Fluoride-resistant Acid Phosphatase Research Articles

Localization of SSeCKS in unmyelinated primary sensory neurons

BackgroundSSeCKS (Src SupprEssed C Kinase Substrate) is a proposed protein kinase C substrate/A kinase anchoring protein (AKAP) that has recently been characterized in the rat peripheral nervous system. It has been shown that approximately 40% of small primary sensory neurons contain SSeCKS-immunoreactivity in a population largely separate from substance P (95.2%), calcitonin gene related peptide (95.3%), or fluoride resistant acid phosphatase (55.0%) labeled cells. In the spinal cord, it was found that SSeCKS-immunoreactive axon collaterals terminate in the dorsal third of lamina II outer in a region similar to that of unmyelinated C-, or small diameter myelinated Aδ-, fibers. However, the precise characterization of the anatomical profile of the primary sensory neurons containing SSeCKS remains to be determined. Here, immunohistochemical labeling at the light and ultrastructural level is used to clarify the myelination status of SSeCKS-containing sensory neuron axons and to further clarify the morphometric, and provide insight into the functional, classification of SSeCKS-IR sensory neurons.MethodsColocalization studies of SSeCKS with myelination markers, ultrastructural localization of SSeCKS labeling and ablation of largely unmyelinated sensory fibers by neonatal capsaicin administration were all used to establish whether SSeCKS containing sensory neurons represent a subpopulation of unmyelinated primary sensory C-fibers.ResultsDouble labeling studies of SSeCKS with CNPase in the dorsal horn and Pzero in the periphery showed that SSeCKS immunoreactivity was observed predominantly in association with unmyelinated primary sensory fibers. At the ultrastructural level, SSeCKS immunoreactivity was most commonly associated with axonal membrane margins of unmyelinated fibers. In capsaicin treated rats, SSeCKS immunoreactivity was essentially obliterated in the dorsal horn while in dorsal root ganglia quantitative analysis revealed a 43% reduction in the number of SSeCKS-labeled cells. This attenuation is concomitant with a decrease in fluoride-resistant acid phosphatase labeled fibers in the spinal cord dorsal horn and small neuronal somata in sensory ganglia.ConclusionThese results demonstrate that SSeCKS is primarily localized within a distinct subpopulation of small diameter, largely unmyelinated C-fiber primary sensory neurons putatively involved in nociception.

Mapping of the fluoride resistant acid phosphatase (FRAP) activity in cervico-thoracic-lumbar spinal dorsal horn in rats

The fluoride resistant acid phosphatase (FRAP) is considered a biomarker enzyme of presence and function in nociceptive fibers. Its well described spinal dorsal horn (DH) superficial lamina location contrasts to its less known cervico-lumbar DH distribution. Thus, 15 male Sprague Dawley rats (300–400 g) were anaesthetized (thiopental 60 mg kg−1 i.p.) and laminectomized (C 1-L 5) to extract and fix (formaline 4% + NaF 1%) spinal cords. Cervical, thoracic and lumbar segments were coronally cut (1 mm slices) and micropunched (1 mmO) at the dorsal horn (both left and right). Samples were diluted in NaF 1% in saline (1000 μL: fragment) and homogenized. Protein concentration of the homogenate was determined by Bradford method to correct variation on tissue sampling and used to compensate enzymatic dilution. FRAP activity was determined by the Gomorri method and expressed as U gr prot−1 L−1. No differences were found between left and right DH FRAP activities (Mann-Whitney U Test, P > 0.05) in any metamera. DH segment values were: cervical 2.53 (2.67–2.40 U gr prot−1 L−1; mean ± 95% CI), thoracic 3.59 (3.96–3.21) and lumbar 2.42 (2.75–2.09). Thoracic FRAP activity was statistically higher (+45.3%) than those from cervical and lumbar DH segments (ANOVA; Ω2 = 0.29; F = 17.78; P = 0.000001; Levene test P = 0.000006). No significant correlation was found among cervicotoraco-lumbar FRAP activity. The increased FRAP activity for thoracic DH could be explained by the thoracic overlapped primary afferents and collateral fibers with cervical or lumbar input added to the basal thoracic FRAP expression.

Prostatic Acid Phosphatase Is Expressed in Peptidergic and Nonpeptidergic Nociceptive Neurons of Mice and Rats

Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4+) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP+) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP+ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP+ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species. Moreover, PAP+ axon terminals in the rat partially overlap with Protein kinase Cγ (PKCγ+) interneurons in dorsal spinal cord whereas PAP+ axon terminals in the mouse terminate dorsal to PKCγ+ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.

Iontophoretically applied microtubule inhibitors induce transganglionic degenerative atrophy of primary central nociceptive terminals and abolish chronic autochtonous pain.

Transcutaneous iontophoresis of microtubule inhibitors (Vinblastin, Vincristin, Formyl-Leurosin) in rats induces depletion of fluoride-resistant acid phosphatase (FRAP) and transganglionic degenerative atrophy (trggl. deg. atr.) of the central terminals of primary nociceptive neurons, probably via blockade of axoplasmic transport in the peripheral sensory nerves. Radiochemical experiments prove that about 0.2% of the microtubule inhibitors applied iontophoretically at the skin reach the level of nociceptive axon terminals. 40 out of 48 patients suffering from chronic intractable pain of diverse etiology (postherpetic, paresthetic, ischaemic and trigeminal neuralgia, alcoholic and diabetic polyneuropathy, meralgia, brachialgia, discopathia, arthropathia and terminal pain) were successfully treated with Vinblastin or Vincristin iontophoresis. Iontophoretically applied microtubule inhibitors do not affect the blood cell count, have no side-effects and do not impair the skin at the site of application.

Prostatic Acid Phosphatase Is an Ectonucleotidase and Suppresses Pain by Generating Adenosine

Thiamine monophosphatase (TMPase, also known as fluoride-resistant acid phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of prostatic acid phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5'-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain.

Mitigation of nociception via transganglionic degenerative atrophy: Possible mechanism of vinpocetine-induced blockade of retrograde axoplasmic transport

Vinpocetine, a derivative of vincamine, widely used in the clinical pharmacotherapy of cerebral circulatory diseases, inhibits retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve, resulting in transganglionic degenerative atrophy (TDA) in the related ipsilateral superficial spinal dorsal horn, as shown in our previous publications. TDA induced by vinpocetine has been demonstrated to be followed by depletion of the marker enzyme fluoride-resistant acid phosphatase (FRAP) and its isoenzyme thiamine monophosphatase (TMP), and by the decrease in the pain-related neuropeptide substance P from laminae I-II-(III) from the segmentally related, ipsilateral substance of Rolando of the spinal cord. In the present paper, we report on the behavioral effects of perineurally administered vinpocetine. Nociception, induced by intraplantar injection of formalin, was mitigated by vinpocetine; increased expression of c-fos in the ipsilateral, segmentally related upper dorsal horn was also prevented. Since vinpocetine is not a microtubule inhibitor, and its chemical structure differs from that of vincristin and vinblastin (used formerly by us in the therapy of intractable, chronic neuropathic pain), its mode of action is enigmatic. We assume that the effect of vinpocetine in blocking retrograde axoplasmic transport of NGF might be related to its interaction with membrane trafficking proteins, such as signalling endosomes and the endocytosis-mediating "pincher" protein. Temporary, locally restricted decrease of nociception, induced by vinpocetine, might be useful in the clinical treatment of intractable, chronic neuropathic pain, since vinpocetine can successfully be applied by transcutaneous iontophoresis.

Morphine acts via μ-opioid receptors to enhance spinal regeneration and synaptic reconstruction of primary afferent fibers injured by sciatic nerve crush

The present study investigated whether morphine can promote regeneration and synaptic reconstruction of the terminals of injured primary afferent fibers in lamina II of the spinal cord in rats following sciatic nerve injury. Fluoride-resistant acid phosphatase (FRAP)-positive terminals in lamina II of the L4 spinal segment after sciatic nerve injury were assessed after treatment with vehicle, morphine, and naloxone plus morphine. Under the electron microscope, types I and II complex terminals of unmyelinated afferent fibers from the dorsal root, simple terminals of interneuronal axons, and terminals of descending axons at lamina II of the L4 spinal segment were documented in the different groups after injury. FRAP-positive terminals in lamina II were depleted after sciatic nerve injury in the vehicle group. Treatment with morphine increased the numbers of FRAP-positive terminals, and this was prevented by naloxone. The present study demonstrates that morphine may promote the regeneration and synaptic reconstruction of the terminals of injured primary unmyelinated afferent fibers in lamina II of spinal cord, by a process mediated by μ-opioid receptors.

Effect of vinpocetine on retrograde axoplasmic transport

Vinpocetine, a derivate of vincamine, is widely used in the clinical pharmacotherapy of cerebral circulatory diseases. Herewith we report on a novel effect of vinpocetine: inhibition of retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve. Blockade of retrograde transport of NGF results in transganglionic degenerative atrophy (TDA) in the segmentally related ipsilateral superficial spinal dorsal horn, which is characterized by depletion of the marker enzymes fluoride-resistant acid phosphatase (FRAP) and thiamine monophosphatase (TMP). At the same time, pain-related neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), are depleted from lamina I-III from the segmentally related, ipsitateral Rolando substance of the spinal cord. On the basis of these experiments it is suggested that vinpocetine may result in a locally restricted decrease of nociception, that might be useful in clinical treatment of intractable pain. Pilot self-experiments support this assumption.

Capsaicin, transient receptor potential (TRP) protein subfamilies and the particular relationship between capsaicin receptors and small primary sensory neurons

A number of subfamilies of the capsaicin receptor, collectively called TRP, have been reported since the discovery of vanilloid receptor 1 (VR1). The term 'TRP' is derived from 'transient receptor potential', which means the transient and rapid defect of reaction following long stimulation with light in the photoreceptor cells of mutant Drosophila. The common features of TRP family members are the centrally situated six transmembrane domain, in which an ion channel is located, three to four ankirin repeats at the N-terminus and a TRP domain comprising 25 amino acids at the C-terminus. The TRP family members are present in animals, including invertebrates and vertebrates, and in the cells in various tissues in individual animals. During evolution, the original TRP seems to have acquired a wide variety of functions related to sensing the inner or outer environment (e.g. to sensing light (Drosophila), osmolarity, protons, temperature, ligands and mechanical force). In mammals, the TRPV subfamily is exclusively expressed in small- to medium-sized primary sensory neurons that also co-express some chemical markers (i.e. isolectin B4 (IB4), fluoride-resistant acid phosphatase (FRAP), the P2X3 purinoceptor (a receptor provoked by ATP-induced nociception) and Ret, a glial cell line-derived neurotrophic factor receptor). There is a paradox in that regardless of the marked or complete loss of noxious, small sensory neurons (polymodal nociceptors) in mice treated with capsaicin during the neonatal period, as well as in VR1 (TRPV1)-deficient knock-out mice, the responses to noxious heat are normal. Regarding the paradox in mice treated with capsaicin as neonates, our explanation is that although capsaicin probably reduces the number of a subgroup of small neurons (IB4-, VR1+), the remaining IB4+ (VR1-) neurons can sense noxious heat normally. One working hypothesis is that mice lacking TRPV1/2 can sense noxious heat under normal conditions, presumably via another still unknown pathway, and TRPV1 has been suggested to be involved in noxious heat transduction under pathological conditions, such as inflammation and tissue injury. Further studies will be required to clarify these complexities. Mice treated with capsaicin as neonates would provide a model to investigate the above paradoxes, as would TRPV1-knock-out mice, although different mechanisms may be operating in the two models.

Molecular plasticity of primary nociceptive neurons: relations of the NGF- c-jun system to neurotomy and chronic pain

Neurotomy is widely used as a model of chronic, intractable pain, the proverbial "crux medicorum". Immunohistochemical aspects of this chronic pain model are discussed in this paper, with the aim of shedding new light on the pathomechanism and possible therapeutical consequences. Central terminals of nociceptive neurons contain substance P, somatostatin and calcitonin generelated peptide or exhibit fluoride resistant acid phosphatase and thiamine monophosphatase enzyme reaction in the superficial dorsal horn of the spinal cord and in analogous structures of the brain stem. These neuropeptides and neuroproteins are expressed by the related dorsal root ganglion cells and transported via orthograde axoplasmic transport via dorsal roots to the central nervous system. Transection of the ipsilateral, segmentally related peripheral sensory nerve results in transganglionic degenerative atrophy of central terminals of primary nociceptive neurons. Transganglionic degenerative atrophy is characterized by marked ultrastructural alterations superficially similar to, but essentially differing from the signs of Wallerian degeneration which ensue after dorsal rhizotomy. Transganglionic degenerative atrophy is accompanied by depletion of marker neuropeptides and enzymes, and later by the expression of vicarious neuropeptides such as vasoactive intestinal polypeptide, neuropeptide Y and galanin and of the enzyme choline acetyl transferase. Consequences of blockade of retrograde axoplasmic transport of the nerve growth factor elicited either by perineural application of microtubule inhibitors or by perineural administration of anti-nerve growth factor are similar to peripheral neurotomy. According to recent studies described in this paper, the blockade of nerve growth factor supply to primary nociceptive neurons induces activation of c-jun in nuclei of primary nociceptive neurons probably responsible for the plasticity of the neuropeptide and neuroprotein synthesizing machinery. In contrast, invasion of and formation of pericellular baskets by noradrenergic axons can be elicited only by axotomy and not by blockade of retrograde axoplasmic transport. Involvement of nerve growth factor and the nerve growth factor-dependent immediate early genes in neuroplasticity of neuropeptidergic primary sensory neurons raise the possibility of a gene therapy of chronic intractable pain.

Inflammation induced increase of fluoride resistant acid phosphatase (FRAP) activity in the spinal dorsal horn in rats

Fluoride-resistant acid phosphatase (FRAP) has been suggested as an enzymatic marker for nociceptive primary afferent terminals in the spinal dorsal horn, however there has not been demonstrated a direct functional relation between FRAP activity and an increased nociceptive transmission. For this purpose, we quantitated FRAP activity in the spinal dorsal horn of the rat in a heat-induced cutaneous inflammatory model. Male Sprague–Dawley rats anaesthetised with thiopental were separated in two groups where the left hindpaw was submerged during 60 s either in water at room temperature (control group) or in water at 60°C (inflammation group) which induce in this group a progressive hindpaw inflammation. After 8 h, the lumbar enlargement of the spinal cord was extracted, cut in slices and 1 mm micropunch fragments were obtained from the right and left dorsal horn. The activity of FRAP was determined using the Gomori colorimetric method and corrected by the protein concentrations. FRAP activity in the left dorsal horn was statistically higher than right dorsal horn in the inflammation group (3.05±0.54 versus 1.91±0.23 u/g per l; P<0.05). Also, FRAP activity from the left dorsal horn of the control and inflammation groups show a significant increase in the last group (3.05±0.54 versus 2.17±0.23 u/g per l; P<0.05). This results demonstrate that FRAP is not only an enzymatic marker for neuronal and fibre integrity of nociceptive primary afferents but also it is associated to the nociceptive afferent activation.

SSeCKS immunolabeling in rat primary sensory neurons

SSeCKS (src suppressed C kinase substrate) is a protein kinase C substrate that may play a role in tumor suppression. Recently described in fibroblasts, testes and mesangial cells, SSeCKS may have a function in the control of cell signaling and cytoskeletal arrangement. To investigate the distribution of SSeCKS throughout the nervous system, representative sections of brain, spinal cord and dorsal root ganglia were processed using immunofluorescence. Labeling of central axonal collaterals of primary sensory neurons was observed in the dorsal horn at all spinal levels. SSeCKS-immunoreactivity was also observed in the cerebellum, medulla and sensory ganglia (including trigeminal ganglia). The pattern and distribution of anti-SSeCKS labeling in dorsal root ganglia and the dorsal horn of the spinal cord was similar to that observed for other markers of small primary sensory neurons. Therefore, the coexistence of SSeCKS with substance P, CGRP and acid phosphatase was examined in sections of sensory ganglia, spinal cord and medulla using double immunofluorescent labeling for SSeCKS and substance P/CGRP or sequential SSeCKS immunofluorescence and acid phosphatase/fluoride-resistant acid phosphatase enzyme histochemistry. A small portion of the SSeCKS-labeled cell bodies appeared to represent a subpopulation of substance P (4.8%) and CGRP (4.7%) containing neurons, while 45.0% contained fluoride-resistant acid phosphatase reactivity. These results indicate that SSeCKS has a restricted distribution within the nervous system and that expression of this protein may reflect the specific signaling requirements of a distinct population of nociceptive sensory neurons.

Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder

In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6–S1 spinal cord segments and in the T13–L2 and L6–S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit–horseradish peroxidase was injected. Cholera toxin B subunit–horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV–VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin–horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit–horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I–horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit–horseradish peroxidase than with wheat germ agglutinin– or isolectin B4–horseradish peroxidase. In the L6–S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with myelinated fibres). Double labelling with other neuronal markers showed that 71%, 43% and 36% of the cholera toxin B subunit-immunoreactive cells were calcitonin gene-related peptide-, isolectin B4-binding- and substance P-positive, respectively. A few cholera toxin B subunit cells showed galanin-immunoreactivity, but none were somatostatin-, vasoactive intestinal polypeptide-, or neuropeptide Y-immunoreactive or contained fluoride-resistant acid phosphatase. The results show that cholera toxin B subunit–horseradish peroxidase is a more effective retrograde and transganglionic tracer for pelvic primary afferents from the urinary bladder than wheat germ agglutinin–horseradish peroxidase and isolectin B4–horseradish peroxidase, but in contrast to somatic nerves, it is transported mainly by unmyelinated fibres in the visceral afferents.

Fluoride-resistant acid phosphatase (FRAP)-positive afferent terminals make synaptic contact with interneuronal soma in the substantia gelatinosa of the mouse spinal dorsal horn.

Fluoride-resistant acid phosphatase (FRAP)-reactive terminals making contact with interneuronal soma are found in the substantia gelatinosa of the mouse spinal dorsal horn. About one half of the interneuronal somata receive FRAP-positive boutons. By electron microscopy, these FRAP-positive terminals appear small, dark, slender, roundish, cap-like, ellipsoid or sinuous and electron-dense, scalloped (fan-like) contours with clear spherical synaptic vesicles of variable size, some large dense-core vesicles and mitochondria. All these features are very similar to those of capsaicin-sensitive terminals. Thus they are considered to be nociceptive primary afferent endings. Therefore, some of the FRAP-positive terminals are suggested to have a modulatory role in the nociceptive circuit in the substantia gelatinosa.

Catechol-O-methyltransferase in rat sensory ganglia and spinal cord.

The localization of catechol-O-methyltransferase immunoreactivity in rat dorsal root ganglia and in the spinal cord and its co-existence with substance P, calcitonin gene-related peptide and fluoride-resistant acid phosphatase in dorsal root ganglion cells was examined with immunohistochemical and histochemical double-staining methods. Analysis of dorsal of dorsal root ganglia at both cervical and lumbar levels revealed catechol-O-methyltransferase immunoreactivity in numerous dorsal root ganglion cells. Double-staining studies showed that catechol-O-methyltransferase and substance P immunoreactivities were located in different cells with a few exceptions, whereas both catechol-O-methyltransferase and calcitonin gene-related peptide immunoreactivities were detected in about 10% of all labeled cells positive for one of the two markers at both levels studied. The great majority of fluoride-resistant alkaline phosphatase-positive cells were also immunoreactive for catechol-O-methyltransferase. Again, no difference was found between cervical and lumbar levels. Catechol-O-methyltransferase immunoreactivity was also found in the neuropil of the dorsal horn of the spinal cord. The staining was most intense in the superficial laminae (I-III) and overlapped partly with substance P and calcitonin gene-related peptide immunoreactivity. Western blotting analysis revealed that soluble catechol-O-methyltransferase was the clearly dominating form of the enzyme in dorsal root ganglia. The distribution pattern of catechol-O-methyltransferase in dorsal horn and sensory neurons suggests that the enzyme may modulate sensory neurotransmission.

Fluoride-Resistant Acid Phosphatase (FRAP)-Positive Primary Afferent Central Terminals in the Mouse Substantia Gelatinosa.

The ultrastructure of the central terminals of nociceptive primary afferent (Cl terminals) neurons in the mouse substantia gelatinosa was studied by the cytochemical method using fluoride-resistant acid phosphatase (FRAP). Small roundish, slender, sinuous and large scalloped terminals showed FRAP reactivity. The FRAP reaction was seen as electron dense granular deposits between spherical synaptic vesicles and around the membrane of the synaptic vesicles. Especially, dark, small sinuous and large scalloped terminals showed intense FRAP reactivity. From the previous studies, these terminals were in accord with the capsaicin-sensitive Cl terminals. Furthermore, larger, roundish terminals with less packed spherical synaptic vesicles, many mitochondria and a few dense cored synaptic vesicles showed FRAP reactivity. Our recent results that Cll terminals are also sensitive to capsaicin support the FRAP activity of the Cll terminals in the present study. Thus, various types of FRAP-positive Cl terminals and some Cll terminals are considered to be central endings of the capsaicin-sensitive nociceptive primary afferents.

Transganglionic transport and binding of the isolectin B4 from Griffonia simplicifolia I in rat primary sensory neurons

The isolectin B4 from Griffonia simplicifolia I binds to a subpopulation of rat small-diameter dorsal root ganglion neurons, and to fibres and presumed terminals in laminae I–II of the spinal cord dorsal horn. In the present study we investigated B4 and B4 conjugated to horseradish peroxidase as potential transganglionic tracers of somatic primary afferent neurons after injection into a peripheral nerve. We also tried to identify the specific subpopulation of dorsal root ganglion neurons that bind and transport B4. Following injection of B4 or B4-horseradish peroxidase into the sciatic nerve, labelled presumed terminals that reached peak labelling at two days were found exclusively in regions of the spinal cord gray matter known to receive unmyelinated primary afferent fibres. Almost all dorsal root ganglion cells that transported B4-horseradish peroxidase also bound B4. Cell counts showed that 51% of the dorsal root ganglion neurons were B4-positive and cell area measurements that these were all in the small size range. An extensive overlap was found between B4 and fluoride-resistant acid phosphatase (85%), and between B4 and calcitonin gene-related peptide (59%). Seventeen per cent of the B4-positive cells were substance P-immunoreactive and 9% were immunoreactive to somatostatin. Minimal overlap was seen between B4-positive cells and cells positive for RT97 (3%), a selective marker of primary afferent neurons with myelinated axons. All somatostatin-immunoreactive cells and almost all (95%) of the fluoride-resistant acid phosphatase-positive cells were contained within the B4-positive population. This comprised also 58% of the cells immunoreactive to calcitonin gene-related peptide and 42% of those immunoreactive to substance P. The results obtained show that B4 binds to a subpopulation of unmyelinated primary afferent neurons, and that B4 and B4-horseradish peroxidase can be used as selective transganglionic tracers of this specific cell subpopulation.

Emerging relationships between cytochemical properties and sensory modality transmission in primary sensory neurons

Primary sensory neurons have been categorized according to a variety of characteristics, including modality responsiveness, somal size, cytology, cytochemistry, and the organization of their central axon collateral aborizations. A major aim in the study of primary afferents has been to determine the relationships between dorsal root ganglia neuronal physiology, anatomy, and chemistry that could provide a basis for a classification scheme more directly relevant to function. Here we briefly review these relationships and examine the utility of specific histochemical and immunohistochemical markers representative of distinct populations of neurons that may transmit particular sensory modalities. In addition, we discuss some of our observations suggesting that one population of dorsal root ganglia neurons contains high levels of cytochrome oxidase, carbonic anhydrase, parvalbumin, and calbindin D28k, while a separate population contains fluoride-resistant acid phosphatase, calcitonin gene-related peptide, and displays immunoreactivity with an antibody that labels the central arborization of a specific class of unmyelinated afferents in the dorsal horn. This may have implications for the combinations of substances contained within neurons with distinct sensory functions.

A histochemical study of acid phosphatases in medullary bone matrix and osteoclasts in laying Japanese quail.

Acid phosphatase activity in medullary bone matrix and osteoclasts of laying Japanese quail was examined histochemically. To avoid nonspecific staining, the reactivity of the enzyme was evaluated using both the azo dye method and the lead salt method and nonembedded thick sections and resin-embedded thin sections. The pH of the incubation medium was also varied from the acid range (pH 5.0 and 6.5) to the alkaline range (pH 8.5). Medullary bone osteoclasts contain both tartrate-resistant acid phosphatase (TRAP) activity and fluoride-resistant acid phosphatase (FRAP) activity, and no significant difference in intensity was detected between active and inactive osteoclasts. The entire matrix of medullary bone was positive for tartrate-resistant, fluoride-sensitive acid phosphatase activity. No reaction product was observed in sections incubated in substrate-free and pH 8.5 media. The results demonstrate the existence of FRAP in medullary bone osteoclasts and suggest that medullary bone matrix includes TRAP throughout the matrix.

Developmental alterations in nociceptive threshold, immunoreactive calcitonin gene‐related peptide and substance P, and fluoride‐resistant acid phosphatase in neonatally capsaicin‐treated rats

This study examined the effect of neonatal administration of capsaicin on nociceptive threshold and the distribution of calcitonin gene-related peptide (CGRP), substance P (SP), and fluoride-resistant acid phosphatase (FRAP) in the dorsal horn of the spinal cord during the course of development (10 days to 12 weeks of age) in the rat. As early as 10 days of age, CGRP-like immunoreactivity was reduced in laminae I, II, and V, as well as in the bundles of fibers situated dorsal and ventral to the central canal. However, beginning on or about 6 weeks of age, the density of CGRP-like immunoreactivity in the superficial laminae and in the bundles dorsal and ventral to the central canal increased. Moreover, thick, nonvaricose CGRP-like immunoreactive fibers appeared in laminae III and IV. These recurring fibers were of primary afferent origin as demonstrated by their disappearance after multiple, unilateral rhizotomies. A similar age-dependent alteration in the density of FRAP activity was also observed. Although virtually absent at 10 days of age after neonatal administration of capsaicin, the density of FRAP activity increased in lamina II by 8 weeks of age. This activity disappeared after multiple, unilateral rhizotomies, indicating that the FRAP activity that reappeared was of primary afferent origin. Neonatal administration of capsaicin also reduced the density of SP-like immunoreactivity in the dorsal horn as early as 10 days of age, although the density of SP-like immunoreactivity showed some recovery after 6 weeks of age. However, unlike CGRP-like immunoreactivity or FRAP activity, the density of SP-like immunoreactivity in capsaicin-treated rats was not detectably altered by multiple, unilateral rhizotomies, indicating that it originated principally from intrinsic dorsal horn neurons. Age-dependent alterations in both thermal and mechanical, but not chemical, nociceptive thresholds were also observed in these same animals. Thus, tail flick latency, hot plate latency, and paw withdrawal threshold were maximally increased at 6 weeks of age, after which time thresholds declined to vehicle-treated values. In contrast, capsaicin-treated animals were uniformly insensitive to ophthalmic administration of capsaicin. The correspondence between developmental alterations in CGRP-like immunoreactivity or FRAP activity and in thermal and mechanical nociceptive thresholds is suggestive of a role of CGRP- or FRAP-containing primary afferents in thermal and mechanical nociception.