sites, and initiator elements. P2X3 is the first member of the P2X gene family to be completely characterised.

A single serine residue confers tetrodotoxin insensitivity on the rat sensory-neuron-specific sodium channel SNS.

Sensory neurons express a sodium channel (SNS) that is highly resistant to block by tetrodotoxin (IC50 = 60 microM). SNS is 65% homologous to the cardiac sodium channel, in which a single hydrophilic residue in the SS2 segment is critical for tetrodotoxin resistance. By site-directed mutagenesis, we have substituted phenylalanine for serine at the equivalent position in SNS: this mutated (S356F) SNS channel is functionally similar to wild-type SNS when expressed in Xenopus oocytes, but is potently blocked by tetrodotoxin and saxitoxin with IC50s of 2.8 nM and 8.2 nM, respectively. These data provide clues to the rational design of selective blockers of SNS with potential as analgesic drugs.

Cloning and characterization of a mouse sensory neuron tetrodotoxin-resistant voltage-gated sodium channel gene, Scn10a.

Small-diameter sensory neurons associated with unmyelinated axons express a tetrodotoxin-insensitive (TTXi) voltage-gated sodium channel (VGSC) that may play an important role in the transmission of nociceptive information to the spinal cord. A TTXi VGSC, named SNS, that accounts for the tetrodotoxin-resistant sodium current described in sensory neurons has been cloned from rat dorsal root ganglia. Using recombinant lambda phage clones encoding a mouse 129/SV genomic library, we have determined the detailed structure of the mouse SNS gene (Scn10a), including the location of exon-intron boundaries and the nucleotide sequence of the exons. The gene consists of 27 exons spanning approximately 90 kb on chromosome 9. Mouse SNS shows 95.3% overall amino acid identity to rat SNS and 98.5% identity throughout the putative transmembrane segments and the intracellular loop linking domains 3 and 4. The sizes of the exons and the exon-intron junction positions of the mouse SNS and the human skeletal muscle VGSC genes are remarkably conserved. These results provide the basis for an evolutionary comparison of sodium channels, the construction and analysis of a mouse SNS null mutant as a direct approach to understanding the biological function of SNS, and the identification of regulatory elements that are responsible for the tissue- and cell-specific expression of SNS.

A role for calcineurin in the desensitization of the P2X3 receptor.

The sensory neurone-specific ATP-gated cation channel P2X3, when expressed in Xenopus oocytes, desensitizes rapidly. Complete removal of extracellular calcium abolishes desensitization. Pretreatment of oocytes with cyclosporin also abolishes P2X3 desensitization. When the calcineurin auto-inhibitory peptide (CaN A457-481) was injected into oocytes, the rate of desensitization of P2X3 decreased on consecutive applications of ATP, suggesting a role for calcineurin in the regulation of desensitization. Truncated P2X3 clones initiating at Met-75 lack the N-terminal intracellular region, but express functional channels in oocytes that do not desensitize. Taken together, these data suggest that P2X3 desensitizes through a calcium-dependent calcineurin-mediated dephosphorylation involving N-terminal residues that are phosphorylated in functional channels.

Structure and distribution of a broadly expressed atypical sodium channel.

A cDNA clone isolated from a rat dorsal root ganglion library encodes a 195 kDa voltage-gated sodium channel-like protein (SCL-11) with homology to the mouse (87%) and human (72%) atypical Na+ channels and rat partial clone NaG (98%). Two dominant mRNAs of 4.5 and 7 kb are expressed. The transcripts are present in lung, Schwann cells, pituitary and tissues containing smooth muscle cells. No functional channels could be detected on oocyte injection with cRNA, consistent with the absence of structural features necessary for voltage-gated sodium channel activity.

Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain.

Increased voltage-gated sodium channel activity may contribute to the hyperexcitability of sensory neurons in inflammatory and neuropathic pain states. We examined the levels of the transcript encoding the tetrodotoxin-resistant sodium channel SNS in dorsal root ganglion neurons in a range of inflammatory and neuropathic pain models in the rat. Local Freund's adjuvant or systemic nerve growth factor-induced inflammation did not substantially alter the total levels of SNS mRNA. When NGF-treated adult rat DRG neurons in vitro were compared with NGF-depleted control neurons, SNS total mRNA levels and the levels of membrane-associated immunoreactive SNS showed a small increase (17 and 25%, respectively), while CGRP levels increased fourfold. SNS expression is thus little dependent on NGF even though SNS transcript levels dropped by more than 60% 7-14 days after axotomy. In the streptozotocin diabetic rat SNS levels fell 25%, while in several manipulations of the L5/6 tight nerve ligation rat neuropathic pain model, SNS levels fell 40-80% in rat strains that are either susceptible or relatively resistant to the development of allodynia. Increased expression of SNS mRNA is thus unlikely to underlie sensory neuron hyperexcitability associated with inflammation, while lowered SNS transcript levels are associated with peripheral nerve damage.

[Cloning genes, specifically expressed in the cerebral cortex and cerebellum of adult rats]. / Klonirovanie genov, spetsificheski ékspressiruiushchikhsia v kore golovnogo mozga i mozzhechke vzroslykh krys.

To isolate genes differentially expressed in the rat brain cortex and cerebellum, a subtractive cDNA cloning procedure requiring only small quantities of poly(A+) RNA, followed by differential screening, was used. Two novel genes, MK and 3L7, with cortex- and cerebellum-specific expression were identified. These genes displayed different expression patterns in the brain cortex, cerebellum, and hippocampus during postnatal development.

[Cloning of genes, differentially expressed in the cerebellum during postnatal rat development]. / Klonirovanie genov, differentsioal'no ékspressiruiushchikhsia v mozzhechke postnatal'nom razvitii krys.

To identify genes differentially expressed in the developing rat cerebellum, a subtractive cDNA cloning procedure, followed by differential screening, was used. Four novel genes, MB, MF, 3E7, and 3C6, the transcriptional activity of which changed by a factor of five during cerebellar development, were isolated. The genes obtained were differentially expressed in different regions of the central nervous system. Variations in the levels of corresponding transcripts in the brain cortex and cerebellum were also recorded during postnatal rat development.

Molecular genetic approaches to nociceptor development and function.

The activation of peripheral nociceptors is the subject of intense scrutiny, because of its significance in pain regulation. Genetic approaches, including homology cloning, difference cloning and transgenic manipulation of mice are providing useful insights into nociceptor function. Recent work suggests that transcriptional regulators (for example, islet-I), which are expressed relatively selectively in sensory neurones, play a crucial role in defining cellular phenotype. Difference cloning has identified genes which encode both ligand-gated and voltage-gated ion channels expressed by small-diameter sensory neurones. The role of inflammatory mediators such as NGF in regulating nociceptor function has been clarified in mis-expression and deletion studies. An understanding of the mechanisms that regulate gene expression in nociceptors should provide new ways to manipulate nociceptor sensitivity, with potential significance for pain therapy.