Fluoride-resistant Acid Phosphatase Research Articles

Peptide- and non-peptide-containing unmyelinated primary afferents: the parallel processing of nociceptive information.

Primary afferent C fibres can be subdivided into a number of subgroups on the basis of cytochemistry or receptor binding characteristics. Numerous peptides have been localized to dorsal root ganglia, yet these appear to be only found in approximately 50% of small perikarya. A large proportion of the remaining small cells do not contain peptides but are identifiable in rodents by their content of a fluoride resistant acid phosphatase. Attempts have been made to correlate particular biochemical types with particular receptive field profiles, with rather modest success. As an alternative we suggest, principally from an analysis of skin afferents, that peptide- and non-peptide-containing afferents are two distinct C fibre pathways innervating similar peripheral structures and conveying similar information, but to different areas within the dorsal horn. Morphological evidence also suggests that these two subsystems form either glomerular or simple synaptic arrangements in the dorsal horn. The significance of parallel pathways for the processing of nociceptive information is briefly discussed.

Structure and expression of differentiation antigens on functional subclasses of primary sensory neurons.

Subpopulations of dorsal root ganglion neurons can be distinguished on the basis of their peripheral receptive properties, spinal terminal arbors and neuropeptide content. We have used monoclonal antibodies (MAbs) to define antigenic determinants on functional populations of DRG neurons projecting to the superficial dorsal horn of the spinal cord. Three MAbs recognize defined carbohydrate epitopes associated with lacto- and globo-series glycolipids that constitute the stage-specific embryonic antigens (SSEAs) 1, 3 and 4. SSEA-3 and SSEA-4 are present in the cytoplasm of about 10% of DRG neurons in adult rat. These neurons are distinct from those that contain substance P, somatostatin or the fluoride-resistant acid phosphatase enzyme, FRAP. SSEA-1 is present in a small percentage of DRG neurons. SSEAs are present on the surface of DRG neurons maintained in dissociated cell culture: 6% are SSEA-1+, 7% are SSEA-3+ and 10-15% are SSEA-4+. MAbs LD2, KH10, TC6 and TD10 identify epitopes expressed coincidently in 25% of small DRG neurons that project to lamina II of the dorsal horn. All somatostatin- but less than 1% of substance P-immunoreactive DRG neurons express these antigens. MAb LA4 labels a distinct population of small DRG neurons that also projects to lamina II. There is extensive overlap between LA4+ neurons and those that contain FRAP. Antigens recognized by these MAbs are expressed on the surface of 10-20% of DRG neurons in culture. Preliminary biochemical studies suggest that these antigens may be glycolipids. Molecules bearing carbohydrate differentiation antigens may be involved in the development and specification of sensory connections in the dorsal horn of the spinal cord.

Neurogenic extravasation and substance P levels are low in muscle as compared to skin in the rat hindlimb

Activation of cutaneous chemosensitive afferents results in the release of substances which increase the permeability of the microcirculation, producing inflammation. That this inflammation is neurogenic is readily demonstrated by antidromic electrical stimulation of afferent fibres. In the present study we have used the technique of dye extravasation to compare both qualitatively and quantitatively neurogenic extravasation skin and skeletal muscle. Plasma extravasation has been found to occur in skeletal muscle after stimulation but only at less than 10% of the levels seen in skin. We have also found that the levels of the C-fibre markers substance P and fluoride-resistant acid phosphatase are greatly reduced in muscle compared with skin nerves. These results show that there are substantial differences in the population of C-fibres supplying muscle compared with those supplying skin.

The postnatal physiological and neurochemical development of peripheral sensory C fibres

The postnatal development of sensory C fibre function was investigated in neonatal rats aged 1–21 days. From birth, flexor-withdrawal reflexes (measured from the hamstring electromyograph) to pinching and heating the skin of the hindfoot were easily recorded under light anaesthesia and in fact were exaggerated in amplitude and duration compared to adult responses. Flexor reflexes to irritant chemicals, however, were not present until day 10–11 of life. In parallel with this late development of specific chemical sensitivity, neurogenic oedema, a C fibre-mediated inflammatory reaction, also did not occur until day 11. Substance P and fluoride-resistant acid phosphatase histochemistry were used to investigate the neurochemical development of sensory C fibres. Substance P was present in the skin, nerve, dorsal root ganglion and spinal cord from birth and fluoride-resistant acid phosphate within 12 h of birth. The adult neurochemical appearance of C-fibre terminals in the dorsal horn was established in a few days. The results show that despite the apparent early anatomical and neurochemical maturity of C fibres, physiological function is not fully established until the second week of life.

Peripheral projections and neuropeptide coexistence in a subpopulation of fluoride-resistant acid phosphatase reactive spinal primary sensory neurons

Combined retrograde axonal tracing with the fluorescent dye Fast Blue and fluoride-resistant acid phosphatase (FRAP) histochemistry revealed that the FRAP-containing sensory neurons project to both somatic and autonomic peripheral nerves. Furthermore, the combination of indirect immunohistochemistry after colchicine treatment and FRAP histochemistry showed that a population of FRAP-containing sensory neurons are also substance P, cholecystokinin or somatostatin positive.

Biochemical and anatomical effects of antibodies against nerve growth factor on developing rat sensory ganglia.

The importance of nerve growth factor (NGF) for the development of sensory ganglia was investigated by injecting rat fetuses (16.50 days of gestation) with a single dose of anti-NGF antiserum. Four months later the treated animals showed a very large decrease in substance P- and somatostatin-like immunoreactivities in dorsal root ganglia and skin with a lesser decrease in trigeminal ganglia. Fluoride-resistant acid phosphatase, substance P-, and somatostatin-like immunoreactivities were greatly decreased in the dorsal horn of the spinal cord. No change in neurotensin- and [Met]enkephalin-like immunoreactivities was observed. The anti-NGF antiserum treatment produced a greater than 90% decrease in the number of unmyelinated dorsal root fibers and a 35% decrease in the total number of myelinated fibers. The loss in myelinated fibers was restricted to small-diameter fibers with no change in large-diameter fibers. No change in taste bud morphology was noted, thereby refuting the proposal that anti-NGF antiserum treatment may represent an animal model for familial dysautonomia. The present results indicate that NGF is a necessary requirement for the normal development of a significant population of prenatal rat dorsal root ganglion cells.

Alterations in the structure, function, and chemistry of C fibers following local application of vinblastine to the sciatic nerve of the rat

Vinblastine, a transport blocker, was applied locally to the sciatic nerve in rats. It was found to be a powerful neurotoxin with a dose-dependent action, destroying all afferents at doses of 5 X 10(-4)M, primarily C fibers at intermediate doses of 2.5 X 10(-4)M, and only at a critically low dose of 10(-4)M was a degeneration-free axon transport blockade, lasting for 4 to 5 days, produced. Such transport block failed to alter thermal responsiveness of the rats as measured behaviorally, by the flexor reflex, or by dorsal horn cell responses. It did, however, significantly reduce both the chemical sensitivity of the C afferents and their ability to produce neurogenic edema. This began 24 hr after treatment and lasted 4 to 5 days. Therefore, it is likely that these functions are dependent on the continuous transport of some compound to the axon terminals from the cell body. This low concentration of local vinblastine treatment also resulted in depletion of fluoride-resistant acid phosphatase from C fiber terminals in the dorsal horn of the spinal cord. Transmission from C fibers to second-order neurons in the spinal cord, however, was totally unaffected. Substance P levels in the spinal terminals was largely unaffected, although in 1 of 5 cases there was depletion. It appears, therefore, that some, but not all, retrograde changes in sensory neurons following peripheral nerve damage can be mimicked by blockade of axon transport. The effects following vinblastine treatment are compared to other peripheral nerve manipulations, such as cut, crush, and application of local capsaicin.

Isolation and characterization of a microvascular fraction from rabbit kidney cortex.

Collagenasic dispersion of rabbit kidney cortex followed by centrifugation on discontinuous sucrose gradient, allowed the simultaneous isolation of a microvascular fraction and of glomerular and tubular fractions. The microvessels were characterized by an overall diameter of 22 micron and the presence of granular and smooth muscle cells. Measurement of cellular renin activity and the muscle specific enzyme creatine kinase showed that these vessels were arteriolar in nature and that they contained the preglomerular arterioles. The glomerular or tubular contamination rates were assessed by means of enzymatic markers. Thus, in the arterioles, potassium fluoride-resistant acid phosphatase was some 10 times lower than in the glomeruli. The specific tubular enzymes gamma-glutamyl-transpeptidase and alkaline phosphatase were about 20 times lower in the arterioles than in the tubular fraction. The possibility of obtaining these fractions may facilitate many types of study on renal hemodynamic and glomerulo-tubular feedback control.

The termination of primary afferents within the rat dorsal horn: evidence for rearrangement following capsaicin treatment.

AbstractThe mode of termination of primary afferent fibres within the superficial dorsal horn of the spinal cord was studied with anterograde tracing methods in normal rats and in animals which had been treated with capsaicin (50 mg/kg. s.c.) shortly after birth. In normal animals following injections of horseradish peroxidase (HRP) into lumbar dorsal root ganglia L4, L5, L6, a Golgi‐like filling of primary afferent fibres was seen consistently within laminae I, II, and III of the dorsal horn. Several types of lamina related arborisations were observed, depending upon the survival time after HRP injection. At the earliest survival time (1–2 days) a punctate granular labelling was found in laminae I and II inner (IIi). At 3 days survival a Golgi like labelling of primary afferent axons occurred and three tiers of arborisation were seen within laminae I, II outer (IIO), and II inner (IIi), respectively. By 7 days after injection this pattern was considerably reduced and there was coarser fibre labelling within laminae I, IIO, and III.When these tracing experiments were repeated in capsaicin‐treated rats in which up to 90%of unmyelinated fibres had been destroyed, evidence for a considerable loss of some inputs and rearrangement of the remaining fibres was found. At 3–5 days survival the axonal labelling within laminae I and IIi was severely depleted yet some input, particularly to lamina IIO, was present. At 7 days survival deep fibre labelling within lamina III now extended well into lamina II.Mapping of the primary afferent input following 3H‐proline injections into the dorsal root ganglion and subsequent autoradiographic processing confirmed that primary afferents terminated throughout the dorsal horn but most heavily in two bands within laminae I and IIi. These two dense layers of termination were not present in capsaicin‐treated animals, suggesting that they corresponded to areas of unmyelinated primary afferent fibre input.These data, taken together with histochemical observations on the distribution of substance P and fluoride‐resistant acid phosphatase (FRAP)‐containing primary afferents, suggests that the earliest‐labelled fibre systems (3days) were unmyelinated and replaced at 7 days by predominantly A6 myelinated fibres. It is further suggested that of the three tiers of C fibre terminations seen at 3 days, the most superficial corresponds in part to those primary afferents that can be stained for substance P within lamina I while the most ventral tier within lamina IIi are those primary afferents containing FRAP. The presence of the two temporally distinct fibre systems also suggests that laminae I and IIo, receive both C and A6 primary afferent input while lamina IIi, receives only C fibre input.

Fluoride-resistant acid phosphatase in the rat adrenal gland

The adrenal medulla of the rat, but not of the mouse, guinea pig or cat, was shown to contain a fluoride-resistant acid phosphatase activity similar to that previously noted in rat spinal cord. By combined immunohistochemistry and FRAP histochemistry it was determined that the FRAP in the rat adrenal was confined to the adrenaline chromaffin cells.

Chronic blockade of sciatic nerve transmission by tetrodotoxin does not produce central changes in the dorsal horn of the spinal cord of the rat

Rat sciatic nerves were treated with tetrodotoxin (TTX) for 4–10 days, by implanting a glass capillary tube filled with TTX into the nerve through the epineurium. Following this treatment the somatotopic organization of receptive fields in the L4 dorsal horn cells was mapped. This was done by making a series of microelectrode tracks through the medial L4 dorsal horn, an area of cord normally responding only to foot stimulation. The map was normal in animals treated with TTX. Dorsal horn levels of fluoride-resistant acid phosphatase, substance P, somatostatin, cholecystokinin-like peptide, neurotensin and neurophysin were also normal as assessed from density of staining. These results are discussed in the light of the positive changes that are seen following chronic sciatic nerve section.

Reversibility of microtubule inhibitor-induced transganglionic degenerative atrophy of central terminals of primary nociceptive neurons

Microtubule inhibitor Vinca alkaloids applied around a peripheral nerve induce transganglionic degenerative atrophy of the central terminals of primary nociceptive neurons. This effect is reversible: 40–50 days later the original histochemical structure of the central terminals is restored. Restoration of fluoride-resistant acid phosphatase activity (the marker enzyme of primary nociceptive neurons) in the Rolando substance is due to regenerative sprouting of the formerly atrophied central terminals. Since peripherally-applied Vinca alkaloids induce transganglionic degenerative atrophy of the central terminals without inducing Wallerian degeneration of the peripheral nerve, and since this effect (virtually a synaptic discoupling) is only temporary, this approach may be used in the treatment of otherwise intractable neuralgias without inducing irreparable alterations.

Fluoride-resistant acid phosphatase-containing neurones in dorsal root ganglia are separate from those containing substance P or somatostatin

The distribution of fluoride-resistant acid phosphatase, substance P and somatostatin were investigated in the dorsal horn of the spinal cord and in dorsal root ganglia. In the dorsal horn, the distribution of fluoride-resistant acid phosphatase closely paralleled that of somatostatin and only partly overlapped with that of substance P. In sensory ganglia, none of the fluoride-resistant acid phosphatasecontaining neurones contained either substance P or somatostatin. The results suggest the existence of a population of fluoride-resistant acid phosphatase-positive sensory neurones which is distinct from neurones containing either of these peptides.

Regenerative synaptoneogenesis in the Mammalian spinal cord: dynamics of synaptochemical restoration in the Rolando Substance after transganglionic degenerative atrophy.

Crush injury of the sciatic nerve, that results in Wallerian degeneration of axons in the peripheral stump, induces, within 10-14 days, transganglionic degenerative atrophy of central terminals of primary nociceptive neurons in the ipsilateral substantia gelatinosa Rolandi of the segmentally related region of the spinal cord. Transganglionic degenerative atrophy is characterized by disappearance of fluoride-resistant acid phosphatase (FRAP) from the Rolando substance, normally exerted by primary nociceptive terminals. From the 40th postoperative day on, FRAP reaction starts to reappear in the formerly depleted Rolando substance. Restoration of FRAP reactivity reflects regenerative sprouting of formerly atrophied primary nociceptive terminals. Growth cones of primary nociceptive axons establish synapses with dendritic growth cones of substantia gelatinosal gelatinosal cells. Synaptoneogenesis in the Rolando substance follows medio-lateral and caudo-rostral gradients.

Effects of capsaicin applied locally to adult peripheral nerve. II. Anatomy and enzyme and peptide chemistry of peripheral nerve and spinal cord

1.(1) Capsaicin solution was applied for 15 min around a 1 cm length of sciatic nerve in the mid upper leg of adult rats.2.(2) Electron microscopic examination of the nerve in the treated region after 14 days shows no signs of degeneration of either myelinated or unmyelinated fibres attributable to the capsaicin.3.(3) Fluoride resistant acid phosphatase FRAP disappears from the central terminals of the treated nerve by 7 days.4.(4) 1.5 mM capsaicin is sufficient to produce a complete reduction of FRAP in the spinal cord.5.(5) The peptides substance P and cholecystokinin (CCK) are markedly depleted in the region of spinal cord terminations of the treated nerve at 14 days.6.(6) Substance P and CCK are not affected in spinal cord regions other than in the unmyelinated afferent terminal zone. Similarly neurotensin and neurophysin which are not present in afferent fibres are not influenced by capsaicin treatment of the sciatic.7.(7) It is concluded that there are chemical changes in the spinal cord terminals of fine afferents after local peripheral capsaicin.

Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after neonatal capsaicin

Administration of a single dose of capsaicin (50 mg/kg; sc) to two-day-old rats resulted in a loss of between 85 to 95% of the small diameter unmyelinated primary afferent fibres from lumbar dorsal roots. The numbers of myelinated fibres in the dorsal roots were not significantly affected. The substance P and somatostatin contents of the dorsal roots were decreased by 85 to 95%. The extent of depletion of these peptides from the dorsal horn of the spinal cord was somewhat less; substance P was decreased by 55% and somatostatin by 19%. Histochemical studies showed that fluoride-resistant acid phosphatase was virtually absent from the dorsal horn of capsaicin-treated animals. There was also a conspicuous absence of substance P-positive fibres from layers I and II of the dorsal horn and of fibres traversing these layers to deeper regions. Colchicine pretreatment revealed the presence of substance P-containing perikarya within layers III–IV. The plexus of substance P-positive elements which normally surrounded these neurons was absent after capsaicin treatment, suggesting that they receive a substance P-containing primary afferent innervation. The neurotoxicity of capsaicin after a single dose of 50 mg/kg in neonatal animals appears to be restricted largely to unmyelinated primary afferent fibres. The present results suggest that both substance P and somatostatin are contained in primary afferent sensory neurons which give rise to unmyelinated fibres. It can be further concluded that about 50% of substance P and only about 20% of the somatostatin in the dorsal horn appears to be of primary afferent origin.

Histochemical Distribution of Enzymes in the Small Intestine of Young Milk-Fed Calves

The histochemical distribution of selected enzymes were examined in the small intestine of 5 about 3-week-old normal calves fed on whole cow’s milk. Alkaline phosphatase and β-D-galactosidase (= lactase) in the epithelial brush border, and non-specific esterase in the cytoplasm showed a strong reaction in the villi of the anterior small intestine and a marked decrease in the posterior regions. Aminopeptidase in the brush border of the villi showed a reverse distribution, with the strongest reaction in the posterior small intestine. Adenosine-triphosphate-splitting enzyme in the epithelial brush border, acid phosphatase and succinate dehydrogenase in the cytoplasm of the epithelial cells gave a relatively uniform reaction in the villi throughout the small intestine. A fluoride-resistant acid phosphatase was demonstrated in the brush border of the villi in the anterior small intestine. The distribution of enzymes demonstrated in this study was generally compatible with the known absorptive functions of the various parts of the small intestine.

Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn

After peripheral nerve injury in rats, naturally occuring fluoride resistant acid phosphatase (FRAP) disappears from the substantia gelatinosa in that part of the dorsal horn in which the injured nerve afferents terminate. We have taken advantage of this fact to establish the spinal distribution of nerves innervating the skin of the hindlimb. The spinal map of the foot, the distal part of the lower lumbar dermatomes, is in the medial part of the substantia gelatinosa. More proximal skin of the thigh and the lower back maps laterally. The zone of disappearance of FRAP after sciatic nerve section did not shrink detectably within the first few months after injury, provided that regeneration of the nerve was prevented. After one year, however, central FRAP activity was at least partially restored. Secondary transection of the sciatic nerve eliminated the new enzyme and transection of neighboring nerves failed to do so. The restored FRAP activity therefore reflects renewed synthesis and transport of enzyme in still injured neurons, and not central sprouting of intact neighboring afferents.

Fine structure and fluoride resistant acid phosphatase activity of electron dense sinusoid terminals in the substantia gelatinosa Rolandi of the rat after dorsal root transection.

Fluoride resistant acid phosphatase (FRAP) activity of the rat substantia gelatinosa Rolandi is confined to electron dense sinusoid terminals under normal conditions. Transection of dorsal roots or removal of dorsal root ganglia results in a rapid degeneration of more than half of the electron dense sinusoid axon terminals. First signs of degeneration ensue 20 hours after surgery; at the 24 hours state osmiophilic degeneration bodies develop that are translocated into glial elements in the course of the second postoperative day. At the same time, light microscopically visible FRAP-activity of the Rolando substance disappears. Electron histochemical investigations reveal that decreased enzyme activity is due to degeneration of FRAP-positive terminals. It is concluded that FRAP-positive terminals, representing the majority of electron dense sinusoids in the Rolando substance, are dorsal root collaterals; the origin of non-degenerating FRAP-negative electron dense terminals remains unknown for the time being.

Histochemical study of a fluoride resistant acid phosphatase reaction in the mouse duodenum.

A fluoride resistant acid phosphatase reaction (pH 5.0) has been demonstrated histochemically in the cuticular border of the mouse duodenum. This reaction was shown to be inactivated by the metal chelating agent, ethylene diamine tetraacetic acid, and reactivated by the following metallic salts : calcium chloride, cobalt(ous) nitrate, cupric sulfate, ferric chloride, ferric ammonium sulfate, ferrous sulfate, lead nitrate, lithium sulfate, magnesium sulfate, mercuric chloride, manganous chloride and zinc acetate. The two ferric salts produced the greatest amount of reactivation and cupric sulfate and manganous chloride produced the least amount of reactivation. These characteristics of inactivation by a metal chelating agent and metal reactivation plus the fluoride resistance of the acid phosphatase reaction present in the cuticular border of the mouse duodenum are features more typical of an alkaline type of phosphatase rather than the acid variety. This suggests that alkaline type phosphatase may be demonstrable histochemically in the cuticular border of the mouse duodenum in the acid pH range.