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Developmental changes in human -aminobutyric acida receptor subunit composition.

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Developmental Changes in
Human y-Armnobutyric AcidA Receptor
3uburnt Composiuon
A. R. Brooks-Kayal, MD, and D. B. Pritchett, PhD
y-Aminobutyric acid (GABA) is the neurotransmitter at most inhibitory synapses in the human central nervous system.
The GABA, receptor, a ligand-gated ion channel, is the site of action of benzodiazepines, the most widely prescribed
neuroactive drugs. It was recently demonstrated that there are multiple subtypes of GABA, receptors. Studies of
rodents have shown that receptor subunits are developmentally controlled. The major a subunit of the adult receptor
is expressed at low levels before birth. This study, using postmortem human tissue, shows that GABA, receptors are
present in significant numbers in the human cerebellum at birth, and the numbers rise threefold by adulthood. TWO
subtypes of benzodiazepine receptors were detected by binding studies in the neonate, whereas only a single subtype
of receptor was detected in the adult cerebellum. Comparison to recombinant human GABA, receptors shows that
receptors containing a1 constitute 50% of the receptors at birth and the percentage rises to over 95% by adulthood.
In both cerebral cortex and cerebellum, a dramatic rise in a1 messenger RNA was observed during development,
suggesting that the complement of GABA, receptors differs in infants and adults. These findings have significant
implications for normal neurodevelopment as well as for the understanding and treatment of pathophysiological states
such as seizures.
Brooks-Kayal AR, Pritchett DB. Developmental changes in human y-aminobutyric aCidA
receptor subunit composition. Ann Neurol 1993;34:687-693
~~~
~~~
The inhibitory effects of the neurotransmitter yaminobutyric acid (GABA) are mediated by two classes
of receptors, GABA,, which are ligand-gated anion
channels, and GABA,, which are coupled to G proteins. The GABA, receptor is the site of action of
several antiepileptic drugs, including barbiturates and
benzodiazepines [l-31. The GABA, receptor is a protein complex composed of multiple receptor subunits.
In the rodent, six a (a1-6), three p (pl-3), three
y(y1-3), and one 6 (6) have been identified [4-83.
Two p subunits have also been isolated from human
retina [C,]. Expression studies of recombinant receptors
using different combinations of subunit complementary D N A s (cDNAs) have demonstrated that changes
in subunit composition confer differences in pharmacological properties. For example, y subunits are required
to confer a vigorous response to benzodiazepines [lo}.
It is the a subunit, however, that dictates the benzodiazepine binding type. Two pharmacologically distinct
classes of benzodiazepine binding sites, type I and type
11, have been identified in the rat brain [ll]. These
two receptor types exhibit different developmental
patterns, and have distinct anatomical patterns of ex-
pression in the rodent [la].Recombinant GABA, receptors that contain a l subunits exhibit type I binding,
and receptors that contain ot2, 013, and a5 subunits
exhibit type 11 binding { S , 7, 10, 13, 141. Brain regions
that express the highest levels of a1 messenger RIVA
(mRNA) [ 151 correspond precisely to regions that exhibit mainly benzodiazepine type I binding [ 16- 181.
Recently, Northern blotting, in situ hybridization,
and quantitative polymerase chain reaction (PCR) techniques demonstrated changes in levels of mRNA encoding GABA, receptor subunits in rat brain during
devclopment. Levels of a t , a6, p2, y2, and 6 mRNAs
increase during development, whereas a3 shows a
postnatal decline. Several other subunits, including a2,
a5, PI, p3, y l , and y3, demonstrate a postnatal peak,
which is followed by a decrease later in development
119-231. Note that the sequence termed a5 by Laurie
and colleagues 1221 is referred to as a4 in the work
by Maclennan and associates r2.3). Developmental
changes in human GABA, receptor subtypes have not
been as extensively investigated. Benzodiazepine binding studies suggest that there is a significant increase in
total receptor density in human frontal cortex between
From the Children’s Hospital of Philadelphia, Children’s Seashore
House, and the Deparrments of Pcdiarrics, Pharmacology, and Ncurology, University of Pennsylvania, Philadelphia, PA.
Address correspondence t o Dr Pritchett, Neuroscience Research,
Room 7 159, Children’s Hospital uf Philadelphia, 34th St and Civic
Center Blvd, Philadelphia, PA 10104-4399.
Received March 31, 1993, and in revised form May 18. Accepted
for publication May 20, 1993.
Copyright
199’1 by the American Ncurological Association 687
weeks 17 and 26 after conception, and another steep
increase postnatally t24, 251. Western blots using an
a 1 subunit-specific monoclonal antibody have suggested that there is an increase in a1 subunit concentration beginning at 33 weeks after conception E251. Neither developmental changes in subunit mRNA levels
nor ontological changes in benzodiazepine binding
types in human brain have been studied previously. In
the current study, changes in benzodiazepine binding
type during human development between 36 weeks
after conception and adulthood are demonstrated. Developmental changes in GABA, receptor al mRNA
expression are shown to occur during the same time
period.
Specimens of frontal cortex and cerebellum were obtained
at autopsy from patients without known neurological disease,
ranging in age from 36 weeks after conception to 81 years.
Tissue was obtained through the National Disease Research
Institute and Department of Pathology of the Hospital of
the University of Pennsylvania. Prior to collection of tissue,
approval for the study was obtained from the Committee
on Studies Involving Human Beings of the University of
Pennsylvania. The postmortem interval ranged from 3 to 22
hours. Age, sex, cause of death, and postmortem interval
were recorded for each case. The cause of death in all of the
pediatric patients was congenital heart disease (n = 5). The
causes of death in the adults included sepsis (n = 2), hypovolemia ( n = 11, and metastatic biliary carcinoma (n = 1).
ment. [d*P)uridine 5 '-triphosphate (650 Ciimmol) was
obtained from New England Nuclear (Boston, MA). Kadiolabeled probe was digested using RNAse-free DNAse (Ambion), then purified by repetitive phenol-chloroform extractions followed by ethanol precipitation. Purified radiolabeled
probe (500,000 cpm) was mixed with either 25 pg of total
R N A extracted from frontal or cerebellar cortex o r subunitspecific R N A sense standards of known quantity synthesized
from the cloned fragment using T7 R N A polymerase. Reactions were then hybridized overnight at 42°C in 80% foracid)
mamide-40 mM piperazine-N,N'-bis(2-ethanesulfonic
(PIPES) ( p H 6.4)-0.4 M NaOAc- 1 mM ethylenediaminetetraacetic acid. Hybrids were digested with RNAse (50
unitsiml of RNAse A + 10,000 unitsiml of RNAse T1) for
45 minutes at 30°C to remove unhybridized probe. RNAse
was inactivated by incubation with proteinase K and 209%
sodium dodecyl sulfate for 15 minut-es at 37"C, followed
by a phenol-chloroform extraction and ethanol precipitation.
Protection products were separated on an 8% denaturing
polyacrylamide gel and visualized by autoradiography. Quantification of radioactivity in protected fragments was performed by exposing the gel to a Molecular Dynamics Phosphor-Image screen and comparing the intensity of protected
bands in brain samples to that of R N A sense standards of
known quantity using the lmige-Quint program. To control
for variations in the amount of intact total R N A included in
each sample, an R h A s e protection assay using a radiolabeled
human cyclophilin RNA probe was performed in parallel
to those using the a1 subunit-specific probe. Quantities of
receptor subunit mRNA were reported in nanograms per
nanogram of cyclophilin mRN A. Linear regression analysis
was used to determine statistical association between age and
a1 mRNA level.
Tissae Handling
Benzodiazepine Binding
Materials and Methods
Postmortem Specimens
Specimens were removed at autopsy and rapidly frozen, without fixation, in liquid nitrogen or o n dry ice, and stored at
- 70°C. Total R N A was isolated from brain samples using an
acid guanidinium thiocyanate-phenol-chloroform extraction
method as described by Chomczynski and Sacchi [26].Tissue
was prepared for binding studies by homogenization in 20
volumes of ice-cold buffer (10 rnM potassium phosphate, pH
7.2, and 100 mM potassium chloride).
Meuszlrement of Messenger RNA Levels
Quantification of GABA, receptor subunit mRNA was performed using the KNAse protection assay 1271.A fragment
of D N A that encoded a 1.1-kb portion (bp 41-1149) of the
human GABA, receptor a1 subunit D N A was cloned into
a pBS+ transcription vector with the 3' end 25 bp downstream from the bacteriophage T3 promoter. The plasmid
was linearized at a BamH1 site 7 5 bp upstream from the 3'
end of the a1 fragment. Probes and control RNAs for the
RNAse protection assay were transcribed with T3 or T7
polymerase by use of a MAXIscript T7IT3 in vitro transcription kit from Ambion (Austin, TX) in accordance with the
manufacturer's instructions. T3 R N A polymerase was used
to synthesize a "P-labeled 100-bp antisense RNA probe.
Seventy-five base pairs were complementary to the human
a1 subunit mRNA and 25 bp were complementary to plasmid sequences adjacent to the 3' end of the cloned a1 frag-
688 Annals of Neurology
Vol 34
No 5
Brain tissue specimens were washed three times by homogenization in 20 volumes of ice-cold buffer ( 10 mM potassium
phosphate, p H 7.2) and centrifugation. In a total volume of
1 rnl of buffer (10 mM potassium phosphate, p H 7.2, 100
aliquots of membrane susmM potassium chloride), 8 0 0 - ~ 1
pension (20 pg of membrane proteiniml) were incubated
with 100 pl of{'H)Ro 15-1788 (70 Ciimmol; New England
Nuclear) and 100 ~1 of either 1 pM clonazepam or varying
concentrations (0.001-100.000 pM) of CL2 18-872. ['HIRo 15-1788 was used at 1 nM final concentration for the
CL2 18-872 binding inhibition studies and at concentrations
from 0.1 to 10.0 nM for Scatchard analysis. After incubation
for 60 minutes at 4"C, the membranes were collected by
rapid filtration on Whatman GF/C filters and immediately
washed two times with 5 ml of ice-cold 10 mM potassium
phosphate buffer, p H 7.2. Radioactivity was measured by
liquid scintillation spectroscopy. Specific binding was defined
as the difference between total binding and binding in the
presence of clonazepam. Protein concentrations were determined according to the method of Lowry and associates f281.
Results
To determine whether changes in benzodiazepine
binding to human tissue paralleled the changes described in rat, radiolabeled benzodiatepines were used
November 1993
0.02
Y
B
B
z
8
0.01
m
1
2
BOUND [3H]Ro 15-1788 pMOLES/rng PROTEIN
4
Fzg 1. Rue in the number of G A B A , receptors during human
cerebellar development. Scatchard aiial~szJof the number of
GABAAreceptors measured by saturatton bindzng of bemodiazepines shows a threejold htghw level 2n adult cerebellum (open
squares) than tn 36-week terebellztrn (filled squares) The radioligand nsed, {jH}Ro 15-1 788. i s expeited to bind uvth the
same 1 nM uffrnity to GABA, receptors containing cu1, d.
123,or as Jubunits
to study binding at GABA, receptors. Benzodiazeyine
binding has been previously demonstrated in human
postmortem tissue. W e obtained tissue at autopsy from
infants and adults who had died from causes not related
to brain dysfunction. Washed membranes prepared
from cerebellar tissue specimens displayed saturable,
high-affinity binding for the benzodiazepine antagonist
[3HJRo 15-1788. Scatchard transformation of the
binding data showed that there was binding to a single,
high-affinity 0.5 nM Kd site in both specimens. B,,
values were 0.6 pmol/mg of protein at 36 weeks and
1.7 pmolimg of protein in the adult tissue (Fig l), a
threefold rise in the total number of GABAA receptors in the cerebellum from birth to adulthood. 'This
rise is similar to the rise seen by others for rat [12)
and human cerebral cortex E24, 251.
Since the radioligand ['H}Ro 15-1788 used to determine the total number of benzodiazepine receptors recognizes several subtypes of GABA, receptors,
binding inhibition data were used to determine
whether there were changes in the level of individual
subtypes over the same developmental time period.
Similar inhibition binding experiments have detected
changes in receptor subtype composition during development in other mammalian species. The triazolopyridazine CL2 18-872 was used to competitively displace
The binding of the I3H)Ro 15-1788. CL218-872
binds with different affinities to the benzodiazepine
binding site on different subtypes of the GABA,
receptor. Two distinct affinities can be measured for
-7
-6
-5
-4
[ CL218-872 ] Log M
F i g 2 . DnJelopmentalchanges in subtypes of the G A B A , recrpto? in human cerebellum. Diiplacement of benzodiazepine binding t o human G A B A , receptm b-y CL218-872. a compound
that is selectice for subtypes of the receptorvdemonjtrates that receptor subtypes change during neurodmelopment of the humun
cerebellum. CL218-872 binds with high af;finityto receptors
containing the a1 subunit. The adult cerebellum (diamonds)
seems t o haw 0.6 the high-affinity binding sites. andfits well
to the one-site theoretical cume shown. The neonatal cerebellum
(squares) has both u high- and a low-affinity site. The lowaffinity binding component presumably is the result of binding
to receptors uiith d ,
cu3, or a5 subunits.
individual receptor subtypes that are expressed in
cells transfected with clones for particular subunits.
{jH}Ro 15-1788 binding was displaced by the use of
CL218-872 at a large number of different concentrations. The concentration-dependent displacemerit of
the ['H)Ro 15-1788 binding (Fig 2) to neonatal brain
membranes covered 4 log units of CL218-872 concentration, and suggests the existence of more than one
affinity site for CL2 18-872. The displacement curve
generated using adult tissue was noticeably different,
indicating that there is only a single affinity site for
CL218-872. The computer program PROPHET was
used to perform curve-fitting analysis for each inhibition curve. The results show that there are two different affinities with inhibition constants of (Ki 93 nM
and 2,622 nM) for CL218-872 in the neonatal cerebellum, and only one in the adult cerebellum (Ki 143
nM). The predicted affinities of these binding sites are
very similar to those described for type I and type 11
benzodiazepine receptors. Based on these inhibition
data, neonatal cerebellum appears to contain 48%' type
I and 52% type I1 benzodiazepine receptors, whereas
adult cerebellum contains only type I sites.
Brooks-Kayal and Pritchett: Human GABAA Receptor Development
689
I
-0
-7
-6
-5
-4
-8
[ CL218-872 1 Log M
jinitji binding sites.
Previous studies of recombinant human GABA, receptors have demonstrated that the high-affinity, type
I binding characteristics are a property of receptors
that contain the c r l subunit. Lower-affinity, or type I1
CL2 18-872 binding sites, are found when recombinant
receptors contain the a 2 or 013 subunit. To determine
if the binding sites detected in adult human brain membranes were the result of the expression of GABA,
receptors that contain a1 subunits, the binding inhibition curves for the human brain membranes were compared to those obtained when membranes from cells
transfected with the a l , p2, and y2 subunits were used.
The similar affinities of CL218-872 binding sites in human adult brain membranes and membrane samples of
transfected cells that express the a1 subunit (Fig 3A)
suggest that GABA, receptors in adult tissue contain
the crl subunit. TOpredict the CY subunit composition
of the neonatal tissue, the CL2 18-872 binding inhibi-
-6
-5
-4
[ CL218-872 ] Log M
B
A
Fig 3. ldentzficution of the G A B A , receptor a subunits in hu?nun brain tisszre by conipariron of CL2 18-872 binding affinities. Human 293 cells were trunsfected with DNA encoding
either the a1 or a? subu~zitin conjunction with PI and y2
subunits. and the affinity of CL.218-872 /or the resulting
GABA, receptors uzas determined. (A)The high-affinity binding .teen in & expressing the a1 mbunit (filled diamonds)
matches the single ufi-nit>rneu~uredin adult himan cerebellum
(open diamonds). (l3)The two-aj$nity site binding inhibition
data seeti in neonatal irrebelliim (open squares) are a combinatioil o f tuio ~ingle-affinit31Binding cuwe-r. Cuwe fitting w i r i g
the program PROPHET generutes a Jtutisticalh better fit of the
neonatal data wing the affinities derivedfrom transfected [Pi)
human enibyonit- kidney 293 cells (American Type Cultwe Collection no. CRL 1 3 7.3) and assuming an equal proportion of
high- ( a l . filled diamonds) and 1ou'- (a.3,filled squares) a$
-7
tion data were compared to similar data derived from
cells transfected with either C Y ~ ,pl, y2 or a3,pl, y2
subunits. Comparison of the inhibition curves (Fig 3B)
derived from cells transfected with pure populations
of type I ( a l )and type I1 (a3)receptors suggests that
the complex inhibition curve detected in neonatal tissue is the result of the expression of approximately
equal percentages of type I and type I1 benzodiazepine
receptors.
The results of the binding studies suggest that during
development, levels of the a1 subunit rise so that it
becomes the predomiriant GABA, benzodiazepine receptor expressed in adult human cerebellum. To more
directly assess whether changes in a. subunit composition occurred during human development, a1 mRNA
levels were measured using the ribonuclease protection
assay {27]. The RNAse protection assay was selected
for several reasons. First, the assay is not as sensitive
to mRNA degradation as is Northern blot analysis.
Second, the assay is insensitive to closely related sequences, such as the other a subunits. Finally, the assay
allows the level of R N A to be measured in absolute
terms, such as micrograms, rather than in relative units,
such as percent change in level. mRNA encoding the
a1 subunit increased significantly between 36 weeks
after conception and adulthood in both cortex ( p =
0.0023) and cerebellum ( p = 0.0079). In cortex, a1
mRNA increased nearly fourfold from the lowest level
of 2.49 pgipg of cyclophyllin mRNA at 36 weeks, to
a peak of 9.51 in adulthood (Fig 4). In cerebellum,
a1 mRNA increased threefold, from 2.65 pg/pg of
cyclophyllin mRNA at 36 weeks, to 7.20 in adulthood
(Fig 5). The time between death and autopsy did not
significantly influence a1 mRNA values ( p = 0.77).
690 Annals of Neurology Vol 34 No 5 November 1993
GABAA a1 subunit
10
9
8
7
6
5
4
3
2
Cyclophilin
1
0
A
B
Fig 4. Ariabsis of a1 messenger R N A (mRNA) levels in human cortex during development. (A)A repmentative autoradiogram of radiolabeled D N A fragments separated on a polyacylamid gel and (B) a graph of the level of a1 mRNA after
conuwsion of rudiouctivitp i n each band t o picogram are shown.
Euch Lane represents an assay perfomed on 25 pg of total RNA
isolated from human brain of postconceptionalages 36. 41,
42.5, 44. and 50 weeh and adult brain (A:lanu 1 , 2. 3, 4 ,
5 . and 6 , respectizdy). Brain R N A from each age was hybridized with antisense probes j i r a1 and cyclophilin as described in
the text. The RNAse protection assay for human cyclophilin. a
l-elfularhousekeeping protein, was pegormed in parallel with
the a1 mRNA assay to control for dzferences in the amount of
intact mRNA in each sample.
GABAA a1 subunit
1
2
3
4
5
6
zl
Cyclophilin
1
A
Fi g 5 . Analysic of a1 messenger RNA imRNAj levels in hu?nun cerebellmn during development. (A) A representatiw autoradiogram of radiolabeled D N A fragments separated on a pobacqdamide gel and (Bi a graph of the h e 1 af a1 mRhTAafter
conversion of radioactiziity in each band t o picograms aw shown.
Each fane rKprKSenlS an assay perfbmed on 23 pg of total RNA
isolated from human brain of postconceptional ages 36, 41,
42.5, 44, and 50 weeh and adult (A:lanes I . 2, 3. 4. 5. and
6. respectively).Brain R N A from each age was hybridized with
antisense probes for a1 and cyclophilin a.r described in the text.
Brooks-Kayal and Pritchett: Human GABA, Receptor Development
691
Discussion
The present study demonstrates changes in benzodiazepine binding during human neurodevelopment with
increases in both total benzodiazepine binding and the
percentage of type 1 benzodiazepine binding between
36 weeks after conception and adulthood. To confirm
that the increase in type I binding was a result of increases in a1 subunit expression, as suggested by recombinant studies, levels of a 1 mRNA were measured
directly and found to increase during human postnatal
development in parallel with the changes in benzodiazepine binding.
The increase in human a 1 mRNA levels seen in
our study resembles the developmental changes in a1
mRNA previously seen in the rat {19-231. Rat a1
mRNA levels reach adult values by the third postnatal
week, whereas according to our study, human a1
mRNA levels do not reach adult values until sometime
after the third postnatal month (50 weeks after conception). This is in keeping with the differences between
the species in the time course of neurodevelopment.
Our findings also agree with those of Reichelt and coworkers [ 2 5 ] , which demonstrated an increase in al
subunit-specific monoclonal antibody binding in human frontal cortex and cerebellum, beginning at gestational week 33 and continuing through 1.75 years postnatally.
Based on the differing interactions between benzodiazepine ligands and the GABAA receptor, two pharmacologically distinct classes of benzodiazepine receptors, designated type I and type 11, have been
identified. Expression studies demonstrate that the defining characteristics of these two GABAA receptor
types depend on the type of a polypeptide present
in the receptor IS, 7, 10, 13, 14f. The current study
demonstrates an increase in type I binding in human
cortex and cerebellum during development that is concurrent with an increase in a1 mRNA levels. This
finding is consistent with the previous studies, which
demonstrated that GABAA receptors that contain a 1
polypeptide display benzodiazepine type I binding.
Further, the increase in type I and decrease in type I1
benzodiazepine binding demonstrated during human
development are similar to the developmental pattern
previously demonstrated in the rat [12).
At birth, the human cerebellum seems to have progressed further in its switch from type I to type I1
benzodiazepine receptors than the rat cerebellum. This
difference correlates with the difference in the levels
of synapse formation in the human and rat cerebellum
at birth. The human cerebellum has a greater percentage of synapses formed before birth than the rat cerebellum does. This comparison between species supports the idea that GABA, receptor subtypes change
with a time course similar to the time course of synapse
formation. In the rat, the appearance of the a1 subunit
692 Annals of Neurology Vol 34
No 5
correlates in time with the formation of synapses. The
nature of the relationship between synapse formation
and the expression of the a1 subunit, however, is not
clear from studies to date. Whether the expression of
the a1 subunit signals for a synapse to be formed or
is instead expressed only after synapses are made could
be answered by further study. Alternatively, it may be
that the similarities in time course of the two processes
are coincidental.
If the functional properties of the GABA, receptor
are largely dictated by the a polypeptide, then developmental changes in a subunit composition, such as demonstrated in this study, may lead to alterations in the
function of central GABAergic systems during human
development. Such a change in GABAergic function
could have significant implications for normal neurodevelopment as well as for pathophysiological states such
a seizures. The GABA, receptor is the site of action of several antiepileptic drugs, including barbiturates and benzodiazepities. Developmental changes in
GABA, receptor subunit composition, distribution, or
abundance may therefore have a significant impact on
the efficacy of these antiepileptic drugs during development. These developmental changes in the GABAA
receptor may have similar implications for the effectiveness in the pediatric population of several new
anticonvulsant agents that are also thought to target
the GABAergic system. In addition, alterations in
GABAergic transmission have been implicated as a
possible etiological factor in epilepsy {29}. It could be
hypothesized that alterations in GABA, receptor activity during development might play a seminal role in
specific developmental patterns seen in certain childhood seizure types, such as infantile spasms and absence and febrile seizures. Additional studies of developmental changes of a1 and other receptor subtypes
are needed to accurately assess the complete significance of alterations in GABA, receptor subunit composition on human brain development and function.
This work was supported by a grant from the National Institute of
Drug Abuse (DA07130-02 to D. B. P.). D r Amy Brooks-Kayal was
a Chtarles A. Dana fellow in neuroscience during the course of these
experiments.
The authors would like to acknowledge the technical advice and
assistance of Robert Williamson, che advice of Dr Eric P. Greenblatr
and Dr Leonard0 Rodriguez Cruz, and rhe assistance of D r Lucy B.
Rorke.
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November 1993
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