Expression and Distribution of Tartrate-resistant Purple Acid Phosphatase in the Rat Nervous System

Experimental Animals

Five 3-week-old Sprague–Dawley rats of unspecified gender were used in each experiment. Rats were kept under controlled light/dark conditions with food and water available ad libitum. The rats were decapitated and tissues were dissected out for partial purification of TRAP and for RNA preparation, as described in the following sections.

Tissues for immunohistochemistry and in situ hybridization histochemistry were dissected out after perfusion via the ascending aorta with 4% paraformaldehyde in 0.1 M Sörensen's phosphate buffer, pH 7.4, immersed in the same fixative for 24 hr, and then embedded in paraffin. Sections (4.5 μm thick) were adhered to slides (SuperFrost; Menzel-Gläser, Braunschweig, Germany) and processed for immunohistochemistry or ISH histochemistry (see below). Before perfusion the animals were anesthetized with a lethal dose of sodium pentobarbital (60 mg/ml).

The use of animals in this study was approved by the local ethical committee of the Karolinska Institute (S 170/98).

Partial Purification of TRAP from Rat Neural Tissues and Bone

TRAP was purified from the rat brain, spinal cord, trigeminal ganglion, and bone essentially as described previously (Ek-Rylander et al. 1997). The dissected tissues were homogenized in 0.15 M KCl containing 0.1% Triton X-100. The homogenates were centrifuged at 3200 × g for 15 min. Protaminsulfate (5%; Sigma, St Louis, MO) was added drop-wise to the supernatants under continuous stirring, to a final concentration of 0.5%. The pH was adjusted to 6.5 with acetic acid, and insoluble material was allowed to precipitate for 30 min. The samples were then centrifuged as above. All procedures up to this point were carried out at 4C. The supernatants were loaded on a CM-cellulose column (4 ml) (CM-52; Whatman, Maidstone, UK) previously equilibrated with 0.1 M sodium acetate buffer, pH 6.5. The column was washed with three bed volumes of 0.15 M sodium acetate, pH 6.5, and the TRAP enzyme was then eluted with 0.4 M sodium acetate, pH 6.5. All steps were performed in the presence of the protease inhibitors E-64 (10 μg/ml), Pefabloc (1 mg/ml), pepstatin A (10 μg/ml) (Boehringer Mannheim; Mannheim, Germany), and EDTA (5 mM). Further purification of the samples was performed for electrophoresis and immunoblotting analysis. The samples were loaded on a phenyl–Sepharose column (Pharmacia Biotech; Uppsala, Sweden) previously calibrated with 0.05 M sodium acetate, pH 5.0, and 144 g/liter ammonium sulfate. Protein was eluted with a gradient from 0.05 M sodium acetate, pH 5.0, containing 144 g/liter ammonium sulfate, to 0.05 M sodium acetate, pH 5.0.

Assay Procedures

The TRAP enzyme activity in each purified sample was assayed in 96-well plates using p-nitrophenylphosphate (pNPP) as substrate in an incubation medium (200 μl) containing (final concentrations): 2.5 mM pNPP (ditris salt; Sigma), 0.1 M sodium acetate buffer, pH 5.8, 0.2 M KCl, 0.1% Triton X-100, 10 mM sodium tartrate, and the reducing agents ascorbic acid (1 mM) and FeCl3 (100 μM). The p-nitrophenol liberated after 1-hr incubation at 37C was converted into p-nitrophenolate by the addition of 50 μl of 0.9 M NaOH, and the absorbance was measured at 405 nm using a Spectramax250 spectrophotometer (Molecular Devices; Sunnyvale, CA). One unit (U) of TRAP activity corresponds to 1 μmol of p-nitrophenol liberated per minute at 37C. To oxidize the reduced enzyme, 10 mM hydrogen peroxide (H₂O₂) was added, and after incubation for 10 min at room temperature (RT), the substrate (pNPP) was added. The concentration of inhibitor required to reduce enzyme activity to 50% of the activity in the absence of inhibitor (IC50) for the oxyanions molybdate, tungstate, and phosphate was calculated using linear regression analysis. K_m for pNPP was calculated using the method of Lineweaver–Burke.

Electrophoresis and Immunoblotting Analysis

Partially purified TRAP (30 mU) from each tissue was subjected to SDS-PAGE in 12% slab minigels according to the procedure described by Laemmli (1970). Proteins were transferred to immuno-PVDF membranes (Bio-Rad; Hercules, CA) using 150 mA for 2 hr. The membranes were rinsed in 20 mM Tris-HCl, pH 7.5, containing 500 mM NaCl and 0.05% Tween-20 (TBST) and then incubated overnight at 4C in 10% dried non-fat milk in TBST. The membranes were then incubated with polyclonal rabbit antirat TRAP antibodies (Ek-Rylander et al. 1997) diluted 1:100 in TBST containing 0.5% dried non-fat milk for 1 hr at RT. After three washes for 10 min each in TBST, the membranes were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch Laboratories, West Grove, PA). The membranes were washed six times for 10 min in TBST and developed for 1 min with Western Blot Chemiluminescence Reagent Plus (NEN Life Science; Boston, MA) according to the manufacturer's protocol. The membranes were then exposed to HyperfilmECL (Amersham Life Science; Poole, UK) using intensifying screens.

Total RNA Preparation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted according to the method of Chomczynski and Sacchi (1987). For RT-PCR analysis, tissues were dissected under aseptic conditions and immediately frozen in liquid nitrogen and then stored at −70C. mRNA was purified using an Oligotex mRNA Mini Kit (QIAGEN; Hilden, Germany). For nested PCR, total RNA was extracted using an RNeasy Mini Kit (QIAGEN).

The RT reaction was performed on 1 μg of mRNA or 5 μg of total RNA (for nested PCR) with 200 U Superscript II RNase H^– Reverse Transcriptase (Gibco; Paisley, UK) at 37C, using oligo d(T)_12–18 (Gibco) as primer, according to the manufacturer's protocol. Negative controls included reactions in which the reverse transcriptase was excluded from the reaction mixture.

PCR amplification was performed on aliquots of cDNA corresponding to 200 ng mRNA or 800 ng total RNA (for nested PCR) using 2.5 U Taq polymerase in the presence of 1.5 mM MgCl₂ and 1 × Q-Solution according to the manufacturer's protocol (QIAGEN). The 5′- TRAP primer (positions 32–47; 5′-TTCTGTTCCAGGAGCTT-3′) and 3′-TRAP primer (positions 600–616; 3′-CAGTCGGTCGTCGGACT-5′) were added (150 ng/reaction) (positions calculated from the rat TRAP cDNA sequence available at GenBank/EMBL Data Bank accession no. M76110). The expected size of the TRAP cDNA fragment is 586 bp.

For nested PCR, the 5′-TRAP primer 5′-CGCCAGAACCGTGCAGA-3′ (positions 160–176) was included in the second amplification, generating a band with a predicted size of 356 bp. The cDNA was amplified in a Perkin–Elmer Cetus Instruments DNA ThermalCycler, running 25, 30, 35, or 40 cycles under the following conditions: 94C, 1 min; 56C, 1 min; 72C, 1 min. In the nested PCR, cDNA was amplified in a Perkin–Elmer Cetus Instruments DNA GeneAmp PCR system 9700 running 40 cycles twice under the following conditions: 94C, 30 sec; 56C, 30 sec; 72C, 30 sec. The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide, 0.15 μg/ml.

PCR analysis for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was performed on all of the samples as an internal control for amplification of equal amounts of RNA. The primers 5′-CTGAACGGGAAGCTCACTGG-3′ and 3′-TGAGGTCCACCACCCTGTTG-5′corresponding to positions 664–683 and 976–957 in the rat GAPDH cDNA sequence (Fort et al. 1985) were used to generate a fragment of the predicted size of 312 bp.

Sequencing of TRAP cDNA PCR Product

Twenty ng of PCR product from the trigeminal ganglion RT-PCR reaction using 5′-TRAP primer (positions 34–49; 5′-TTCTGTTCCAGGAGCTT-3′) and 3′-TRAP primer (positions 602–618; 3′-CAGTCGGTCGTCGGACT-5′) was ligated into a pCR 2.1 vector and then transformed into TOP 10 F′ cells using a TA Cloning Kit (Invitrogen; Leek, The Netherlands) according to the manufacturer's protocol. Cells were plated onto Luria-Bertani-41 (LB) plates containing ampicillin (50 g/ml) and 5-bromo-4-chloro-3-indolyl · β-d-galactopyranoside (X-gal) (40 μl; 40 mg/ml) and screened for colonies containing vector with insert using blue/white selection.

Plasmids from selected colonies were miniprepared according to the protocol of Maniatis (Fritsch et al. 1989), with the exceptions that 400 μg lysozyme was added in the first step and that the plasmid DNA was precipitated twice in isopropanol. The accuracy of the insert was confirmed using PCR with the same primers as for nested PCR, and then sequenced at Cybergene (Huddinge, Sweden).

Immunohistochemistry

Paraffin sections of brain, spinal cord, spinal ganglia, trigeminal ganglion, celiac–superior mesenteric ganglion complex, superior cervical ganglion, and long bones were deparaffinized and rehydrated, and then incubated in a microwave oven in citrate buffer, pH 6.0 (1.8 mM citric acid, 9.8 mM sodium citrate) at 1100 W for 3 min and then at 160 W for another 12 min. After cooling to RT, immunostaining was achieved using 200 μl polyclonal antiserum, diluted 1:500, against TRAP as previously described (Ek-Rylander et al. 1997; Nordahl et al. 1998). After washing with 50 mM Tris-HCl, pH 7.6, 150 mM NaCl (TBS) three times for 10 min, the sections were exposed to 200 μl biotinylated goat anti-rabbit IgG (E0432; DAKO, Copenhagen, Denmark) diluted 1:300 for 30 min at RT and then washed as above. To amplify the signal, the sections were incubated with Strept-ABComplex/HRP (K0355; DAKO) according to the manufacturer's protocol for 30 min at RT. After washing, the sections were incubated for 5 min at RT with the substrate diaminobenzidine (DAB; Sigma) (600 μg/ml) in TBS containing 0.03% H₂O₂.

Incubation of parallel sections with primary antibodies preabsorbed with 1 μg purified PAP antigen or with normal swine serum (X0901; DAKO) diluted 1:500 served as controls.

Some of the sections were counterstained with Ehrlich hematoxylin before dehydration and mounting.

The localization of TRAP immunoreactivity and the anatomic structures were identified by using a stereotaxic atlas (Paxinos and Watson 1986; Paxinos et al. 1999a, b).

DIG Labeling of RNA Probe

The plasmid pT7T3 19U (Pharmacia) containing rat TRAP cDNA fragment of 832 bp (Ek-Rylander et al. 1991) was linearized with HindIII (for the sense probe) or with EcoRI (for the antisense probe). The labeling was performed with digoxygenin-UTP (DIG RNA Labeling Kit; Boehringer Mannheim) according to the manufacturer's protocol, with the exception that T3 RNA polymerase was used instead of SP6 polymerase.

In Situ Hybridization Histochemistry

Paraffin sections prepared as described above were heated overnight at 55C. After deparaffinization and rehydration, the sections were washed in PBS (0.145 M NaCl, 0.01 M Na2PO₄). After treatment with pepsin (1.5 mg/ml) (Boehringer Mannheim) in 0.02 M HCl at 37C for 20 min and washing in PBS, the sections were postfixed in 2% paraformaldehyde in PBS for 5 min at RT and then washed in PBS. The sections were prehybridized in 2 × SSC (1 × SSC = 150 mM NaCl, 15 mM sodium citrate)/50% formamide at 42C for 2 hr. Hybridization was performed in 30 μl of a solution containing 2 × SSC, 2 × Denhardt's, 1 mM EDTA, 0.25 mg/ml tRNA, 100 mg/ml dextran sulfate, 50% formamide, and 2.5 ng/ml probe at 42C overnight. The sections were washed twice in 2 × SSC for 10 min and then three times in 0.1 × SSC for 15 min at RT. The detection was performed using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim) according to the manufacturer's protocol. After counterstaining with Ehrlich hematoxylin, the sections were dehydrated and mounted. Sections exposed to DIG-labeled sense probes served as negative controls.

Partial Purification and Characterization of TRAP Activity from Rat Neural Tissue

TRAP was partially purified from trigeminal ganglion, spinal cord, brain and, as a comparison, from rat bone, according to an established procedure (Ek-Rylander et al. 1997). Addition of reducing agents, such as 1 mM sodium ascorbate and 0.1 mM FeCl₃, converts the inactive enzyme to the active Fe(III)Fe(II) mixed-valent form. The yields of activated TRAP enzyme activity (U/g tissue) varied significantly among the tissues (Table 1). The recoveries from trigeminal ganglia, spinal cord, and brain amounted to 10, 4, and 0.4%, respectively, compared to the recovery of TRAP activity from bone. After oxidation with H₂O₂, virtually no TRAP activity was detectable in any of the enzyme preparations analyzed (Table 1). The K_m of the isolated TRAP enzyme for hydrolysis of the substrate, pNPP, was in the range of 0.23–0.56 mM and was not significantly different between the various tissues. These values are similar to those previously determined for highly purified preparations of recombinant rat TRAP (Ek-Rylander et al. 1997).

To examine the sensitivity to different oxyanion inhibitors of TRAP from the trigeminal ganglion, spinal cord, brain, and bone, the IC₅₀ values for the known PAP/TRAP inhibitors molybdate, tungstate, and phosphate were determined (Table 1). The IC₅₀ values were derived from linear regression analysis. The sensitivity of TRAP enzyme activity for these inhibitors was similar in the different tissues, and the rank order of potency was molybdate>tungstate>phosphate.

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Table 1

Biochemical properties of TRAP partially purified from rat long bones and neural tissue

	Bone	Trigeminal ganglia	Spinal cord	Brain
U/g tissue (reduced)	7.1 ± 3.8	0.75 ± 0.26	0.31 ± 0.14	0.03 ± 0.01
Reduced/unreduced	28	14	17	17
% activity after
reduction/oxidation	0.3	0	0	0.7
Km	0.23 ± 0.04	0.56 ± 0.10	0.56 ± 0.19	0.38 ± 0.01
IC50 (μM) MoO4 2–	39 ± 8	58 ± 22	30 ± 14	10 ± 4
IC50 (μM) WO4 2–	239 ± 63	241 ± 70	163 ±70	21 ± 2.6
IC50 (μM) PO4 3–	1030 ± 200	1620 ± 680	1250 ± 110	900 ± 100

Identification of TRAP Protein and mRNA in Neural Tissue

Immunoblotting analysis of partially purified TRAP (30 mU) from the trigeminal ganglion, spinal cord, and brain showed the appearance of a protein band with the expected size of 35–37 kD in the absence of β-mercaptoethanol as the disulfide reductant (Figure 1). Recombinant rat TRAP (100 ng) expressed from a Baculovirus vector was used as a positive control (Ek-Rylander et al. 1997). To show the presence and relative abundance of TRAP mRNA in bone, trigeminal ganglion, spinal cord and brain, RT-PCR was performed and aliquots were analyzed after 25, 30, 35, and 40 cycles (Figure 2A). A PCR product of 586 bp, corresponding to the theoretical size, was detected, and the relative expression levels were consistent with those observed for enzymatic activity in the different tissues (Table 1). Sequencing of the 586-bp product obtained from the trigeminal ganglion showed complete identity with the bone TRAP cDNA sequence. A larger fragment, 1078 bp, was detected after amplification of cDNA from spinal cord and brain. The levels of GAPDH mRNA was similar in the different samples, ensuring that amplification of equal amounts of RNA had been achieved (Figure 2B). No product was amplified when reverse transcriptase had been omitted, demonstrating the absence of DNA contamination of the different RNA preparations.

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Figure 1

Western blotting analysis of TRAP purified from neural tissue and bone in the absence of reducing agents. Thirty mU of purified TRAP enzyme activity from trigeminal ganglia, spinal cord, and brain was applied to a 12% non-reducing SDS-PAGE, blotted to PVDF, and incubated with polyclonal rabbit antibodies to TRAP. Baculovirus-expressed rat TRAP (100 ng) was used as a positive control. Pre-stained SDS-PAGE standards were electrophoresed in separate lanes.

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Figure 2

RT-PCR analysis of TRAP mRNA expression in neural tissue and bone. (A) Aliquots were removed at different cycles of the PCR reaction to compare the levels of TRAP in neural tissues and bone. Arrows mark the larger-sized band, whereas TRAP marks the band of the expected size. (B) GAPDH was used as an internal control for the PCR reaction. (C) Nested PCR of the 40-cycle reaction in A.

Nested RT-PCR of cDNA from bone, trigeminal ganglion, spinal cord, and brain was performed with 40 cycles, resulting in a PCR product of 356 bp corresponding to the theoretical size (Figure 2C).

Immunohistochemical Localization of TRAP

TRAP-like immunoreactivity was encountered in neuronal cell bodies in both the central (Figures 3–7) and peripheral (Figures 8 and 9) nervous systems of untreated rats. The signal was seen in the cytoplasm of the cells but was not uniformly distributed. Instead, the signal appeared in aggregated structures (see Figures 5 and 7–9). Immunoreactive material could also be seen in neuronal processes close to the cell soma (Figure 5A). A very weak immunoreactive signal was observed in nerve fibers in the dorsal horn of the spinal cord. TRAP-immunoreactive material was also seen in glial cells, e.g., in the white matter of corpus callosum (Figures 3A–3C).

Central Nervous System. Wide distribution of TRAP-like immunoreactivity was observed in neurons in the rat brain and spinal cord. A large number of neuron populations were labeled but with a signal of variable intensity. The overall distribution is shown in the set of color micrographs (Figures 3A–3C) representing different levels of the brain. A strong signal was noted in the neurons in the septum and diagonal bands (Figure 3A), ventral pallidum, cerebral cortex, and piriform cortex (Figures 3A–3C). Intensely stained cells were also observed in the triangular septal nucleus. In the cerebral cortex, the most intense staining was seen in neurons in Layers V and VIb, somewhat less intense in Layer VIa, less in Layers II and III, and very weak in Layer I (Figures 3A–3C). All of the pyramidal cells in hippocampal fields CA3 and CA4 (Figures 3B–3C, 6, and 7B) were intensely immunoreactive, whereas in the CA1 (Figures 6 and 7A) and CA2 (Figure 6) fields a strong signal was limited to scattered neurons. A strong signal could be demonstrated in neurons in the hilar region of the hippocampus, whereas the majority of the granule cells of the dentate gyrus exhibited a weaker signal (Figures 6 and 7B). A few scattered granule cells in the dentate gyrus had a stronger TRAP immunoreactivity (Figure 7B). Strong TRAP-like immunoreactivity could be seen in neurons in the exterior globus pallidus (Figures 3B and 8D) and the caudate nucleus (Figures 3B and 8B), particularly in larger cells (Figure 8B), and in the interior globus pallidus (Figures 3B and 8A) and medial habenula (Figure 3B). Neurons in the thalamus and hypothalamus were intensely stained, with particularly strong labeling in the reticular, rhomboid, anterodorsal, and ventral posterolateral thalamic nuclei (Figure 3B). Moderate staining was observed in the dorsal nucleus of the hippocampal commissure, ventromedial hypothalamic nucleus, and basolateral amygdaloid nucleus. Weak labeling was observed in the dorsomedial and ventrolateral laterodorsal thalamic nuclei. There was intense TRAP-like immunoreactivity in neurons of the substantia nigra pars compacta and reticulata (Figures 3C and 8C), neurons of the Darkschewitsch nucleus, the lateral mammillary nucleus, the magnocellular red nucleus, and the postcommissurial magnocellular nucleus (Figure 3C).

In the pons and medulla oblongata, intensely stained neurons were present, e.g., in the dorsal part of the central gray and the deep gray layer of the superior colliculus (Figure 5A), the spinal trigeminal nucleus, the inferior olive, and the parvocellular reticular nucleus alpha (Figure 4B). A strong signal was seen in the neurons in the spinal cord (Figure 4C), particularly within α-motor neurons in the ventral spinal cord (Figures 4C and 5B). A very weak signal was noted in nerve fibers in the dorsal horn of the spinal cord. In the olfactory bulb, intense staining of nerve cells was observed in the mitral cell layer and external plexiform layer (Figure 4A). Scattered cells were also detected in the internal plexiform and granular layers and in the glomerular layer. Medium immunoreactivity could be seen in the mammillary nucleus. In the cerebellum, intense labeling was observed in the Purkinje cells, and in scattered neurons in the molecular and granular layer (Figures 4B, 9A, and 9B).

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Figure 3

Micrographs of frontal sections of the rat brain at 0.7 mm anterior bregma (A) and 2.1 (B) and 4.8 mm (C,D) posterior to bregma after incubation with antiserum to TRAP (A–C) or TRAP antiserum preabsorbed with antigen (D). All sections were counterstained with Ehrlich's hematoxylin. Intensely TRAP-immunoreactive cells are observed in the septum and diagonal bands and in the piriform cortex (A). Similarly, strong labeling can be seen in all of the pyramidal cells of the hippocampal fields CA3 and CA4 (B,C) and in scattered cells in the CA1 and CA2 fields (C), whereas only a weak signal is seen in the granule cells of the dentate gyrus (B,C). In the cerebral cortex, neurons in Layers V and VIb are most strongly labeled (B). Strong TRAP-like immunoreactivity is also seen in neurons in the interior and exterior globus pallidus and in the reticular, rhomboid, and anterodorsal thalamic nuclei (B). Neurons with intense labeling can also be seen in the substantia nigra pars compacta, the magnocellular red nucleus, the postcommissurial magnocellular nucleus, the lateral mammillary nucleus, and the Darkschewitsch nucleus (C). No labeling is observed in the section incubated with control serum (D). ad, anterodorsal thalamic nucleus; ca3, pyramidal cell layer of CA3 in the hippocampus; cpu, caudate putamen; dk, Darkschewitsch nucleus; egp, exterior globus pallidus; igp, internal globus pallidus; lm, lateral mammillary nucleus; mhb, medial habenula; pir, piriform cortex; rh, rhomboid thalamic nucleus; rt, reticular thalamic nucleus; sn, substantia nigra; vdb, ventral diagonal band; I, V, and VIb, layers of the cerebral cortex. Bar = 500 μm.

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Figure 4

Micrographs of sections of the rat olfactory bulb (A), medulla oblongata (B), and the thoracic spinal cord (C,D) after incubation with antiserum to TRAP (A–C) or hybridization with an antisense cRNA probe to TRAP mRNA (D). The sections in A and B were counter-stained with Ehrlich's hematoxylin. Strong TRAP-like immunoreactivity can be seen in neurons in the mitral cell layer and external plexiform layer of the olfactory bulb and in scattered cells in the internal granular and plexiform layers and the glomerular layer (A). TRAP immunoreactivity of varying intensity is found in many of the nuclei in the medulla oblongata, e.g., the PCRtA, IO, MVe and Sp5 (B). Both TRAP-like immunoreactivity (C) and TRAP mRNA (D) can be seen in nerve cells in the gray matter of the spinal cord. Gl, glomerular layer; EPl, external plexiform layer; IGr, internal granular layer; IO, inferior olive; Mve, medial vestibular nucleus; PCRtA, parvicellular reticular nucleus alpha; Sp5, spinal trigeminal nucleus. Bars: A,C,D = 250 μm; B = 500 μm.

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Figure 5

Micrographs of sections of rat brain stem (A), spinal cord (B,C), and cerebral cortex (D,E) after incubation with antiserum to TRAP (A,B,D,E) or hybridization with an antisense cRNA probe to TRAP mRNA (C). Neurons with intense immunoreactivity for TRAP are observed in the trochlear nucleus and with medium level of labeling in the CG, the DpG, and the Pn (A). TRAP-like immunoreativity can be seen as rounded or irregularly shaped structures in the cytoplasm of neuronal cells, particularly the large α-motor neurons (B). A similar pattern of intense labeling for TRAP mRNA can be seen after ISH histochemistry. Note also the immunoreactive material and hybridization signal in the first part of the axon (arrows in B and C, respectively). (C) Higher magnification of part of Figure 4 D. TRAP-immunoreactive neurons are observed in all layers of the cerebral cortex (D). Both pyramidal cells (short arrows pointing to the same neurons in D and E) and granule cells (long arrows pointing to the same cell in D and E). (E) Higher magnification of part of D. 4, trochlear nucleus; Aq, aqueduct; CG, central gray; DpG, deep gray layer of the superior colliculus; Pn, pontine nucleus. Bars: A = 500 μm; B–D = 50 μm; E = 25 μm.

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Figure 6

Micrograph montage of a frontal section of the rat hippocampus after incubation with antiserum against TRAP. Intense TRAP-like immunoreactivity can be seen in pyramidal cells of the hippocampal fields CA3 and CA4, in scattered pyramidal cells in the CA1 and CA2 fields, and in neurons in the hilar region. Weaker staining is seen in the majority of pyramidal cells in the CA1 and CA2 fields and in the granule cells in the dentate gyrus. Scattered TRAP-positive cells can also be seen in the hippocampal fields stratum oriens and radiatum and the molecular layer of the dentate gyrus. CA1, CA2, and CA3, pyramidal cell layers of the CA1, CA2, and CA3 fields of the hippocampus; DG, dentate gyrus of the hippocampus. Bar = 50 μm.

Peripheral Nervous System. TRAP-like immunoreactivity was observed in all neuronal cell bodies in both sensory ganglia, and paravertebral and prevertebral sympathetic ganglia (Figures 10 and 11). Medium to large nerve cells in sensory ganglia, including the trigeminal ganglion (Figures 10A and 10C) and dorsal root ganglia (Figures 11C and 11F), were intensely labeled, whereas smaller neurons exhibited a weaker staining intensity. Similarly, in the superior cervical ganglion (Figures 11A and 11D) and the celiac ganglion (Figures 11B and 11E), larger neurons were more intensely stained than smaller nerve cells, although the difference in cell size was not as marked as in the sensory ganglia.

Controls. No signal could be seen in sections incubated with primary antiserum preabsorbed with the antigen (see Figures 3D, 9C, and 10D) or after omission of the primary antiserum (not shown).

Histochemical Localization of TRAP mRNA by In Situ Hybridization

Central Nervous System. The distribution of TRAP mRNA expression closely resembled that of the TRAP-like immunoreactivity in the spinal cord (Figures 4D and 5C). In the cerebellum, a strong signal was seen in Purkinje cells (Figures 9D and 9E), and intense labeling was also observed in some cells in the molecular layer (Figures 9D and 9E).

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Figure 7

Micrographs of sections of the CA1 field (A), CA4 field (B), and dentate gyrus (B) of rat hippocampus after incubation with antiserum to TRAP. Intense immunoreactivity for TRAP can be seen in scattered cells in the pyramidal cell layer of the CA1 field and the granule cell layer of the dentate gyrus (arrowheads on some of these cells in A and B), whereas the majority of the cells in these layers are weakly stained (arrows on some in A and B). Many intensely stained pyramidal cells are observed in the CA4 field (B). Bar = 50 μm.

Peripheral Nervous System. In the trigeminal ganglion, TRAP mRNA expression was most marked in small to medium-sized neurons, whereas the larger neurons were weakly labeled (Figure 10B and 10E).

Controls. No signal could be seen after hybridization with labeled sense probe (Figure 9F) or after treatment with RNase before the hybridization (not shown).