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Molecular Brain Research 139 (2005) 367 – 371 www.elsevier.com/locate/molbrainres
Short Communication
a1-Adrenoreceptor in human hippocampus: Binding and receptor subtype mRNA expression
Patricia Szot*, Sylvia S. White, J. Lynne Greenup, James B. Leverenz, Elaine R. Peskind, Murray A. Raskind
Northwest Network Mental Illness Research, Education and Clinical Center (S-116), VA Puget Sound Health Care System, S-116 MIRECC, 1660 S. Columbian Way, Seattle, WA 98108, USA Department of Psychiatry and Behavioral Science, University of Washington, Seattle, WA 98195, USA Accepted 1 June 2005 Available online 20 July 2005
Abstract a1-Adrenoreceptors (AR), of which three subtypes exist (a1A-, a1B- and a1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, a1-ARs are found in the hippocampus where animal studies have shown the ability of a1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of a1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (a1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogenously. Human a1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while a1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. a1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal a1-AR localization between rat and humans and further describe a difference in the localization of the a1A- and a1D-AR mRNA subtype between rats and humans. Published by Elsevier B.V.
Theme: Neurotransmitters, modulators, transporters and receptors Topic: Signal transduction: gene expression Keywords: Norepinephrine; Hippocampus; Prazosin; a1A-Adrenoreceptor; a1D-Adrenoreceptor; In situ; Receptor binding
The a1-adrenoreceptor (AR) is a G-protein-coupled receptor that mediates norepinephrine (NE) signaling via the phosphatidylinositol pathway [5,8]. a1-ARs are localized postsynaptic to noradrenergic terminals where they modulate the release of other neurotransmitters [13,15]. Receptor autoradiography has shown a wide distribution of a1-AR binding sites in the CNS of rodents [11,17,26,27]. Cloning of the a1-AR has revealed three different subtypes
* Corresponding author. Northwest Network Mental Illness Research, Education and Clinical Center (S-116), VA Puget Sound Health Care System, S-116 MIRECC, 1660 S. Columbian Way, Seattle, WA 98108, USA. Fax: +1 206 768 5456. E-mail address: [email protected] (P. Szot). 0169-328X/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.molbrainres.2005.06.013
which are classified as a1A (formerly a1c)-, a1B- and a1DAR (formerly a1a/a1d) [9,12,20,21,24,25]. Expression of each of the a1-AR subtypes has been thoroughly characterized in rodents [6,14,19]. However, a comparison of a1AR expression between humans and rats has not been thoroughly investigated, even though earlier studies indicated differences in a1-AR binding in the hippocampus between rats and humans [3,17,27]. In the hippocampus, a1ARs modulate the activity of many neurons and interneurons in all regions of the hippocampus [2,4,7], ultimately affecting long-term potentiation and memory [22], at least in rodents. Extrapolation of rodent a1-AR function to humans requires knowledge of the distribution of a1-AR subtypes in the human hippocampus. The purpose of this study was to
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examine by receptor autoradiography the binding distribution of the a1-AR in human dorsal hippocampus and to determine by in situ hybridization the localization of each of the a1-AR subtypes (a1A-, a1B- and a1D-AR) mRNA. Hippocampal tissue from eight control (4 men and 4 women) human subjects from the Alzheimer’s Disease Research Center (ADRC) were used for these experiments. The ADRC has received approval for use of tissue from human subjects. The subjects were normal antemortem with no neurological or psychiatric illness and had no brain neuropathology at postmortem examination. The subjects prior to death did not use alcohol or drugs of abuse. Age (mean T SD) for men was 70.3 T 6.6 years (range 55– 84) and 71.0 T 11.1 years (range 38 –85) for women; with postmortem delay of 8.3 T 1.7 h for men and 7.9 T 1.9 h for women. The fresh medial temporal tissue block for each individual was dissected into 1-cm-thick coronal blocks, snap frozen in liquid nitrogen cooled isopentane and stored at À70 -C. Serial coronal dorsal hippocampal sections were cut on cryostat onto Fisher Superfrost slides (Fisher Scientific, Houston, TX) and stored at À70 -C. a1-AR binding sites were measured using 3H-prazosin (a1-AR antagonist; Perkin Elmer, Boston, MA). Briefly, slides were thawed at room temperature for 10 min and then 400 Al/slide of incubation buffer (¨0.2 nM 3H-prazosin in 50 mM Tris buffer, 1 mM EDTA, pH 7.4) was placed over the tissue. Non-specific binding was defined in the presence of 10 AM phentolamine. Slides were incubated for 40 min at room temperature, washed twice for 2 min in ice-cold 50 mM Tris-buffer, pH 7.4, dipped in ice-cold distilled water to remove the salts and then rapidly dried under a stream of
cool air. Slides were apposed to Biomax MR film (Eastman Kodak Co., Rochester, NY) for 8 weeks. For each human, 4 consecutive slides containing the hippocampus were run (each slide contained one section of hippocampal tissue), three slides for total binding and the fourth for non-specific binding. Specific binding was total binding minus nonspecific binding in the same region. Specific binding for 3Hprazosin constituted ¨90% of total binding. Tissue preparation and labeling of the a1A-, a1B- and a1D-AR oligonucleotide probes was performed as previously described for oligonucleotide labeling [23]. For each human subject 3 consecutive slides (each slide containing one section of dorsal hippocampal tissue unilateral) were labeled with either the a1A-, a1B- or a1D-AR probes. The a1A-AR probe consisted of three separate oligonucleotide probes to the following nucleotides of the published human sequence [9,20]: (a) 1– 45, (b) 1102 – 1156 and (c) 1435– 1483. The a1B-AR oligonucleotide probe consisted of three separate oligonucleotide probes to the following nucleotides of the human a1B-AR sequence (PubMed NM_000679.2): (a) 247– 297, (b) 334– 384 and (c) 910 – 960. The a1D-AR oligonucleotide probe consisted of three separate oligonucleotide probes to the following nucleotides of the human a1D-AR sequence [21,25]: (a) 587 –635, (b) 990 – 1038 and (c) 1668 –1716. The oligonucleotide probes were 3V -end-labeled with [33P]-dATP (Perkin Elmer, Boston, MA) using terminal deoxyribonucleotidyl transferase (Invitrogen, Carlsbad, CA) and then purified with MicroSpin G-25 columns (Amersham Biosciences, Piscataway, NJ). The a1A-AR probe contained 1.4 Â 106 cpm/50 Al, the a1B-AR probe contained
Fig. 1. a1-AR sites in human hippocampus. (A) 3H-Prazosin binding pattern (dark line indicates the location of the hippocampal fissure), (B) a1A- and (C) a1DAR mRNA expression in adjacent sections of the same human hippocampus. (D) Cartoon composite of 3H-prazosin binding in relation to where a1A (dark line)- and a1D-AR mRNA (hatched area) is expressed in the same subject. GCL; granule cell layer of the dentate gyrus, CA1 and CA3; pyramidal cell layer of the hippocampus.
P. Szot et al. / Molecular Brain Research 139 (2005) 367 – 371
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0.5 Â 106 cpm/50 Al and the a1D-AR probe contained 0.4 Â 106 cpm/50 Al. The labeled probes were applied to the tissue with HybriSlip hybridization covers (Grace Bio-Labs, Bend, OR) and placed in a moist chamber and incubated overnight at 37 -C for each a1-AR subtype probe. Following incubation of slides, coverslips were removed and sections were washed three times in 1Â SSC (1Â SSC = 150 mM NaCl/15 mM Na-citrate) for 20 min at 65 -C for each of the a1-AR subtypes, then washed for 1 h in 1Â SSC at room temperature. After sections were dehydrated the slides were apposed to Hyperfilm h-max (Amersham) for 4 days for a1A-AR, 11 days for a1B-AR and 7 days for a1DAR. Film was developed as previously described [23]. To determine the localization of labeling over cell bodies, the a1A- and a1D-AR-labeled slides were coated with NTB2 Nuclear Tract Emulsion (undiluted; Eastman Kodak Co) and stored at 4 -C for 7 days for a1A-AR and 14 days for a1DAR, developed as previously described [23] and stained with cresyl violet. To determine which regions of the human hippocampus were labeled with 3H-prazosin, the binding pattern on film was compared to thionine-stained sections and the hippocampal fissure located. Based on the thionine-stained sections and the location of the hippocampal fissure which separates the molecular layer of the dentate gyrus from the hippocampus [1] with respect to 3H-prazosin binding (fissure seen as dark line in Fig. 1A), 3H-prazosin appears to label all three layers of the dentate gyrus (DG: molecular, granular and polymorphic [hilus]) and the stratum lucidum of the CA3 (Fig. 1A) [1]. This description of 3H-prazsoin in the human hippocampus is in agreement with previously published data [3,17,27]. a1A-AR mRNA expression in human dorsal hippocampus was observed only in the granular cell layer (GCL) of the dentate gyrus (Fig. 1B). Localization of a1A-AR labeling to the GCL is verified from emulsioncoated slides using low power magnification under light and dark field (Figs. 2A and B). High power magnification of a1A-AR labeling in the GCL is found over cell bodies (Fig. 2C). The labeling of a1A-AR mRNA in GCL cannot be considered ‘‘background’’ labeling over a dense neuronal population because it differs from what is considered
Fig. 3. Labeling of a1D-AR mRNA in the human hippocampus. Low power light (A)- and dark-field (B) images of a1D-AR mRNA labeling in the pyramidal cell layer of CA1. High power light-field image of a1D-AR mRNA labeling over neuronal cell bodies in the CA1 (C; arrows indicate labeled cell bodies) and background labeling over the GCL of the dentate gyrus (D). GCL = granule cell layer. Scale bar = 200 Am.
‘‘background’’ labeling observed with the a1D-AR probe in the GCL of the dentate gyrus (Figs. 3A, B and D). a1D-AR mRNA expression was observed only in the pyramidal cell layer (CA1 –CA3) of the hippocampus (Fig. 1C). Localization of a1D-AR labeling to the pyramidal cell layer is verified from emulsion-coated slides using low power magnification under light and dark field (Figs. 3A and B). High power magnification of a1D-AR labeling in the pyramidal cell layer is found over cell bodies (Fig. 3C). Expression of the a1B-AR subtype was undetectable in any region of the hippocampus (data not shown). Fig. 1D is a cartoon composite demonstrating the relationship of 3Hprazosin binding to the expression of a1A- and a1D-AR mRNA expression in an individual’s hippocampus. These results show a1-AR binding to occur in specific regions of the hippocampus—the dentate gyrus and stratum lucidum. This binding pattern is in agreement with previously published binding studies in the human hippo-
Fig. 2. Labeling of a1A-AR mRNA in the human hippocampus. Low power light (A)- and dark-field (B) images of a1A-AR mRNA labeling in the GCL. (C) High power light-field image of a1A-AR mRNA labeling over neuronal cell bodies in the GCL of the dentate gyrus. GCL = granule cell layer. Scale bar = 200 Am.
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campus [3,17,27]. However, Biegon et al. [3] incorrectly identified the CA2 and CA1 region [1]. Zilles et al. [27] indicated a greater density of 3H-prazosin in the molecular layer as compared to the rest of the dentate gyrus, but it is difficult to see how this information was concluded from the poorly digitized autoradiograph images. As previously described [17,25] the pattern of 3Hprazosin binding in the human hippocampus is different from that obtained in the rat hippocampus ([11,17,27]; Szot, unpublished observation). Postmortem delay (PMD) does not explain the difference in 3H-prazosin binding in the hippocampus between rats and humans because non-human primates with no PMD had a similar 3H-prazosin binding pattern to that observed here in the human hippocampus [17], indicating a phylogenetic change in a1-AR binding sites in the dorsal hippocampus in primates. The difference in distribution of 3H-prazosin binding in humans from rats suggests that prazosin may exhibit a species-specific behavioral and cognitive response. A difference between humans and rats also extends to the localization of the a1A- and a1D-AR mRNA. As shown here for the first time in the human hippocampus, a1A-AR mRNA is detected only in the GCL of the dentate gyrus and a1D -AR mRNA expression is detected only in the pyramidal cell layer (CA1 –3) of the hippocampus. In the rat hippocampus, previously published data localizing the a1A- and a1D-AR mRNA expression has shown these two subtypes to be expressed in the same regions: the pyramidal cell layer (CA1 – 3) of the hippocampus and GCL of the dentate gyrus ([6,14,19]; Szot, unpublished observation). However, neither rats nor humans express the a1B-AR mRNA in the hippocampus ([6,14,19]; Szot, unpublished observation). The difference in expression of a1A- and a1D-AR mRNA between rats and humans may account for the difference in 3 H-prazosin binding. Zilles et al. [27] suggested that 3Hprazosin binding in the rat and human hippocampus was due to the a1A- and a1B-AR. However, the present data and previously published work in rats [6,14,19] indicate that the a1B-AR mRNA is not expressed in the hippocampus; therefore, it could be suggested that 3H-prazosin may be binding just to the a1A-AR. This hypothesis is supported in the human hippocampus where expression of the a1A-AR mRNA in the GCL of the dentate gyrus would explain the binding of 3H-prazosin in the dentate gyrus and stratum lucidum. Neurons in the GCL innervate the molecular cell layer (dendrites), and axonal projections (mossy fibers) innervate the polymorphic (hilus) and stratum lucidum of the CA3, areas in the human hippocampus where 3Hprazosin was observed. It is unclear why 3H-prazosin did not label the regions where the a1D-AR mRNA was expressed in the human hippocampus, considering prazosin is classified as a nonselective a1-AR antagonist at the concentration used. Classification of prazosin as a non-selective a1-AR antagonist is based on cell culture studies. Membrane binding
studies from tissue homogenates have indicated 3H-prazosin to bind with a greater selectivity to the a1A- and a1B-ARs [10,16], indicating the inability of 3H-prazosin to bind to the a1D-AR in tissue. The exact consequence of this species-specific difference in a1-AR binding and expression of subtypes is unknown. There are differences in agonist selectivity, coupling efficiency, and cellular localization between a1-AR subtypes [18], suggesting that the effects of NE release in the hippocampus may differ between humans and rats. Since hippocampal a1-ARs are involved in learning and memory [22] as well as psychiatric diseases, these findings suggest that extrapolation of a1-AR-mediated results from rat to human should be made with caution.
References
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Short Communication
a1-Adrenoreceptor in human hippocampus: Binding and receptor subtype mRNA expression
Patricia Szot*, Sylvia S. White, J. Lynne Greenup, James B. Leverenz, Elaine R. Peskind, Murray A. Raskind
Northwest Network Mental Illness Research, Education and Clinical Center (S-116), VA Puget Sound Health Care System, S-116 MIRECC, 1660 S. Columbian Way, Seattle, WA 98108, USA Department of Psychiatry and Behavioral Science, University of Washington, Seattle, WA 98195, USA Accepted 1 June 2005 Available online 20 July 2005
Abstract a1-Adrenoreceptors (AR), of which three subtypes exist (a1A-, a1B- and a1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, a1-ARs are found in the hippocampus where animal studies have shown the ability of a1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of a1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (a1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogenously. Human a1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while a1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. a1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal a1-AR localization between rat and humans and further describe a difference in the localization of the a1A- and a1D-AR mRNA subtype between rats and humans. Published by Elsevier B.V.
Theme: Neurotransmitters, modulators, transporters and receptors Topic: Signal transduction: gene expression Keywords: Norepinephrine; Hippocampus; Prazosin; a1A-Adrenoreceptor; a1D-Adrenoreceptor; In situ; Receptor binding
The a1-adrenoreceptor (AR) is a G-protein-coupled receptor that mediates norepinephrine (NE) signaling via the phosphatidylinositol pathway [5,8]. a1-ARs are localized postsynaptic to noradrenergic terminals where they modulate the release of other neurotransmitters [13,15]. Receptor autoradiography has shown a wide distribution of a1-AR binding sites in the CNS of rodents [11,17,26,27]. Cloning of the a1-AR has revealed three different subtypes
* Corresponding author. Northwest Network Mental Illness Research, Education and Clinical Center (S-116), VA Puget Sound Health Care System, S-116 MIRECC, 1660 S. Columbian Way, Seattle, WA 98108, USA. Fax: +1 206 768 5456. E-mail address: [email protected] (P. Szot). 0169-328X/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.molbrainres.2005.06.013
which are classified as a1A (formerly a1c)-, a1B- and a1DAR (formerly a1a/a1d) [9,12,20,21,24,25]. Expression of each of the a1-AR subtypes has been thoroughly characterized in rodents [6,14,19]. However, a comparison of a1AR expression between humans and rats has not been thoroughly investigated, even though earlier studies indicated differences in a1-AR binding in the hippocampus between rats and humans [3,17,27]. In the hippocampus, a1ARs modulate the activity of many neurons and interneurons in all regions of the hippocampus [2,4,7], ultimately affecting long-term potentiation and memory [22], at least in rodents. Extrapolation of rodent a1-AR function to humans requires knowledge of the distribution of a1-AR subtypes in the human hippocampus. The purpose of this study was to
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examine by receptor autoradiography the binding distribution of the a1-AR in human dorsal hippocampus and to determine by in situ hybridization the localization of each of the a1-AR subtypes (a1A-, a1B- and a1D-AR) mRNA. Hippocampal tissue from eight control (4 men and 4 women) human subjects from the Alzheimer’s Disease Research Center (ADRC) were used for these experiments. The ADRC has received approval for use of tissue from human subjects. The subjects were normal antemortem with no neurological or psychiatric illness and had no brain neuropathology at postmortem examination. The subjects prior to death did not use alcohol or drugs of abuse. Age (mean T SD) for men was 70.3 T 6.6 years (range 55– 84) and 71.0 T 11.1 years (range 38 –85) for women; with postmortem delay of 8.3 T 1.7 h for men and 7.9 T 1.9 h for women. The fresh medial temporal tissue block for each individual was dissected into 1-cm-thick coronal blocks, snap frozen in liquid nitrogen cooled isopentane and stored at À70 -C. Serial coronal dorsal hippocampal sections were cut on cryostat onto Fisher Superfrost slides (Fisher Scientific, Houston, TX) and stored at À70 -C. a1-AR binding sites were measured using 3H-prazosin (a1-AR antagonist; Perkin Elmer, Boston, MA). Briefly, slides were thawed at room temperature for 10 min and then 400 Al/slide of incubation buffer (¨0.2 nM 3H-prazosin in 50 mM Tris buffer, 1 mM EDTA, pH 7.4) was placed over the tissue. Non-specific binding was defined in the presence of 10 AM phentolamine. Slides were incubated for 40 min at room temperature, washed twice for 2 min in ice-cold 50 mM Tris-buffer, pH 7.4, dipped in ice-cold distilled water to remove the salts and then rapidly dried under a stream of
cool air. Slides were apposed to Biomax MR film (Eastman Kodak Co., Rochester, NY) for 8 weeks. For each human, 4 consecutive slides containing the hippocampus were run (each slide contained one section of hippocampal tissue), three slides for total binding and the fourth for non-specific binding. Specific binding was total binding minus nonspecific binding in the same region. Specific binding for 3Hprazosin constituted ¨90% of total binding. Tissue preparation and labeling of the a1A-, a1B- and a1D-AR oligonucleotide probes was performed as previously described for oligonucleotide labeling [23]. For each human subject 3 consecutive slides (each slide containing one section of dorsal hippocampal tissue unilateral) were labeled with either the a1A-, a1B- or a1D-AR probes. The a1A-AR probe consisted of three separate oligonucleotide probes to the following nucleotides of the published human sequence [9,20]: (a) 1– 45, (b) 1102 – 1156 and (c) 1435– 1483. The a1B-AR oligonucleotide probe consisted of three separate oligonucleotide probes to the following nucleotides of the human a1B-AR sequence (PubMed NM_000679.2): (a) 247– 297, (b) 334– 384 and (c) 910 – 960. The a1D-AR oligonucleotide probe consisted of three separate oligonucleotide probes to the following nucleotides of the human a1D-AR sequence [21,25]: (a) 587 –635, (b) 990 – 1038 and (c) 1668 –1716. The oligonucleotide probes were 3V -end-labeled with [33P]-dATP (Perkin Elmer, Boston, MA) using terminal deoxyribonucleotidyl transferase (Invitrogen, Carlsbad, CA) and then purified with MicroSpin G-25 columns (Amersham Biosciences, Piscataway, NJ). The a1A-AR probe contained 1.4 Â 106 cpm/50 Al, the a1B-AR probe contained
Fig. 1. a1-AR sites in human hippocampus. (A) 3H-Prazosin binding pattern (dark line indicates the location of the hippocampal fissure), (B) a1A- and (C) a1DAR mRNA expression in adjacent sections of the same human hippocampus. (D) Cartoon composite of 3H-prazosin binding in relation to where a1A (dark line)- and a1D-AR mRNA (hatched area) is expressed in the same subject. GCL; granule cell layer of the dentate gyrus, CA1 and CA3; pyramidal cell layer of the hippocampus.
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0.5 Â 106 cpm/50 Al and the a1D-AR probe contained 0.4 Â 106 cpm/50 Al. The labeled probes were applied to the tissue with HybriSlip hybridization covers (Grace Bio-Labs, Bend, OR) and placed in a moist chamber and incubated overnight at 37 -C for each a1-AR subtype probe. Following incubation of slides, coverslips were removed and sections were washed three times in 1Â SSC (1Â SSC = 150 mM NaCl/15 mM Na-citrate) for 20 min at 65 -C for each of the a1-AR subtypes, then washed for 1 h in 1Â SSC at room temperature. After sections were dehydrated the slides were apposed to Hyperfilm h-max (Amersham) for 4 days for a1A-AR, 11 days for a1B-AR and 7 days for a1DAR. Film was developed as previously described [23]. To determine the localization of labeling over cell bodies, the a1A- and a1D-AR-labeled slides were coated with NTB2 Nuclear Tract Emulsion (undiluted; Eastman Kodak Co) and stored at 4 -C for 7 days for a1A-AR and 14 days for a1DAR, developed as previously described [23] and stained with cresyl violet. To determine which regions of the human hippocampus were labeled with 3H-prazosin, the binding pattern on film was compared to thionine-stained sections and the hippocampal fissure located. Based on the thionine-stained sections and the location of the hippocampal fissure which separates the molecular layer of the dentate gyrus from the hippocampus [1] with respect to 3H-prazosin binding (fissure seen as dark line in Fig. 1A), 3H-prazosin appears to label all three layers of the dentate gyrus (DG: molecular, granular and polymorphic [hilus]) and the stratum lucidum of the CA3 (Fig. 1A) [1]. This description of 3H-prazsoin in the human hippocampus is in agreement with previously published data [3,17,27]. a1A-AR mRNA expression in human dorsal hippocampus was observed only in the granular cell layer (GCL) of the dentate gyrus (Fig. 1B). Localization of a1A-AR labeling to the GCL is verified from emulsioncoated slides using low power magnification under light and dark field (Figs. 2A and B). High power magnification of a1A-AR labeling in the GCL is found over cell bodies (Fig. 2C). The labeling of a1A-AR mRNA in GCL cannot be considered ‘‘background’’ labeling over a dense neuronal population because it differs from what is considered
Fig. 3. Labeling of a1D-AR mRNA in the human hippocampus. Low power light (A)- and dark-field (B) images of a1D-AR mRNA labeling in the pyramidal cell layer of CA1. High power light-field image of a1D-AR mRNA labeling over neuronal cell bodies in the CA1 (C; arrows indicate labeled cell bodies) and background labeling over the GCL of the dentate gyrus (D). GCL = granule cell layer. Scale bar = 200 Am.
‘‘background’’ labeling observed with the a1D-AR probe in the GCL of the dentate gyrus (Figs. 3A, B and D). a1D-AR mRNA expression was observed only in the pyramidal cell layer (CA1 –CA3) of the hippocampus (Fig. 1C). Localization of a1D-AR labeling to the pyramidal cell layer is verified from emulsion-coated slides using low power magnification under light and dark field (Figs. 3A and B). High power magnification of a1D-AR labeling in the pyramidal cell layer is found over cell bodies (Fig. 3C). Expression of the a1B-AR subtype was undetectable in any region of the hippocampus (data not shown). Fig. 1D is a cartoon composite demonstrating the relationship of 3Hprazosin binding to the expression of a1A- and a1D-AR mRNA expression in an individual’s hippocampus. These results show a1-AR binding to occur in specific regions of the hippocampus—the dentate gyrus and stratum lucidum. This binding pattern is in agreement with previously published binding studies in the human hippo-
Fig. 2. Labeling of a1A-AR mRNA in the human hippocampus. Low power light (A)- and dark-field (B) images of a1A-AR mRNA labeling in the GCL. (C) High power light-field image of a1A-AR mRNA labeling over neuronal cell bodies in the GCL of the dentate gyrus. GCL = granule cell layer. Scale bar = 200 Am.
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campus [3,17,27]. However, Biegon et al. [3] incorrectly identified the CA2 and CA1 region [1]. Zilles et al. [27] indicated a greater density of 3H-prazosin in the molecular layer as compared to the rest of the dentate gyrus, but it is difficult to see how this information was concluded from the poorly digitized autoradiograph images. As previously described [17,25] the pattern of 3Hprazosin binding in the human hippocampus is different from that obtained in the rat hippocampus ([11,17,27]; Szot, unpublished observation). Postmortem delay (PMD) does not explain the difference in 3H-prazosin binding in the hippocampus between rats and humans because non-human primates with no PMD had a similar 3H-prazosin binding pattern to that observed here in the human hippocampus [17], indicating a phylogenetic change in a1-AR binding sites in the dorsal hippocampus in primates. The difference in distribution of 3H-prazosin binding in humans from rats suggests that prazosin may exhibit a species-specific behavioral and cognitive response. A difference between humans and rats also extends to the localization of the a1A- and a1D-AR mRNA. As shown here for the first time in the human hippocampus, a1A-AR mRNA is detected only in the GCL of the dentate gyrus and a1D -AR mRNA expression is detected only in the pyramidal cell layer (CA1 –3) of the hippocampus. In the rat hippocampus, previously published data localizing the a1A- and a1D-AR mRNA expression has shown these two subtypes to be expressed in the same regions: the pyramidal cell layer (CA1 – 3) of the hippocampus and GCL of the dentate gyrus ([6,14,19]; Szot, unpublished observation). However, neither rats nor humans express the a1B-AR mRNA in the hippocampus ([6,14,19]; Szot, unpublished observation). The difference in expression of a1A- and a1D-AR mRNA between rats and humans may account for the difference in 3 H-prazosin binding. Zilles et al. [27] suggested that 3Hprazosin binding in the rat and human hippocampus was due to the a1A- and a1B-AR. However, the present data and previously published work in rats [6,14,19] indicate that the a1B-AR mRNA is not expressed in the hippocampus; therefore, it could be suggested that 3H-prazosin may be binding just to the a1A-AR. This hypothesis is supported in the human hippocampus where expression of the a1A-AR mRNA in the GCL of the dentate gyrus would explain the binding of 3H-prazosin in the dentate gyrus and stratum lucidum. Neurons in the GCL innervate the molecular cell layer (dendrites), and axonal projections (mossy fibers) innervate the polymorphic (hilus) and stratum lucidum of the CA3, areas in the human hippocampus where 3Hprazosin was observed. It is unclear why 3H-prazosin did not label the regions where the a1D-AR mRNA was expressed in the human hippocampus, considering prazosin is classified as a nonselective a1-AR antagonist at the concentration used. Classification of prazosin as a non-selective a1-AR antagonist is based on cell culture studies. Membrane binding
studies from tissue homogenates have indicated 3H-prazosin to bind with a greater selectivity to the a1A- and a1B-ARs [10,16], indicating the inability of 3H-prazosin to bind to the a1D-AR in tissue. The exact consequence of this species-specific difference in a1-AR binding and expression of subtypes is unknown. There are differences in agonist selectivity, coupling efficiency, and cellular localization between a1-AR subtypes [18], suggesting that the effects of NE release in the hippocampus may differ between humans and rats. Since hippocampal a1-ARs are involved in learning and memory [22] as well as psychiatric diseases, these findings suggest that extrapolation of a1-AR-mediated results from rat to human should be made with caution.
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