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Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunohistochemistry
Neuroscience Letters 254 (1998) 137–140
Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunohistochemistry Kang Tsou a,*, M. Isabel Nogueron c, Shanmugam Muthian c, M. Clara San˜udo-Pen˜a a, Cecilia J. Hillard c, Dale G. Deutsch d, J. Michael Walker a,b a
Schrier Research Laboratory, Department of Psychology, Brown University, Providence, RI 02912, USA b Department of Neuroscience, Brown University, Providence, RI 02912, USA c Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA d Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA Received 18 June 1998; received in revised form 10 August 1998; accepted 10 August 1998
Abstract The distribution in the rat brain of fatty acid amide hydrolase (FAAH) an enzyme that catalyzes the hydrolysis of the endogenous cannabinoid anandamide was studied by immunohistochemistry. An immunopurified, polyclonal antibody to the C terminal region of FAAH was used in these studies. The large principal neurons, such as pyramidal cells in the cerebral cortex, the pyramidal cells the hippocampus, Purkinje cells in the cerebellar cortex and the mitral cells in the olfactory bulb, showed the strongest FAAH immunoreactivity. These FAAH-containing principal neurons except the mitral cells in the olfactory bulb are in close proximity with cannabinoid CB1 receptors as revealed by our previous immunohistochemical study. Moderately or lightly stained FAAH-containing neurons were also found in the amygdala, the basal ganglia, the deep cerebellar nuclei, the ventral posterior nuclei of the thalamus, the optic layer and the intermediate white layer of the superior colliculus and the red nucleus in the midbrain, and motor neurons of the spinal cord. These data demonstrate that FAAH is heterogeneously distributed and this distribution exhibits considerable, although not complete, overlap with the distribution of cannabinoid CB1 receptors in rat brain. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Fatty acid amide hydrolase; N-arachidonylethanolamine; Cannabinoids; Amidohydrolase; 2-Arachidonylglycerol
The putative endogenous cannabinoid receptor ligand, Narachidonylethanolamine (anandamide) is hydrolyzed to free arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH) [5,12,13]. Alternate substrates of the enzyme include other long chain, unsaturated N-acyl ethanolamines [4], primary amides of fatty acids such as oleamide [3] and 2-arachidonylglycerol [7]. Brain FAAH is membrane-associated [9] and is inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) [5]. FAAH has been purified from liver, sequenced and cloned [2]. The sequence information reveals a likely transmembrane domain at the N terminal which is consistent with the presence of enzymatic activity in membrane fractions. It has been shown that FAAH can also catalyze the synthesis of * Corresponding author. P.O. Box 1853. Tel.: +1 401 8632605; fax: +1 401 8631300; e-mail: [email protected]
anandamide in membrane preparations in the presence of very high concentrations of ethanolamine [5,6,11]; however, FAAH has not been shown to participate in the synthesis of anandamide in intact cells. The distribution of FAAH activity in the brain is not homogeneous; regions with high neuronal cannabinoid receptor (CB1) density including the cerebellum, cortex and hippocampus [8] exhibit high amidohydrolase specific activity [9]. In a recent in situ hybridization study carried out using adult rat brain, FAAH mRNA was observed in neuronal cells with the most prominent signals in the neocortex, hippocampal formation, amygdala and cerebellum [14]. The purpose of the studies outlined here is to study the distribution of FAAH protein in the brain on a cellular level using immunohistochemistry. A peptide corresponding to residues 561–579 of the Cterminus of FAAH [2] was synthesized at the Protein and Nucleic Acid Shared Facility of the Medical College of
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00700- 9
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Wisconsin. Polyclonal antisera was raised in rabbits to a KLH-conjugated preparation of the peptide using conventional methods. The IgG fraction was further purified by affinity chromatography. To determine the specificity of the antibodies, Western blots were performed using rat brain membrane fractions prepared as described previously [9] or using lysates of COS-7 cells stably transfected with FAAH [1]. Fifteen micrograms of brain membrane protein or 10 mg of COS-7 cell lysate were fractionated using 4– 12% gradient gels and transferred onto nitrocellulose. Blots were incubated with affinity purified pre-immune or immune sera (1:250 in blocking solution) overnight at 4°C; antibody binding was visualized using either an HRP-conjugated secondary antibody (1: 1500) or a biotinylated secondary antibody (1:5000) and HRP-conjugated streptavidin (1:1500) followed by enhanced chemiluminescence (ECL, Amersham, UK). Blocking experiments were performed by preincubation of the sera with 5 mg of immunizing peptide for 4 h at 4°C. The immunohistochemical protocol was carried out using brains from male, Sprague–Dawley rats (300–350 g, Charles River, n = 5) as described previously [15]. Briefly, the floating slices (40 mm) were incubated with the affinity purified FAAH antibody (1:100 dilution in 50 mM KPBS, 0.4% Triton, 1% bovine serum albumin) at 4°C for 48 h. The sections were washed with 50 mM KPBS, incubated with biotinylated goat anti-rabbit IgG (1:200) at room temperature for 1 h followed by avidin-biotin complex (Vector Elite, Burlingame, CA). Visible reaction product was produced by treating the sections with 0.04% diaminobenzidine (DAB), 2.5% nickel sulfate and 0.01% H2O2, dissolved in 0.1 M sodium acetate. Controls for immunohistochemistry included the preabsorption (1 h) and co-incubation of the antibody with the immunizing peptide (5, 2.5 or 1.25 mg/ml) and incubation with preimmune serum (1:100). Staining was blocked completely by these two procedures. Antibodies to the carboxy terminus of FAAH recognized a single band of approximately 60 kDa molecular weight in the rat forebrain (Fig. 1) in good agreement with the expected molecular weight of FAAH [2]. No immunoreactivity was seen with either pre-immune serum or immune
Fig. 1. Western blot using affinity purified FAAH antisera. Lanes 1–3: 15 mg of forebrain membranes incubated with affinity purified preimmune sera (lane 1), antisera (lane 2) or antisera preincubated with 5 mg of immunizing peptide. Lanes 4–6: 10 mg COS-7 cell lysates from untransfected cells (lane 4), cells transfected with FAAHpcDNA3 (lane 5) or pcDNA only (lane 6).
Fig. 2. Mitral cells in the olfactory bulb showing strong FAAH immunoreactivity. (A). Mitral cells (arrow) are the only cell type showing FAAH immunoreactivity in the olfactory bulb. (B). Mitral cells with dendrites in the external plexiform layer of the olfactory bulb. Note the dendrites extend most toward the glomeruli but some dendrites extend laterally. A few axons can be seen in the granule cell layer. Scale bar, 200 mm (A), 50 mm (B).
serum preincubated with immunizing peptide. The specificity of the antibody for FAAH was determined comparing Western blots of cell lysates from COS-7 cells stably expressing FAAH with control cells. The antibody labels a protein of approximately 60 kDa in COS-7 cells transfected with FAAH but no signal is observed in untransfected COS-7 cells or cells transfected with vector only (Fig. 1). Membrane proteins from brainstem, cerebellum, striatum, hypothalamus and cerebellar cortex were probed using the FAAH antibody. FAAH immunoreactivity was detected in all regions tested, the intensity of the signals (greatest to least) is cerebellum, cortex, striatum brainstem and hypothalamus (data not shown). This relative distribution is in agreement with the FAAH activity in the same regions [9]. Immunohistochemical studies were carried out using the FAAH antibody. FAAH immunoreactive neurons could be found throughout the central nervous system, however, the most striking finding was that FAAH immunoreactivity was most intense in the large, principal cells of different brain areas. For example, intense staining was seen in the pyramidal cells in the neocortex and the piriform cortex, the pyramidal cells in the hippocampus, the Purkinje cells in the cerebellum and the mitral cells in the olfactory bulb. The staining appeared in puncta, suggestive of an intracellular membrane localization of the protein. In the olfactory bulb, a single layer of mitral cells and their dendrites in the external plexiform layer were clearly stained (Fig. 2). Most of the dendrites extended to the glomeruli, but some extended laterally. Mitral cells and their dendrites in the more caudal accessory olfactory bulb were also clearly stained. In the hippocampal formation, only the pyramidal cells in the stratum pyramidale of CA1 and CA3 and its hilar extension were stained. The granule cells in the dentate were unstained. No immunoreactive interneurons were found in the hippocampal formation (Fig. 3A). The staining in the pyramidal cell bodies was punctate but darker in the periphery. The cell nuclei were generally unstained. The proximal
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(Fig. 4D). No cell bodies in the granular layer were stained. In the deep cerebellar nuclei, both neurons and fibers were moderately stained. The lateral and basal lateral amygdaloid nuclei showed moderate to strong FAAH immunoreactivity. The caudate nucleus contained an even distribution of lightly stained, medium sized neurons. In the subthalamus, small clustered neurons were stained. In the globus pallidus, entopeduncular nucleus and substantia nigra, moderately stained neurons were also seen. In the thalamus, FAAH immunoreactive neurons were seen in the ventral posterior lateral nucleus and the ventral posterior medial nucleus, as well as in the lateral and medial geniculate nuclei. In the hypothalamus, small cells in the arcuate nucleus and dorsal medial nucleus were moderately stained. In the superior colliculus a distinctive pattern of labeled cells in two bands, in the optic layer and the intermediate white layer was observed. Larger FAAH immunoreactive neurons were also seen in the red nucleus. The staining in the pons and medulla oblongata was generally light except the pontine nuclei, in which moderately stained neurons were seen. In the spinal cord, beaded fibers were seen in the Lissauer’s tract. The small neurons in the superficial dorsal horn and the motor neurons in the ventral horn were moderately stained. In the sagittal sections of the brain, corpus callusum and anterior commissure show clear and even immunoreactivity.
Fig. 3. Pyramidal neurons are the only cell type showing FAAH immunoreactivity in the hippocampal formation. (A). Pyramidal neurons in CA1 and CA3 (arrows) are intensely stained. The granule cell layer of the dentate is unstained. (B). Higher magnification of the pyramidal cell bodies and their dendrites in the CA1. Note the cell nuclei are unstained and the staining is punctate. (C). Higher magnification of the pyramidal neurons of CA3 in the hilar region of the dentate gyrus. These neurons are less densely packed and their dendrites extend in different directions. Scale bar, 200 mm (A), 50 mm (B,C).
dendrites running perpendicularly in the stratum radiatum were clearly stained (Fig. 3B). The distal dendrites could be traced to the stratum lacunosum moleculare. The pyramidal cells in the hilus were more dispersed with their dendrites pointing in different directions (Fig. 3C). Very fine FAAH immunoreactive fibers were seen in the alveus and the dorsal and ventral hippocampal commissure. In the neocortex, the large pyramidal neurons and their apical and basal dendrites in the layer V were prominently stained (Fig. 4B). Another group of smaller pyramidal neurons were also seen in layers II and III (Fig. 4A). In the piriform cortex, pyramidal cells in layer II were prominently stained. In the cerebellar cortex, only the Purkinje cells were stained (Fig. 4C,D). The characteristic dendrites (arbor vitae) of the Purkinje cells are clearly visible. Small axons of the Purkinje cells could be traced in the granular layer
Fig. 4. FAAH immunoreactive neurons in the neocortex and the cerebellar cortex. (A). Large pyramidal neurons in layer V and smaller pyramidal neurons in layer II and III in the neocortex showing strong FAAH immunostaining. (B). Higher magnification showing the large pyramidal neurons and their apical and basal dendrites in layer V of the neocortex. Note the staining is punctate. (C). Purkinje cells are the only cell type showing FAAH immunoreactivity in the cerebellar cortex. The granule cell layer is not stained. (D). Higher magnification showing Purkinje cell bodies and their dendrites (arbor vitae). Note a few lightly stained axons of Purkinje cells in the granule cell layer. Scale bar, 200 mm (A), 100 mm (C), 50 mm (B,D).
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These studies demonstrate that FAAH protein has a heterogeneous distribution in the brain and this distribution generally coincides with the localization of mRNA for FAAH demonstrated by in situ hybridization [14], although several areas with strong mRNA signals had weak or negligible staining for FAAH protein. For example, message was seen in the cells of the dentate gyrus of the hippocampal formation and in the granule cell layer of the cerebellum, however, no immunohistochemical staining was seen in these regions. These findings imply that FAAH protein expression is regulated at a step that is post-transcriptional. FAAH immunoreactivity was found to be preferentially localized in large principal neurons. In some areas such as the hippocampus, the cerebellar cortex and the olfactory bulb, the large principal neurons are the only cell type stained. In the cerebellar cortex, the exclusive localization of FAAH in Purkinje cells agrees well with the fact that cerebellar granule cells in culture have little FAAH activity (Greenberg and Hillard, unpublished observations). Although the large principal neurons were the most prominently stained cells by the FAAH antibody, many smaller neurons were moderately or lightly stained. The punctate appearance of FAAH immunoreactivity in neurons is consistent with the intracellular membrane bound location of the enzyme [9]. Although FAAH can catalyze anandamide synthesis in vitro, it is likely that its primary function in vivo is catabolic. Degradative enzymes for several neurotransmitters, including monoamine oxidase A and B which degrade norepinephrine and serotonin, are preferentially localized in noradrenergic and serotonergic neurons, respectively [10]. By analogy, the large principal cells intensely stained for FAAH may represent anandamide-producing neurons. It is of great interest to note that some FAAHcontaining neurons identified in the present work are in close proximity to neurons with strong CB1 receptor immunoreactivities demonstrated in our previous immunohistochemical study [15]. For example, the pyramidal cells in the hippocampus are surrounded by a dense plexus of CB1 immunoreactive fibers, the cell bodies of the Purkinje cells in the cerebellum are surrounded by a dense triangular basket form of CB1 immunoreactivity, and the pyramidal neurons in the neocortex are themselves both CB1 and FAAH immunoreactive. This close proximity supports the hypothesis that FAAH plays a role in the inactivation of anandamide at its site of action, the CB1 receptor. However, in other regions of the brain, the correlation between cells expressing CB1 receptors and cells expressing FAAH is not obvious. For example, the FAAH-containing mitral cells and CB1-containing interneurons in the olfactory bulb [15] are not in close proximity. In addition, strong CB1 immunoreactivity was found in globus pallidus and in substantia nigra reticulata, regions that contained a few FAAHpositive neurons. The fact that FAAH has other noncannabinoid substrates [3] or the possibility that anadamide has another route for metabolism may explain the discrepancy.
These studies were funded by grants from the NIH (NS33247, DA10536 to JMW and KT, K02MH01083, DA10043 to JMW, DA09155 and DA08098 to CJH, DA07318 to DGD). [1] Arreaza, G., Devane, W.A., Omeir, R.L., Sajnani, G., Kunz, J., Cravatt, B.F. and Deutsch, D.G., The cloned rat hydrolytic enzyme responsible for the breakdown of anandamide also catalyzes its formation via the condensation of arachidonic acid and ethanolamine, Neurosci. Letts., 234 (1997) 59–62. [2] Cravatt, B.F., Giang, D.K., Mayfield, S.P., Bolger, D.L., Lerner, R.A. and Guila, N.B., Molecular characterization of an enzyme that degrades neuromodulatory fatty acid amides, Nature, 384 (1996) 84–87. [3] Cravatt, B.F., Prospero-Garcia, O., Siuzdak, G., Gilul, N., Henriksen, S., Bolger, D.L. and Lerner, R.A., Chemical characterization of a family of brain lipids that induce sleep, Science, 268 (1995) 1506–1509. [4] Desarnaud, F., Cadas, H. and Piomelli, D., Anandamide amidohydrolase activity in rat brain microsomes, J. Biol. Chem., 270 (1995) 6030–6035. [5] Deutsch, D.G. and Chin, S.A., Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist, Biochem. Pharmacol., 46 (1993) 791–796. [6] Devane, W.A. and Axelrod, J., Enzymatic synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, by brain membranes, Proc. Natl. Acad. Sci. USA, 91 (1994) 6698– 6701. [7] Goparaju, S.K., Ueda, N., Yamaguchi, H. and Yamamoto, S., Anandamide amidohydrolase reacting with 2-arachidonylglycerol, another cannabinoid receptor ligand, FEBS Letts., 422 (1998) 69–73. [8] Herkenham, M., Lynn, A.B., Little, M.D., Johnson, M.R., Melvin, L.S., DeCosta, B.R. and Rice, K.C., Cannabinoid receptor localization in brain, Proc. Natl. Acad. Sci. USA, 87 (1990) 1932– 1936. [9] Hillard, C., Wilkison, D.M., Edgemond, W.S. and Campbell, W.B., Characterization of the kinetics and distribution of N-arachidonylethanolamine (anandamide) hydrolysis by rat brain, Biochim. Biophys. Acta, 1257 (1995) 249–256. [10] Kitahama, K., Arai, R., Maeda, T. and Jouvet, M., Demonstration of monoamine oxidase type B in serotonergic and type A in noradrenergic neurons in the cat dorsal pontine tegmentum by an improved histochemical technique, Neurosci. Lett., 71 (1986) 19–24. [11] Kruszka, K.K. and Gross, R.W., The ATP- and CoA-independent synthesis of anandamide, a cannabinoid receptor agonist, J. Biol. Chem., 269 (1994) 14345–14348. [12] Natarajan, V., Schmid, P.C., Reddy, P.V. and Schmid, H.H., Catabolism of N-acylethanolamine phospholipids by dog brain preparations, J. Neurochem., 42 (1984) 1613–1619. [13] Schmid, P.C., Zuzarte-Augustin, M.L. and Schmid, H.H., Properties of rat liver N-acylethanolamine amidohydrolase, J. Biol. Chem., 260 (1985) 14145–14149. [14] Thomas, E.A., Cravatt, B.F., Danielson, P.F., Gilula, N.B. and Sutcliffe, J.G., Fatty acid amide hydrolase, the degradative enzyme for anandamide and oleamide, has selective distribution in neurons within the rat central nervous system, J. Neurosci. Res., 50 (1997) 1047–1052. [15] Tsou, K., Brown, S., Sanudo-Pena, M.C., Mackie, K. and Walker, J.M., Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system, Neuroscience, 83 (1998) 393–411.
Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunohistochemistry Kang Tsou a,*, M. Isabel Nogueron c, Shanmugam Muthian c, M. Clara San˜udo-Pen˜a a, Cecilia J. Hillard c, Dale G. Deutsch d, J. Michael Walker a,b a
Schrier Research Laboratory, Department of Psychology, Brown University, Providence, RI 02912, USA b Department of Neuroscience, Brown University, Providence, RI 02912, USA c Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA d Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA Received 18 June 1998; received in revised form 10 August 1998; accepted 10 August 1998
Abstract The distribution in the rat brain of fatty acid amide hydrolase (FAAH) an enzyme that catalyzes the hydrolysis of the endogenous cannabinoid anandamide was studied by immunohistochemistry. An immunopurified, polyclonal antibody to the C terminal region of FAAH was used in these studies. The large principal neurons, such as pyramidal cells in the cerebral cortex, the pyramidal cells the hippocampus, Purkinje cells in the cerebellar cortex and the mitral cells in the olfactory bulb, showed the strongest FAAH immunoreactivity. These FAAH-containing principal neurons except the mitral cells in the olfactory bulb are in close proximity with cannabinoid CB1 receptors as revealed by our previous immunohistochemical study. Moderately or lightly stained FAAH-containing neurons were also found in the amygdala, the basal ganglia, the deep cerebellar nuclei, the ventral posterior nuclei of the thalamus, the optic layer and the intermediate white layer of the superior colliculus and the red nucleus in the midbrain, and motor neurons of the spinal cord. These data demonstrate that FAAH is heterogeneously distributed and this distribution exhibits considerable, although not complete, overlap with the distribution of cannabinoid CB1 receptors in rat brain. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Fatty acid amide hydrolase; N-arachidonylethanolamine; Cannabinoids; Amidohydrolase; 2-Arachidonylglycerol
The putative endogenous cannabinoid receptor ligand, Narachidonylethanolamine (anandamide) is hydrolyzed to free arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH) [5,12,13]. Alternate substrates of the enzyme include other long chain, unsaturated N-acyl ethanolamines [4], primary amides of fatty acids such as oleamide [3] and 2-arachidonylglycerol [7]. Brain FAAH is membrane-associated [9] and is inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) [5]. FAAH has been purified from liver, sequenced and cloned [2]. The sequence information reveals a likely transmembrane domain at the N terminal which is consistent with the presence of enzymatic activity in membrane fractions. It has been shown that FAAH can also catalyze the synthesis of * Corresponding author. P.O. Box 1853. Tel.: +1 401 8632605; fax: +1 401 8631300; e-mail: [email protected]
anandamide in membrane preparations in the presence of very high concentrations of ethanolamine [5,6,11]; however, FAAH has not been shown to participate in the synthesis of anandamide in intact cells. The distribution of FAAH activity in the brain is not homogeneous; regions with high neuronal cannabinoid receptor (CB1) density including the cerebellum, cortex and hippocampus [8] exhibit high amidohydrolase specific activity [9]. In a recent in situ hybridization study carried out using adult rat brain, FAAH mRNA was observed in neuronal cells with the most prominent signals in the neocortex, hippocampal formation, amygdala and cerebellum [14]. The purpose of the studies outlined here is to study the distribution of FAAH protein in the brain on a cellular level using immunohistochemistry. A peptide corresponding to residues 561–579 of the Cterminus of FAAH [2] was synthesized at the Protein and Nucleic Acid Shared Facility of the Medical College of
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Wisconsin. Polyclonal antisera was raised in rabbits to a KLH-conjugated preparation of the peptide using conventional methods. The IgG fraction was further purified by affinity chromatography. To determine the specificity of the antibodies, Western blots were performed using rat brain membrane fractions prepared as described previously [9] or using lysates of COS-7 cells stably transfected with FAAH [1]. Fifteen micrograms of brain membrane protein or 10 mg of COS-7 cell lysate were fractionated using 4– 12% gradient gels and transferred onto nitrocellulose. Blots were incubated with affinity purified pre-immune or immune sera (1:250 in blocking solution) overnight at 4°C; antibody binding was visualized using either an HRP-conjugated secondary antibody (1: 1500) or a biotinylated secondary antibody (1:5000) and HRP-conjugated streptavidin (1:1500) followed by enhanced chemiluminescence (ECL, Amersham, UK). Blocking experiments were performed by preincubation of the sera with 5 mg of immunizing peptide for 4 h at 4°C. The immunohistochemical protocol was carried out using brains from male, Sprague–Dawley rats (300–350 g, Charles River, n = 5) as described previously [15]. Briefly, the floating slices (40 mm) were incubated with the affinity purified FAAH antibody (1:100 dilution in 50 mM KPBS, 0.4% Triton, 1% bovine serum albumin) at 4°C for 48 h. The sections were washed with 50 mM KPBS, incubated with biotinylated goat anti-rabbit IgG (1:200) at room temperature for 1 h followed by avidin-biotin complex (Vector Elite, Burlingame, CA). Visible reaction product was produced by treating the sections with 0.04% diaminobenzidine (DAB), 2.5% nickel sulfate and 0.01% H2O2, dissolved in 0.1 M sodium acetate. Controls for immunohistochemistry included the preabsorption (1 h) and co-incubation of the antibody with the immunizing peptide (5, 2.5 or 1.25 mg/ml) and incubation with preimmune serum (1:100). Staining was blocked completely by these two procedures. Antibodies to the carboxy terminus of FAAH recognized a single band of approximately 60 kDa molecular weight in the rat forebrain (Fig. 1) in good agreement with the expected molecular weight of FAAH [2]. No immunoreactivity was seen with either pre-immune serum or immune
Fig. 1. Western blot using affinity purified FAAH antisera. Lanes 1–3: 15 mg of forebrain membranes incubated with affinity purified preimmune sera (lane 1), antisera (lane 2) or antisera preincubated with 5 mg of immunizing peptide. Lanes 4–6: 10 mg COS-7 cell lysates from untransfected cells (lane 4), cells transfected with FAAHpcDNA3 (lane 5) or pcDNA only (lane 6).
Fig. 2. Mitral cells in the olfactory bulb showing strong FAAH immunoreactivity. (A). Mitral cells (arrow) are the only cell type showing FAAH immunoreactivity in the olfactory bulb. (B). Mitral cells with dendrites in the external plexiform layer of the olfactory bulb. Note the dendrites extend most toward the glomeruli but some dendrites extend laterally. A few axons can be seen in the granule cell layer. Scale bar, 200 mm (A), 50 mm (B).
serum preincubated with immunizing peptide. The specificity of the antibody for FAAH was determined comparing Western blots of cell lysates from COS-7 cells stably expressing FAAH with control cells. The antibody labels a protein of approximately 60 kDa in COS-7 cells transfected with FAAH but no signal is observed in untransfected COS-7 cells or cells transfected with vector only (Fig. 1). Membrane proteins from brainstem, cerebellum, striatum, hypothalamus and cerebellar cortex were probed using the FAAH antibody. FAAH immunoreactivity was detected in all regions tested, the intensity of the signals (greatest to least) is cerebellum, cortex, striatum brainstem and hypothalamus (data not shown). This relative distribution is in agreement with the FAAH activity in the same regions [9]. Immunohistochemical studies were carried out using the FAAH antibody. FAAH immunoreactive neurons could be found throughout the central nervous system, however, the most striking finding was that FAAH immunoreactivity was most intense in the large, principal cells of different brain areas. For example, intense staining was seen in the pyramidal cells in the neocortex and the piriform cortex, the pyramidal cells in the hippocampus, the Purkinje cells in the cerebellum and the mitral cells in the olfactory bulb. The staining appeared in puncta, suggestive of an intracellular membrane localization of the protein. In the olfactory bulb, a single layer of mitral cells and their dendrites in the external plexiform layer were clearly stained (Fig. 2). Most of the dendrites extended to the glomeruli, but some extended laterally. Mitral cells and their dendrites in the more caudal accessory olfactory bulb were also clearly stained. In the hippocampal formation, only the pyramidal cells in the stratum pyramidale of CA1 and CA3 and its hilar extension were stained. The granule cells in the dentate were unstained. No immunoreactive interneurons were found in the hippocampal formation (Fig. 3A). The staining in the pyramidal cell bodies was punctate but darker in the periphery. The cell nuclei were generally unstained. The proximal
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(Fig. 4D). No cell bodies in the granular layer were stained. In the deep cerebellar nuclei, both neurons and fibers were moderately stained. The lateral and basal lateral amygdaloid nuclei showed moderate to strong FAAH immunoreactivity. The caudate nucleus contained an even distribution of lightly stained, medium sized neurons. In the subthalamus, small clustered neurons were stained. In the globus pallidus, entopeduncular nucleus and substantia nigra, moderately stained neurons were also seen. In the thalamus, FAAH immunoreactive neurons were seen in the ventral posterior lateral nucleus and the ventral posterior medial nucleus, as well as in the lateral and medial geniculate nuclei. In the hypothalamus, small cells in the arcuate nucleus and dorsal medial nucleus were moderately stained. In the superior colliculus a distinctive pattern of labeled cells in two bands, in the optic layer and the intermediate white layer was observed. Larger FAAH immunoreactive neurons were also seen in the red nucleus. The staining in the pons and medulla oblongata was generally light except the pontine nuclei, in which moderately stained neurons were seen. In the spinal cord, beaded fibers were seen in the Lissauer’s tract. The small neurons in the superficial dorsal horn and the motor neurons in the ventral horn were moderately stained. In the sagittal sections of the brain, corpus callusum and anterior commissure show clear and even immunoreactivity.
Fig. 3. Pyramidal neurons are the only cell type showing FAAH immunoreactivity in the hippocampal formation. (A). Pyramidal neurons in CA1 and CA3 (arrows) are intensely stained. The granule cell layer of the dentate is unstained. (B). Higher magnification of the pyramidal cell bodies and their dendrites in the CA1. Note the cell nuclei are unstained and the staining is punctate. (C). Higher magnification of the pyramidal neurons of CA3 in the hilar region of the dentate gyrus. These neurons are less densely packed and their dendrites extend in different directions. Scale bar, 200 mm (A), 50 mm (B,C).
dendrites running perpendicularly in the stratum radiatum were clearly stained (Fig. 3B). The distal dendrites could be traced to the stratum lacunosum moleculare. The pyramidal cells in the hilus were more dispersed with their dendrites pointing in different directions (Fig. 3C). Very fine FAAH immunoreactive fibers were seen in the alveus and the dorsal and ventral hippocampal commissure. In the neocortex, the large pyramidal neurons and their apical and basal dendrites in the layer V were prominently stained (Fig. 4B). Another group of smaller pyramidal neurons were also seen in layers II and III (Fig. 4A). In the piriform cortex, pyramidal cells in layer II were prominently stained. In the cerebellar cortex, only the Purkinje cells were stained (Fig. 4C,D). The characteristic dendrites (arbor vitae) of the Purkinje cells are clearly visible. Small axons of the Purkinje cells could be traced in the granular layer
Fig. 4. FAAH immunoreactive neurons in the neocortex and the cerebellar cortex. (A). Large pyramidal neurons in layer V and smaller pyramidal neurons in layer II and III in the neocortex showing strong FAAH immunostaining. (B). Higher magnification showing the large pyramidal neurons and their apical and basal dendrites in layer V of the neocortex. Note the staining is punctate. (C). Purkinje cells are the only cell type showing FAAH immunoreactivity in the cerebellar cortex. The granule cell layer is not stained. (D). Higher magnification showing Purkinje cell bodies and their dendrites (arbor vitae). Note a few lightly stained axons of Purkinje cells in the granule cell layer. Scale bar, 200 mm (A), 100 mm (C), 50 mm (B,D).
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These studies demonstrate that FAAH protein has a heterogeneous distribution in the brain and this distribution generally coincides with the localization of mRNA for FAAH demonstrated by in situ hybridization [14], although several areas with strong mRNA signals had weak or negligible staining for FAAH protein. For example, message was seen in the cells of the dentate gyrus of the hippocampal formation and in the granule cell layer of the cerebellum, however, no immunohistochemical staining was seen in these regions. These findings imply that FAAH protein expression is regulated at a step that is post-transcriptional. FAAH immunoreactivity was found to be preferentially localized in large principal neurons. In some areas such as the hippocampus, the cerebellar cortex and the olfactory bulb, the large principal neurons are the only cell type stained. In the cerebellar cortex, the exclusive localization of FAAH in Purkinje cells agrees well with the fact that cerebellar granule cells in culture have little FAAH activity (Greenberg and Hillard, unpublished observations). Although the large principal neurons were the most prominently stained cells by the FAAH antibody, many smaller neurons were moderately or lightly stained. The punctate appearance of FAAH immunoreactivity in neurons is consistent with the intracellular membrane bound location of the enzyme [9]. Although FAAH can catalyze anandamide synthesis in vitro, it is likely that its primary function in vivo is catabolic. Degradative enzymes for several neurotransmitters, including monoamine oxidase A and B which degrade norepinephrine and serotonin, are preferentially localized in noradrenergic and serotonergic neurons, respectively [10]. By analogy, the large principal cells intensely stained for FAAH may represent anandamide-producing neurons. It is of great interest to note that some FAAHcontaining neurons identified in the present work are in close proximity to neurons with strong CB1 receptor immunoreactivities demonstrated in our previous immunohistochemical study [15]. For example, the pyramidal cells in the hippocampus are surrounded by a dense plexus of CB1 immunoreactive fibers, the cell bodies of the Purkinje cells in the cerebellum are surrounded by a dense triangular basket form of CB1 immunoreactivity, and the pyramidal neurons in the neocortex are themselves both CB1 and FAAH immunoreactive. This close proximity supports the hypothesis that FAAH plays a role in the inactivation of anandamide at its site of action, the CB1 receptor. However, in other regions of the brain, the correlation between cells expressing CB1 receptors and cells expressing FAAH is not obvious. For example, the FAAH-containing mitral cells and CB1-containing interneurons in the olfactory bulb [15] are not in close proximity. In addition, strong CB1 immunoreactivity was found in globus pallidus and in substantia nigra reticulata, regions that contained a few FAAHpositive neurons. The fact that FAAH has other noncannabinoid substrates [3] or the possibility that anadamide has another route for metabolism may explain the discrepancy.
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