RESUMO
Previous reports have demonstrated that small- to medium-diameter dorsal root ganglia (DRG) cells in rats can be subgrouped into individual cell types by patterns of voltage-activated currents. These cell types have consistent responses to algesic compounds and maintain characteristic histochemical phenotypes. Using immunocytochemical methods, we have now examined expression of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related acid sensitive K+ (TASK) channels, TASK-1, TASK-2 and TASK-3, in nine electrophysiologically identified small- to medium-diameter DRG cell types. The immunoreactivity in DRG cells was diverse, with all nine cell types expressing one to all three TASK channels. Some cells expressed TASK-1 (types 1, 4, 6 and 9), some TASK-2 (types 2, 4, 5, 6, 7 and 9), and some TASK-3 (types 1, 2, 3, 4, 5, 6 and 8). The co-expression of TASK-1 and TASK-3 in cell types 1, 4 and 6 suggests that these sensory afferents might contain functional heterodimeric channels. In peripheral sensory afferents, TASK channels have been implicated in the pain sensory transduction pathway, and can be modulated by anesthetics and neuroprotective agents. This study seeks to identify TASK channel populations in electrophysiologically characterized populations of putative nociceptive afferents.
Assuntos
Capsaicina/farmacologia , Gânglios Espinais/citologia , Expressão Gênica/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Acetilcolina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Estimulação Elétrica/métodos , Imuno-Histoquímica/métodos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Proteínas do Tecido Nervoso , Neurônios Aferentes/classificação , Técnicas de Patch-Clamp/métodos , Canais de Potássio de Domínios Poros em Tandem/classificação , Ratos , Ratos Sprague-DawleyRESUMO
The development of opiate analgesics that do not produce adverse side effects is hampered by the difficulty in developing drugs that are tissue/sensory cell-specific. Previously, our laboratory has demonstrated that small- and medium-diameter dorsal root ganglia (DRG) cells can be subclassified into at least nine distinct cell types based upon their patterns of voltage activated currents [Petruska, J.C., Napaporn, J., Johnson, R.D., Gu, J.G., Cooper, B.Y., 2000. Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents. J. Neurophysiol. 84 (5), 2365-2379; Petruska, J.C., Napaporn, J., Johnson, R.D., Cooper, B.Y., 2002. Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion. Neuroscience 115 (1), 15-30.] Based on their responses to algesic compounds and histochemical phenotype, eight of the nine subtypes are likely nociceptors. In the present study, we examined the immunoreactivity (IR) of delta-, kappa- and mu-opioid receptors (DOR, KOR and MOR, respectively), in 164 electrophysiologically subclassified DRG neurons. The expression of opioid receptors in the DRG cell types was diverse. Type 1 (25-30 microm cell diameter) and type 9 (35-45 microm) expressed MOR-IR, but were negative for DOR-IR and KOR-IR. Type 2 (25-30 microm) co-expressed DOR-IR and MOR-IR, but did not express KOR-IR. Type 3 (15-20 microm), the non-nociceptive cell type, was not immunoreactive. Type 4 (35-45 microm), type 6 (35-45 microm), and type 7 (15-20 microm) expressed all three opioid receptors. Type 5 (35-45 microm) and type 8 (35-45 microm), co-expressed KOR-IR and MOR-IR, but did not express DOR-IR. The co-expression of opioid receptors in some of the cell types suggests that these sensory afferents might contain heteromeric opioid receptors. Additionally, the diverse expression patterns of opioid receptors between cell types and the consistency of these patterns maintained within each cell type provides further evidence of distinct functional properties of DRG nociceptors.
Assuntos
Gânglios Espinais/metabolismo , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Dor/metabolismo , Receptores Opioides/metabolismo , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/metabolismo , Animais , Capsaicina/farmacologia , Tamanho Celular , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios Aferentes/classificação , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/citologia , Nociceptores/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/fisiopatologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores Opioides/efeitos dos fármacos , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/efeitos dos fármacos , Receptores Opioides mu/metabolismoRESUMO
We examined the properties of a proton sensitive current in acutely dissociated, capsaicin insensitive nociceptive neurons from rat dorsal root ganglion (DRG). The current had features consistent with K(+) leak currents of the KCNK family (TASK-1, TASK-3; TWIK-related acid sensing K(+)). Acidity and alkalinity induced inward and outward shifts in the holding current accompanied by increased and decreased whole cell resistance consistent with a K(+) current. We used alkaline solutions to open the channel and examine its properties. Alkaline evoked currents (AECs; pH 10.0-10.75), reversed near the K(+) equilibrium potential (-74 mV), and were suppressed 85% in 0 mM K(+). AECs were insensitive to Cs(+) (1 mM) and anandamide (1 microM), but blocked by Ba(++) (1 mM), quinidine (100 microM) or Ruthenium Red (10 microM). This pharmacology was identical to that of rat TASK-3 and inconsistent with that of TASK-1 or TASK-2. The TASK-like AEC was not modulated by PKA (forskolin, kappa opioid agonists U69593 and GR8696, somatostatin) but was inhibited by PKC activator phorbol-12-myristate-13 acetate (PMA). When acidic solutions were used, we were able to isolate a Ba(++) and Ruthenium Red insensitive current that was inhibited by Zn(++). This Zn(++) sensitive component of the proton sensitive current was consistent with TASK-1. In current clamp studies, acidic pH produced sensitive changes in resting membrane potential but did not influence excitability (pH 7.2-6.8). In contrast, Zn(++) produced substantial changes in excitability at physiological pH. Alkaline solutions produced hyperpolarization followed by proportional burst discharges (pH 10.75-11.5) and increased excitability (at pH 7.4). In conclusion, multiple TASK currents were present in a DRG nociceptor and differentially contributed to distinct discharge mechanisms.
Assuntos
Neurônios/metabolismo , Nociceptores/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Potenciais Evocados/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Zinco/metabolismoRESUMO
We determined the co-expression of immunoreactivity (IR) for ATP-receptor subunits (P2X1, P2X2, and P2X3), neuropeptides, neurofilament (NF), and binding of the isolectin B(4) from Griffonia simplicifolia type one (GS-I-B(4)) in adult dorsal root ganglion neurons. P2X1-IR was expressed primarily in small DRG neurons. Most P2X1-IR neurons expressed neuropeptides and/or GS-I-B(4)-binding, but lacked NF-IR. P2X1-IR overlapped with P2X3-IR, though each was also found alone. P2X2-IR was expressed in many P2X3-IR small neurons, as well as a group of medium to large neurons that lacked either P2X3-IR or GS-I-B(4)-binding. A novel visible four-channel fluorescence technique revealed a unique population of P2X2/3-IR neurons that lacked GS-I-B(4)-binding but expressed NF-IR. Co-expression of P2X1, and P2X3 in individual neurons was also demonstrated. We examined P2X subunit-IR on individual recorded neurons that had been classified by current signature in vitro. Types 1, 2, 4 5, and 7 expressed distinct patterns of P2X-IR that corresponded to patterns identified in DRG sections, and had distinct responses to ATP. Types with rapid ATP currents (types 2, 5, and 7) displayed P2X3-IR and/or P2X1-IR. Types with slow ATP currents (types 1 and 4) displayed P2X2/3-IR. Type 1 neurons also displayed P2X1-IR. This study demonstrates that the correlation between physiological responses to ATP and the expression of particular P2X receptor subunits derived from expression systems is also present in native neurons, and also suggests that novel functional subunit combinations likely exist.
Assuntos
Neurônios Aferentes/química , Lectinas de Plantas , Receptores Purinérgicos P2/análise , Trifosfato de Adenosina/farmacologia , Animais , Anticorpos , Capsaicina , Feminino , Gânglios Espinais/química , Gânglios Espinais/citologia , Imuno-Histoquímica , Lectinas , Masculino , Nociceptores/química , Nociceptores/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/imunologia , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3RESUMO
Triple fluorescent histochemistry was used to describe the types of overlap in visceral sensory neurons (nodose ganglion) for the labeling of the isolectin B4 from Griffonia simplicifolia type one (GS-I-B4) and their immunoreactivity (IR) for two of the ATP receptor subunits (P2X1/3 or P2X2/3). The vast majority of nodose neurons expressed GS-I-B4-binding and most of these displayed P2X receptor IR. Most of the P2X-IR was co-expressed on these individual nodose neurons (P2X1/P2X3 or P2X2/P2X3). A very small subpopulation of neurons that were GS-I-B4 negative but P2X positive displayed a very high relative intensity of P2X3-IR. The functional role that these expression patterns play in visceral sensory processing is currently unclear.
Assuntos
Lectinas/farmacocinética , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Gânglio Nodoso/metabolismo , Dor/metabolismo , Receptores Purinérgicos P2/metabolismo , Fibras Aferentes Viscerais/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Feminino , Imuno-Histoquímica , Neurônios Aferentes/citologia , Nociceptores/citologia , Gânglio Nodoso/citologia , Dor/fisiopatologia , Ratos , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Fibras Aferentes Viscerais/citologiaRESUMO
Recordings were made from small and medium diameter dorsal root ganglia (DRG) neurons that expressed transient receptor potential (TRP) proteins. Physiologically characterized skin nociceptors expressed either TRPV1 (type 2) or TRPV2 (type 4) in isolation. Other nociceptors co-expressed both TRP proteins and innervated deep tissue sites (gastrocnemius muscle, distal colon; type 5, type 8) and skin (type 8). Subpopulations of myelinated (type 8) and unmyelinated (type 5) nociceptors co-expressed both TRPs. Cells that expressed TRPV1 were excellent transducers of intense heat. Proportional inward currents were obtained from a threshold of approximately 46.5 to approximately 56 degrees C. In contrast, cells expressing TRPV2 alone (52 degrees C threshold) did not reliably transduce the intensity of thermal events. Studies were undertaken to assess the capacity of skin and deep nociceptors to exhibit sensitization to repeated intense thermal stimuli [heat-heat sensitization (HHS)]. Only nociceptors that expressed TRPV2, alone or in combination with TRPV1, exhibited HHS. HHS was shown to be Ca(2+) dependent in either case. Intracellular Ca(2+) dependent pathways to HHS varied with the pattern of TRP protein expression. Cells co-expressing both TRPs modulated heat reactivity through serine/threonine phosphorylation or PLA(2)-dependent pathways. Cells expressing only TRPV2 may have relied on tyrosine kinases for HHS. We conclude that heat sensitization in deep and superficial capsaicin and capsaicin-insensitive C and Adelta nociceptors varies with the distribution of TRPV1 and TRPV2 proteins. The expression pattern of these proteins are specific to subclasses of physiologically identified C and A fiber nociceptors with highly restricted tissue targets.
Assuntos
Músculos/inervação , Nociceptores/fisiologia , Pele/inervação , Canais de Cátion TRPV/biossíntese , Animais , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Colo/inervação , Colo/metabolismo , Colo/fisiologia , Histocitoquímica , Temperatura Alta , Masculino , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Músculo Liso/inervação , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Músculos/metabolismo , Músculos/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Amielínicas/fisiologia , Proteínas de Neurofilamentos/biossíntese , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Pele/metabolismoRESUMO
We contrasted the physiology and peripheral targets of subclassified nociceptive and nonnociceptive afferents that express acid-sensing ion channel (ASIC)-like currents. The threshold for current activation was similar in eight distinct cell subclasses regardless of functional modality (pH 6.8). When potency was determined from concentration-response curves, nonnociceptors exhibited currents with significantly greater potency than that of all but one class of nociceptors (pH50 = 6.54 and 6.75 vs. 6.20-6.34). In nonnociceptive cells, acid transduction was also confined to a very narrow range (0.1-0.3 vs. 0.8-1.4 pH units for nociceptors). Simultaneous whole cell recording and ratiometric imaging of three peptidergic nociceptive classes were consistent with the expression of Ca2+ -permeable ASICs. Sensitivity to psalmotoxin and flurbiprofen indicated the presence of Ca2+ -permeable ASIC1a. Immunocytochemistry on these subclassified populations revealed a differential distribution of five ASIC proteins consistent with Ca2+ permeability and differential kinetics of proton-gated currents (type 5: ASIC1a, 1b, 2a, 2b, 3; type 8a: ASIC1a, 1b, 3; type 8b: ASIC1a, 1b, 2a, 2b, 3). Using DiI tracing, we found that nociceptive classes had discrete peripheral targets. ASIC-expressing types 8a and 9 projected to hairy skin, but only types 8a and 13 projected to glabrous skin. Non-ASIC-expressing types 2 and 4 were present only in hairy skin. We conclude that ASIC-expressing nociceptors differ from ASIC-expressing nonnociceptors mainly by range of proton reactivity. ASIC- as well as non-ASIC-expressing nociceptors have highly distinct cutaneous targets, and only one class was consistent with the existence of a generic C polymodal nociceptor (type 8a).
Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Cabelo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios Aferentes/fisiologia , Prótons , Pele/inervação , Canais de Sódio/fisiologia , Canais Iônicos Sensíveis a Ácido , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio/análise , Canais de Cálcio/fisiologia , Canais de Sódio Degenerina , Eletrofisiologia , Canais Epiteliais de Sódio , Gânglios Espinais/química , Gânglios Espinais/fisiologia , Imuno-Histoquímica , Canais Iônicos/análise , Canais Iônicos/fisiologia , Masculino , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Neurônios Aferentes/química , Nociceptores/química , Nociceptores/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Canais de Sódio/análiseRESUMO
Nociceptive cells of the dorsal root ganglion (DRG) were subclassified, in vitro, according to patterns of voltage-activated currents. The distribution and form of nicotinic ACh receptors (nAChRs) were determined. nAChRs were present on both capsaicin-sensitive and -insensitive nociceptors but were not universally present in unmyelinated nociceptors. In contrast, all A delta nociceptors (types 4, 6, and 9) expressed slowly decaying nAChR. Three major forms of nicotinic currents were identified. Specific agonists and antagonists were used to demonstrate the presence of alpha7 in two classes of capsaicin-sensitive, unmyelinated nociceptors (types 2 and 8). In type 2 cells, alpha7-mediated currents were found in isolation. Whereas alpha7 was co-expressed with other nAChR in type 8 cells. These were the only classes in which alpha7 was identified. Other nociceptive classes expressed slowly decaying currents with beta4 pharmacology. Based on concentration response curves formed by nicotinic agonists [ACh, nicotine, dimethyl phenyl piperazinium (DMPP), cytisine] evidence emerged of two distinct nAChR differentially expressed in type 4 (alpha3beta4) and types 5 and 8 (alpha3beta4 alpha5). Although identification could not be made with absolute certainty, patterns of potency (type 4: DMPP > cytisine > nicotine = ACh; type 5 and type 8: DMPP = cytisine > nicotine = ACh) and efficacy provided strong support for the presence of two distinct channels based on an alpha3beta4 platform. Studies conducted on one nonnociceptive class (type 3) failed to reveal any nAChR. After multiple injections of Di-I (1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) into the hairy skin of the hindlimb, we identified cell types 2, 4, 6, 8, and 9 as skin nociceptors that expressed nicotinic receptors. We conclude that at least three nicotinic AChR are diversely distributed into discrete subclasses of nociceptors that innervate hairy skin.