We used a "current signature" method to subclassify acutely dissociated dorsal root ganglion (DRG) cells into nine subgroups. Cells subclassified by current signature had uniform properties. The type 1 cell had moderate capsaicin sensitivity (25.9 pA/pF), powerful, slowly desensitizing (tau = 2,300 ms), ATP-activated current (13.3 pA/pF), and small nondesensitizing responses to acidic solutions (5.6 pA/pF). Type 1 cells expressed calcitonin gene-related peptide immunoreactivity (CGRP-IR), manifested a wide action potential (7.3 ms), long duration afterhyperpolarization (57.0 ms), and were IB4 positive. The type 2 cell exhibited large capsaicin activated currents (134.9 pA/pF) but weak nondesensitizing responses to protons (15.3 pA/pF). Currents activated by ATP and alphabeta-m-ATP (51.7 and 44.6 pA/pF, respectively) had fast desensitization kinetics (tau = 214 ms) that were distinct from all other cell types. Type 2 cells were IB4 positive but did not contain either substance P (SP) or CGRP-IR. Similar to capsaicin-sensitive nociceptors in vivo, the afterhyperpolarization of the type 2 cell was prolonged (54.7 ms). The type 3 cell expressed, amiloride-sensitive, rapidly desensitizing (tau = 683 ms) proton-activated currents (127.0 pA/pF), and was insensitive to ATP or capsaicin. The type 3 cell was IB4 negative and contained neither CGRP nor SP-IR. The afterhyperpolarization (17.5 ms) suggested nonnociceptive function. The type 4 cell had powerful ATP-activated currents (17.4 pA/pF) with slow desensitization kinetics (tau = 2, 813 ms). The afterhyperpolarization was prolonged (46.5 ms), suggesting that this cell type might belong to a capsaicin-insensitive nociceptor population. The type 4 cell did not contain peptides. The type 7 cell manifested amiloride-sensitive, proton-activated currents (45.8 pA/pF) with very fast desensitization kinetics (tau = 255 ms) and was further distinct from the type 3 cell by virtue of a nondesensitizing amiloride-insensitive component (6.0 pA/pF). Capsaicin and ATP sensitivity were relatively weak (4.3 and 2.9 pA/pF, respectively). Type 7 cells were IB4 positive and contained both SP and CGRP-IR. They exhibited an exceptionally long afterhyperpolarization (110 ms) that was suggestive of a silent (mechanically insensitive) nociceptor. We concluded that presorting of DRG cells by current signatures separated them into internally homogenous subpopulations that were distinct from other subclassified cell types.

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The distribution of tetrodotoxin (TTX)-sensitive and -insensitive Na ϩ currents and their modulation by serotonin (5HT) and prostaglandin E 2 (PGE 2 ) was studied in four different types of dorsal root ganglion (DRG) cell bodies (types 1, 2, 3, and 4), which were previously identified on the basis of differences in membrane properties (Cardenas et al., 1995). Types 1 and 2 DRG cells expressed TTX-insensitive Na ϩ currents, whereas types 3 and 4 DRG cells exclusively expressed TTX-sensitive Na ϩ currents. Application of 5HT (1-10 M) increased TTX-insensitive Na ϩ currents in type 2 DRG cells but did not affect Na ϩ currents in type 1, 3, or 4 DRG cells. The 5HT receptor involved resembled the 5HT 4 subtype. It was activated by 5-methoxy-N,Ndimethyltryptamine (10 M) but not by 5-carboxyamidotryptamine (1 M), (ϩ)-8-hydroxydipropylaminotetralin (10 M), or 2-methyl-5HT (10 M), and was blocked by ICS 205-930 with an EC 50 of ϳ2 M but not by ketanserin (1 M). PGE 2 (4 or 10 M) also increased Na ϩ currents in varying portions of cells in all four groups.The effect of 5HT and PGE 2 on Na ϩ currents was delayed for 20-30 sec after exposure to 5HT, suggesting the involvement of a cytosolic diffusible component in the signaling pathway. The agonist-mediated increase in Na ϩ current, however, was not mimicked by 8-chlorophenylthio-cAMP (200 M), suggesting the possibility that cAMP was not involved.The data suggest that the 5HT-and PGE 2 -mediated increase in Na ϩ current may be involved in hyperesthesia in different but overlapping subpopulations of nociceptors. Key words: serotonin; PGE 2 ; capsaicin; nociceptor; tetrodotoxin; cAMP; dorsal root ganglion; 5HT 4 receptorPrimary hyperesthesia is thought to be a consequence of the release of proinflammatory mediators in the vicinity of nociceptor endings. Proinflammatory agents such as serotonin (5HT), prostaglandins, and adenosine are derived from a number of sources (Cooper and Sessle, 1992). Interactions between such agents and endings of thinly myelinated and unmyelinated nociceptive afferents induce activity, decrease threshold, and increase suprathreshold mechanothermal reactivity (Handwerker, 1976;Kumazawa and Mizumura, 1980;Mense, 1981;Martin et al., 1987;Schaible and Schmidt, 1988;Lang et al., 1990;Grubb et al., 1991;Herbert and Schmidt, 1992).Contributions to hyperesthesia by proinflammatory agents are possibly achieved by modulation of membrane currents involved in the initiation and repolarization of action potentials in nociceptive endings, as well as induction of edema in the surrounding matrix (Cooper, 1993). The study of acutely isolated dorsal root ganglion (DRG) cell bodies may be usef ul in determining the roles of various ion channels in the actions of proinflammatory agents on nociceptor f unction. A number of studies have shown that afferent cell bodies exhibit properties of nociceptor endings in vitro. These include expression of currents sensitive to proinflammatory mediators as well as features of peripheral transduction mechanisms (Baccaglini and Hogan...

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.

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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.

A limitation in the administration of parenteral products is the pain caused upon injection. Injection site pain has been predominately associated with intravenous, intramuscular, and subcutaneous administration. It becomes important for the formulation scientist to have a basic understanding of the physiology underlying the pain process, as well as the pharmaceutical factors associated with injection site pain. Initially, this review will provide the reader with a primer on the mediation of pain in the periphery and a compilation of those drugs that have been associated with pain on injection. In addition, this review will present important considerations and general formulation approaches or methods that have been used to overcome pain on injection. Finally, a brief overview of the various experimental systems used to investigate injection site pain is discussed.

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