Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion.

Whole cell patch recordings were obtained from medium diameter (35-45 microm) dorsal root ganglion neurons. Using electrophysiological parameters, we were able to subclassify acutely dissociated dorsal root ganglion cells into three uniform (types 5, 6 and 9) and one mixed class (type 8) of neurons. All subtypes (types 5, 6, 8 and 9) had broad action potentials (7.0+/-0.2, 5.2+/-0.4, 7.3+/-0.5 and 6.0+/-0.4 ms) and exceptionally long afterhyperpolarizations (112+/-9, 178+/-19, 124+/-31 and 204+/-33 ms). Long afterhyperpolarizations have been linked to mechanically insensitive (silent) nociceptors by other laboratories [Djouhri et al., J. Physiol. 513 (1998) 857-872]. Chemosensitivity varied among cell classes. Cell types 5, 8 and 9 were capsaicin sensitive (45+/-13, 87+/-30 and 28+/-13 pA/pF; 5 microM) groups, while the type 6 cell was capsaicin insensitive. All cell types expressed ASIC-like (acid sensing ion channel) amiloride sensitive, proton-activated currents with a threshold of pH 6.8 and a peak near pH 5.0. All medium sized cells were sensitive to ATP (50 microM) and exhibited the 'mixed' form of ATP-gated current [Burgard et al., J. Neurophysiol. 82 (1999) 1590-1598; Grubb and Evans, Eur. J. Neurosci. 11 (1999) 149-154]. Immunohistochemistry performed on individual cells indicated the expression of both P2X(1) and P2X(3) subunits. Electrophysiologically defined classes were histochemically uniform. All types were examined for the presence of substance P (SP), calcitonin gene related peptide (CGRP) and binding of isolectin B4 (IB4). All subtypes expressed CGRP immunoreactivity. Types 5 and 8 co-expressed SP and CGRP immunoreactivity and also bound IB4. Subtypes 6 and 9 were positive for neurofilament m. It is likely that these cells represent major classes of myelinated and unmyelinated peptide expressing nociceptors.

Co-expression of P2X receptor subunits on rat nodose neurons that bind the isolectin GS-I-B4.

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.

Distribution of P2X1, P2X2, and P2X3 receptor subunits in rat primary afferents: relation to population markers and specific cell types.

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.

Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents.

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.

P2X1 receptor subunit immunoreactivity and ATP-evoked fast currents in adult rat dorsal root ganglion neurons.

Triple fluorescent staining for P2X1 and P2X3 subunits and isolectin I-B4 (IB4) were performed on acutely dissociated rat DRG neurons. Immunoreactivity for P2X1 and P2X3 subunits was present separately or together in DRG neurons. P2X1 immunoreactivity was present in both IB4-positive and IB4-negative cells. When combining patch-clamp recordings with immunostaining for the P2X1 and P2X3 subunits on single recorded cells, ATP-evoked fast currents were shown to be present on DRG neurons that have immunoreactivity for the P2X3 subunit only, the P2X1 subunit only, or both P2X1 and P2X3 subunits. These results raised a possibility that, in addition to the P2X3 receptor subunit, the P2X1 subunit may also contribute to functional P2X receptors with fast kinetics in DRG neurons.

Distribution patterns of different P2x receptor phenotypes in acutely dissociated dorsal root ganglion neurons of adult rats.

P2x receptors may be used to detect ATP release from tissues during physiological and pathological conditions. We used whole-cell patch clamp recordings to study the expression of P2x receptor phenotypes, their distribution patterns, and their sensitivity to alphabetamATP and suramin in dorsal root ganglion (DRG) neurons acutely dissociated from adult rats. Based on the onset and decay rates of 10 microM ATP-evoked currents, we showed three types of P2x currents: fast, slow, and mixed. Each of these P2x receptor phenotypes had a distinct distribution pattern among DRG neurons. The fast P2x currents were predominantly expressed in small-diameter, isolectin-B4 (IB4)-positive, and capsaicin-sensitive DRG neurons. The slow P2x currents were expressed in both small and medium DRG neurons, and about half of them were IB4 positive. The mixed P2x currents were also expressed in both small and medium-sized DRG neurons, and most of these neurons were IB4-positive neurons. The slow and mixed P2x current groups had both capsaicin-sensitive and -insensitive DRG neurons. All phenotypes revealed with 10 microM ATP could be inhibited by 30 microM suramin. All DRG neurons with fast or mixed P2x currents were also sensitive to 10 microM alphabetamATP, and alphabetamATP evoked currents similar to those induced by ATP. The group expressing slow P2x currents could be further divided into alphabetamATP-sensitive and -insensitive groups. Thus, the relationships among P2x receptor phenotypes, cell sizes, IB4 positivity, and capsaicin sensitivity are more complicated than previously thought, and different P2x receptors may be involved in both nociceptive and non-nociceptive functions.

Anodally focused polarization of peripheral nerve allows discrimination of myelinated and unmyelinated fiber input to brainstem nuclei.

We investigated the ability of a novel direct current (DC) polarization technique to block selectively the conduction in peripheral myelinated nerve fibers and allowing propagation in only unmyelinated fibers. In anesthetized adult rats, distal branches of the sciatic nerve (caudal cutaneous sural and tibial nerves) were exposed for electrical stimulation of A- and C-fibers. Two specially fabricated trough electrodes of different size and surface area were placed onto the sciatic nerve. Through these proximal electrodes a controlled ramped DC was timed to coincide with the arrival of A- and C-fiber action potentials, evoked electrically at the distal nerves or naturally from the foot or ankle, with the intent of blocking propagation in A-fibers while allowing C-fiber throughput. Neuronal recordings were made both peripherally (proximal sciatic nerve fascicles or L5 dorsal roots) and centrally (single cells in the nucleus gracilis or nucleus reticularis gigantocellularis). The DC polarization was shown to block conduction in myelinated A-fibers effectively, while allowing conduction in the unmyelinated C-fibers, without activation of fibers via the DC polarization itself. This was dependent upon the following factors: electrode polarity, onset rate of polarization, peak amplitude of polarization, distance between polarizing electrodes, size difference between polarizing electrodes, and gross nerve size. These experiments demonstrate that anodally focused DC polarization, applied utilizing two trough electrodes of different sizes, is capable of effectively, reversibly, and reproducibly blocking conduction in myelinated A-fibers evoked either electrically or naturally, while still allowing conduction to occur in the unmyelinated C-fiber population. In the context of experimental usage, we have demonstrated blocking of low-threshold A-fiber, but not C-fiber, mediated inputs to the caudal brainstem. This technique should find wide application in studies involving the processing of information conveyed centrally by the unmyelinated C-fiber afferent population, including discriminating afferent responses to peripheral stimuli, the role of C-fiber input in reflex activity, and the plasticity following injury or other manipulations.

Localization of unmyelinated axons in rat skin and mucocutaneous tissue utilizing the isolectin GS-I-B4.

The alpha-D-galactose specific isolectin I-B4 from Griffonia simplicifolia (GS-I-B4) labels CNS microglia and certain peripheral neurons, including a subpopulation of small dark, type B dorsal root ganglion cells, some post-ganglionic sympathetic axons, and nearly all peripheral gustatory axons. The innervation patterns of GS-I-B4 reactive sensory ganglion cells are unknown for many peripheral target tissues, including their probable primary target, the skin. The present study describes the distribution of GS-I-B4 reactive axons in hairy and glabrous hindpaw skin and in the glans penis of rats, using both single and double-labelling histochemical techniques. Neuronal processes were identified using (1) histochemistry with horseradish peroxidase conjugated GS-I-B4 or (2) immunohistochemistry against PGP 9.5 to identify all axons, and biotinylated lectin histochemistry with avidin-FITC to identify the subpopulation of GS-I-B4 reactive axons. GS-I-B4 strongly labelled unmyelinated cutaneous sensory afferents, as well as some sympathetic efferents and visceral afferents. lectin reactive axons were seen to innervate the upper hair shaft epidermis in hairy skin, and were abundant in the shallow dermis in hairy and glabrous skin and glans penis. Lectin reactive axons were also abundant in the lamina propria and distal urethral epithelium of the penis. These results provide new evidence for the cutaneous sensory role of GS-I-B4 reactive primary afferents, as well as evidence to support the contention that the lectin is a specific marker for a subpopulation of unmyelinated axons and not simply a marker for the myelination state of an axon.

Passive hemagglutination procedure for group A streptococcal polysaccharide.

A passive hemagglutination procedure for detection of antibody to group A beta-hemolytic streptococcus polysaccharide is described. The polysaccharide was conjugated to human gamma globulin and the complex adsorbed onto stabilized human erythrocytes. Antigen coated cells were used to measure antibody levels in normal human sera and in rabbit antisera.