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 local-
ized 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: szot@u.washington.edu (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 a1D-
AR (formerly a1a/a1d) [9,12,20,21,24,25]. Expression of
each of the a1-AR subtypes has been thoroughly charac-
terized in rodents [6,14,19]. However, a comparison of a1-
AR expression between humans and rats has not been
thoroughly investigated, even though earlier studies indi-
cated differences in a1-AR binding in the hippocampus
between rats and humans [3,17,27]. In the hippocampus, a1-
ARs modulate the activity of many neurons and interneu-
rons 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
368 P. Szot et al. / Molecular Brain Research 139 (2005) 367 – 371

examine by receptor autoradiography the binding distribu-
tion 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 70 -C. Serial coronal dorsal hippocampal sections were
cut on cryostat onto Fisher Superfrost slides (Fisher
Scientific, Houston, TX) and stored at 70 -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 non-
specific binding in the same region. Specific binding for 3H-
prazosin constituted ¨90% of total binding.
Tissue preparation and labeling of the a1A-, a1B- and
a1D-AR oligonucleotide probes was performed as previ-
ously 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 oligonu-
cleotide 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 (Amer-
sham Biosciences, Piscataway, NJ). The a1A-AR probe
contained 1.4  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) a1D-
AR 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 – 371 369

0.5  106 cpm/50 Al and the a1D-AR probe contained 0.4 

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 1 SSC (1 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 1 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 a1D-
AR. 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 a1D-
AR, developed as previously described [23] and stained Fig. 3. Labeling of a1D-AR mRNA in the human hippocampus. Low power
with cresyl violet. light (A)- and dark-field (B) images of a1D-AR mRNA labeling in the
To determine which regions of the human hippocampus 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
were labeled with 3H-prazosin, the binding pattern on film
labeled cell bodies) and background labeling over the GCL of the dentate
was compared to thionine-stained sections and the hippo- gyrus (D). GCL = granule cell layer. Scale bar = 200 Am.
campal 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 ‘‘background’’ labeling observed with the a1D-AR probe
hippocampus [1] with respect to 3H-prazosin binding in the GCL of the dentate gyrus (Figs. 3A, B and D).
(fissure seen as dark line in Fig. 1A), 3H-prazosin appears a1D-AR mRNA expression was observed only in the
to label all three layers of the dentate gyrus (DG: molecular, pyramidal cell layer (CA1 –CA3) of the hippocampus (Fig.
granular and polymorphic [hilus]) and the stratum lucidum 1C). Localization of a1D-AR labeling to the pyramidal cell
of the CA3 (Fig. 1A) [1]. This description of 3H-prazsoin in layer is verified from emulsion-coated slides using low
the human hippocampus is in agreement with previously power magnification under light and dark field (Figs. 3A
published data [3,17,27]. and B). High power magnification of a1D-AR labeling in the
a1A-AR mRNA expression in human dorsal hippo- pyramidal cell layer is found over cell bodies (Fig. 3C).
campus was observed only in the granular cell layer Expression of the a1B-AR subtype was undetectable in any
(GCL) of the dentate gyrus (Fig. 1B). Localization of region of the hippocampus (data not shown). Fig. 1D is a
a1A-AR labeling to the GCL is verified from emulsion- cartoon composite demonstrating the relationship of 3H-
coated slides using low power magnification under light and prazosin binding to the expression of a1A- and a1D-AR
dark field (Figs. 2A and B). High power magnification of mRNA expression in an individual’s hippocampus.
a1A-AR labeling in the GCL is found over cell bodies (Fig. These results show a1-AR binding to occur in specific
2C). The labeling of a1A-AR mRNA in GCL cannot be regions of the hippocampus—the dentate gyrus and stratum
considered ‘‘background’’ labeling over a dense neuronal lucidum. This binding pattern is in agreement with
population because it differs from what is considered 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.
370 P. Szot et al. / Molecular Brain Research 139 (2005) 367 – 371
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 3H-
prazosin 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 3H-
prazosin 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 3H-
prazosin 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 non-
selective a1-AR antagonist at the concentration used.
Classification of prazosin as a non-selective a1-AR antag-
onist 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 sub-
types [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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