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Morphologic and neurochemical studies of embryonic brain development in murine trisomy 16
155
Developmental Brain Research, 15 (1984) 155-166
Elsevier BRD 50081
Morphologic and Neurochemical Studies of Embryonic Brain Development Murine Trisomy 16 HARVEY S. SINGER, MICHAEL TIEMEYER, Departments of Neurology,
Neuroscience,
JOHN C. HEDREEN,
JOHN GEARHART
in
and JOSEPH T. COYLE
Pathology, Pediatrics, Pharmacology and Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, MD (U.S.A.)
(Accepted April lOth, 1984) Key words: murine trisomy 16 -
chromosomal imbalance - brain development-synaptic
neurochemistry - neurotransmitters
Telencephalic and diencephalicbrainstem regions from embryonic trisomy-16 mice (Ts16) between gestational days 15-18 were analyzed for alterations of morphologic and neurochemical parameters and compared to phenotypically normal littermates. Mean trisomic wet weights from both regions were significantly diminished (> 20%) and total protein content was reduced. Ratios of the thickness of the ventricular (germinal) zone to the thickness of the whole cortex were increased, suggesting a delay in neuronal differentiation. Pre- and postsynaptic markers for GABAergic, cholinergic, catecholaminergic and serotonergic transmitter systems were compared. A significant impairment of the trisomic brain catecholaminergic and serotonergic system development was observed, based upon regional reductions in norepinephrine, dopamine and serotonin content. Choline acetyltransferase activity in the diencephalonbrainstem was reduced by 21-26% in contrast to normal levels within the cerebral hemispheres. Presynaptic GABAergic markers were not affected in the Ts16 embryos. It is concluded that although a genetic imbalance involving chromosome 16 in the mouse embryo produces a delay in neurogenesis, it has a more selective effect on the catecholaminergic, serotonergic and cholinergic systems than on GABAergic neurons. INTRODUCTION
Chromosomal imbalance or aneuploidy is a frequent outcome of conception in mammals. The genetic imbalance of autosomal trisomies, usually results in embryonic and fetal death or, in those who survive the perinatal period, mental retardation. Primarily because of the difficulty of obtaining brain tissue, little is known about morphologic and neurochemical consequences of autosomal trisomies within the central nervous system. Recently, selective breeding schemes have been utilized in mice to obtain a variety of autosomal trisomies with high frequency=.%. Among these trisomies, trisomy 16 (Ts16) is of particular interest since it may represent a model system for the study of neurobiologic alterations observed in Down’s syndrome (DS)i6JJJ. Several lines of evidence support the hypothesis that murine trisomy 16 may provide a useful animal model for understanding the pathophysiology of DS.
Correspondence:
Current evidence of homology between the two chromosomal disorders includes synteny of several gene loci, common morphogenic features and a high rate of fetal wastage. Genes for superoxide dismutase-1, phosphoribosyl glycinamide synthetase and the cell surface receptor for interferon have been mapped to the distal segment of both the mouse chromosome 16 and human chromosome 21*,93. The full extent of genetic homology between the murine chromosome 16 and human chromosome 21 remains to be determined. Morphologically, for example, the cardiovascular atrio-ventricular defects characteristic of DS occur frequently in murine Ts1632. In these studies, we have analyzed morphologic and synaptic neurochemical parameters of brain development in prenatal Ts16 mice and their unaffected littermates with the objective of determining possible selective effects related to a trisomic condition. The catecholaminergic, serotonergic, GABAergic, and cholinergic neuronal systems were evaluated since all
H. S. Singer, Pediatric Neurology, Meyer S-109, The Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore,
MD 21205, U.S.A. 0165-3806/84/$03.00 0 1984 Elsevier Science Publishers B.V.
have been implicated in the Down’s syndrome~.i.7.19,‘1.7.i(i.ih.~ll.~l,
neuropathology We
served that the effect of chromosomal
have
of ob_
aneuploidy,
ventricular (germinal) zone to the thickness whole cortex was determined. Measurements obtained
bilaterally
along a line located midway be-
represented by Ts16 in the mouse. is one of generalized retardation of neuronal development. Findings
tween the dorsolateral
from the analysis of neurochemical
drawn perpendicular
cephalic
markers
and diencephalonibrainstem
ever, indicate that certain neuronal severely affected than others. MATERIALS
in telen-
regions,
how-
systems are more
AND METHODS
Litters containing mating
normal
doubly
heterozygous
Ts16 progeny
C57BLi6J for
female
were obtained
by
mice with males
Robertsonian
transloca-
tions, Rb (16.17) 7BnriRb (9.16) YRma. Since we and others*3 have observed that Ts16 fetuses may remain viable only until the late prenatal period, the progeny of such matings were studied on gestational days (DG) 1.5-18 (plug day = DG 0). Pregnant females were killed by cervical dislocation. The conceptuses were removed quickly and placed in phosphate-buffered isotonic saline. pH 7.4. Placental membranes were removed with the aid of a dissecting microscope. The fetuses were then examined for the phenotypic characteristics of Ts16; those with some or all of the following characteristics were selected for study: developmental growth retardation, moderate hypoplasia, anasarca, hygroma of the neck and back, open eyelids, bifid sternum, persistent omphalocoele, and petechiae. No macerated or dead fetuses or deciduomas (moles) were colFor each trisomic fetus selected, a lected. phenotypically normal littermate was collected to serve as its paired control. Each animal was decapitated and the brain was carefully dissected and weighed. Histology Brains from selected trisomic (n = 3) and littermate control (n = 3) animals (DG 15-18) were fixed by immersion in Bouin’s solution for 6-18 h, passed through repeated changes of 70% ethanol until free of yellow color, and then dehydrated, cleared and embedded in paraffin. Tissue blocks were cut in the coronal plane at lo-pm intervals and every 20th section was stained with cresyl violet. As a measure of cortical development, the ratio of the thickness of the
edge of the ganglionic
nence and the dorsalmost
to the pial and ventricular
da1 section which contained
emi-
part of the lateral ventricle
faces. Four levels so analyzed connection
of the were
included
sur-
the most cau-
the septum with its dorsal
to cortex and corpus callosum.
three succeeding
more posterior
sections.
section contained
rostra1 hippocampus.
and the The third
Neurochemical analyses For
the
hemispheres
neurochemical (‘cerebral
analyses, hemispheres’)
telencephalic were
sepa-
rated and the brainstem was transected at the level of the rudimentary cerebellum (‘diencephalon/brainstem’). Occasionally, assays were performed on the entire brain, including cerebral hemispheres, diencephalon, and dissected brainstem (‘whole brain’). The tissues were dried by blotting, then frozen on dry ice and maintained at -70 “C until assay. Measurements of the content of neurotransmitter synthesizing enzymes and endogenous neurotransmitters, as well as receptor binding assays, were carried out on paired samples derived from Ts16 and littermate control animals. Neurotransmitter synthetic enzymes were analyzed after sonication in 10 ~01s. of ice-cold 50 mM Tris-HCl, pH 7.4, containing 0.2% (v/v) aton X-100. Homogenates were centrifuged at 12,000g at 4 “C for 15 min and portions of these supematant fractions were assayed. Activity of choline ace tyitransferase, the enzyme that catalyzes the synthesis of acetylcholine, (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) was measured by the method of Bull and Oderfeld-Nowake. Glutamate decarboxylase, the enzyme that synthesizes the inhibitory amino acid GABA, (GAD; L-glutamate l-carboxylase, EC 4.1.1.15) was assayed by a modification of the method of Wilson et al.42 with l-DL-[r4C]glutamic acid as the substrate. Tyrosine hydroxylase, the initial and rate-limiting enzyme in the synthesis of catecholamines, (TH; tyrosine 3-hydroxylase. EC 1.14.16.2) activity was determined by the method of Coylet” except that D,t_-methyl-5,6,7,8_tetrahydropterine was used as the cofactor. Tissue protein was measured by the method of Lowry et al.31 with bo-
157 tant fluid for amino acid determination
was treated
vine serum albumin as standard. The specific binding of [3H]quinuclidinyl
benzilate
with o-phthalaldehyde
(0.3 nM QNB; 29 Ci/mmol;
Nuclear)
od of Hill et al.26 before being separated
New England
to muscarinic cholinergic receptors, was measured by a modification of the method of Yamamura and Snydera.
Non-specific
binding is determined
by incu-
formance Associates;
liquid
chromatography
Waltham,
tos fluorescence
and ethanethiol
by the methby high per-
(HPLC;
MA) and measured
detector.
Biogenic
perchloric
acid supernatant
fraction
by HPLC
and quantified
Waters by a Kra-
amines
in the
were separated
bating membranes with [3H]QNB in the presence of the muscarinic agonist oxotremorine (0.1 mM). The gamma-amino butyric acid (GABA) receptor was
tion according to the method of Zaczek and Coyle45.
studied (6 nM;
Statisticalanalyses
by analyzing the binding of [3H]muscimol 10.3 Ci/mmol; New England Nuclear) to
membrane
preparationsz.
[3H]L-glutamic
The
specific
acid was measured
binding
by the method
Slevin et al.39 which was a modification
of of
of the method
Significance
by electrochemical
of differences
assays in the Ts16 embryos mate controls
were assessed
detec-
for the neurochemical and their matched by an analysis
litterof vari-
of Foster and Robertsls. Serotonin receptors were measured by utilizing the binding of [3H]D-lysergic acid diethylamide (4.25 nM; 58.7 Ci/mmol; New England Nuclear) according to the method of Bennett
ance. Synthesizing enzyme activity, neurotransmitter content and receptor-ligand binding were analyzed for a gene dose effect (Ts16 vs control) and a possible interaction of aneuploidy with rate of accu-
and SnyderJ. Beta adrenergic receptors were assayed by incubating crude membranes with [3H]dihydroalprenolol(18 nM; 49.4 Ci/mmol; New England Nuclear) by the method of Alexander et al.1. Biogenic amines and amino acids were measured in frozen tissues obtained from cerebral hemispheres and diencephalon/brainstem regions. For each determination, 4 paired samples (Ts16 and control litter-
mulation during maturation. Statistical comparison of values on individual gestational days was assessed by a two-sample and paired t-test.
mate) were collected for DG 17 and 18. Samples were assayed in duplicate. Tissues were homogenized in either 20 ~01s. of methanol (amino acid determination) or 10 ~01s. of ice-cold 0.1 N perchloric acid containing 0.5 ,uM dihydroxybenzylamine as an internal standard (monoamines). Homogenates were centrifuged at 10,000 g for 15 min at 5 “C. Superna-
RESULTS Morphologic observations Both the cerebral hemispheres and the diencephalon/brainstem regions of Ts16 mice were grossly smaller than those of littermate controls. An analysis of variance of brain weight showed a significant gene dose effect in both the cerebral hemispheres (P < 0.001) and diencephalon/brainstem (P < 0.001) regions. A gene dose-maturational rate of accumulation interaction (P < 0.02) was present in the cere-
TABLE I Mean wet weights of dissected embryonic and adult structures
Results are given as means f S.E.M. of measurements on several animals in each group (number in parentheses). Analysis of variance showed a significant gene dose effect in both the cerebral hemisphere (P < 0.001) and diencephalon/brainstem (P < 0.001). A significant interaction effect between gene dose and rate of weight change was noted in the hemisphere (P < 0.02). Two-sample t-test analyses were also significant. Days of gestation
Cerebral hemispheres Normal 21.6 + 2
15 (5)
16 (7) 17 (15) 18 (15) Adult (8) * p
25.4 + 32.4 f 46.8 + 190.9 + P
1.2 1.8 0.9 20.6
P
Diencephalonlbrainsrm Ts16
Normal
Ts16
17.2 f 1.5’
25.4 f. 1.3 29.4 + 0.8 36.4 + 0.8 43.3 f 1.0 195.2 ?I 33.8
17.3 23.4 26.6 31.2
19.6 f l.l** 24.4 f 0.9*** 34.3 + 0.9***
f f f +
1.3** 1.2** 0.5*** 2.2***
158 bral hemispheres. In addition, the mean wet weights of the trisomic cerebral hemispheres and diencephalon/brainstem were significantly reduced (P < 0.05 to < 0.001) on DG 15 through 18 (Table I). Total content of protein was also significantly reduced in the Tsl6 brain (Fig. 1). Analysis of variance showed a gene dose effect for protein content in cerebral hemispheres (P < 0.002) and diencephalon/brainstem (P < 0.001). There was no significant gene dose-accumulation rate interaction. Examination of Nissl-stained sections of the trisomic telencephalon disclosed a delay in neuronal development when compared to paired controls (Fig. 2). In the control embryo as the cerebral cortex matured, cortical thickness increased, concomitant with a reduction in the size of the germinal zone. The average ratio of the thickness of the germinal zone to that of the whole cortex in the normal embryo was 0.220 at D G 15, 0.149 at D G 17 and 0.141 at D G 18. In contrast, comparable ratios in the trisomic cerebral hemisphere, were 0.309, 0.211 and 0.206 on D G 15, 17 and 18, respectively. No qualitative differ-
ences in cell groups or their architectonic arrangement were observed in any of the brain sections, from the rostral end of the forebrain to the level of the locus coeruleus and cerebellum. The cholinergic neurons in the basal forebrain were not cytologically distinct from other cells in either the trisomic or littermate control animals at these ages.
Synaptic neurochemistry The development of afferent and intrinsic cortical neuronal systems was assayed by monitoring pre- and postsynaptic neurochemical markers in the cerebral hemispheres and diencephalon/brainstem. Catecholaminergic markers: tyrosine hydroxylase (TH) activity, a presynaptic marker for both noradrenergic and dopaminergic systems, was not detected on D G 15 in either the cerebral hemispheres (Table II) or diencephalon/brainstem (Table III). On D G 18 the specific activity for TH in trisomic and control hemispheres represented 34% of their adult activity. TH activity tended to be reduced in the brain regions of the trisomic embryo although a sta-
Cerebral Hemispheres
Dlencephalon/Brainstem
.o 3
,//'t
3
!.
tS e st
o
Q. v
o~ E
2
sss S
2i
fs ff"
C
o 0
$ C
o
n
s j s s s ' ~
1
j SS°
1
" ~
I
15
0 .... 0 Normal e----e Ts16 I 16
I 17
Gestational Day
I 18
t
,
~
Normal
~ Ts16
I 15
I 16
....
I 17
I 18
Gestational Day
Fig. 1. Total protein content in embryonic Ts16 and control mouse cerebral hemispheres and diencephalon/brainstem. Values shown are the means ± S.E.M. (bars) for 4-5 animals in each group. Analysis of variance showed a significantgene dose effect in both the cerebral hemispheres (P < 0.002) and diencephalon/brainstem (P < 0.00l). Two-sample t-test analyses were also significant (P < o.o5)*.
159
P
p
¢
V
v Fig. 2. Nissl-stained sections of cortex from Ts16 (A) and control (B) embryosat 17 days of gestation. Sectionswere obtained from an area midwaybetween the dorsolateral edge of the ganglioniceminence and the dorsalmost part of the lateral ventricle; p, pial; v, ventricular surface. Note, the reduced cortical thickness and larger germinal zone in the Tsl6 section. Magnificationin both x 260.
tistically significant difference was not achieved. Norepinephrine (NE) concentrations were higher in the diencephalon/brainstem region whereas dopamine (DA) was higher in the telencephalic region. Analysis of variance showed a significant gene dose effect for NE (P < 0.05) and dopamine (P < 0.05) in the hemispheres and for NE (P < 0.05) in the diencephalon/brainstem. There was no statistical interaction between gene dose and maturational rate of accumulation. In 13 out of the 14 experimental catecholaminergic points the mean values in the Tsl6 were lower than that measured in littermate controls
(P < 0.02 Wilcoxon rank order). Specific [3H]dihydroalprenoloi binding was not detected in the fetal mouse brains. Serotonergic markers. 5-Hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured on gestational days 17 and 18 (Table IV). In the cerebral hemispheres, neither compound was significantly reduced. However, in the diencephalon/brainstem region analysis of variance showed a gene dose effect for 5-HT (P < 0.01); no interaction with rate of accumulation was observed. In addition, values for 5-HT and 5-HIAA in
160
this region were significantly reduced on DG 17 (two sample t-test, P < 0.05). Specific binding for [3H]LSD was not detected in embryonic brains. Putative amino acid neurotransmitter markers. Presynaptic markers assayed for GABAergic neurons included glutamate decarboxylase (GAD) activity and the concentration of G A B A . GAD activity was detected on all gestational days studied and reached
19-20% of adult activity in the hemispheres on DG 18 (Table V). No significant differences were found between Tsl6 and littermate control embryos. On DG 18, levels of G A B A (nmol/mg prot) in the trisomic cerebral hemispheres (n = 4) and diencephalon/brainstem (n = 4) did not differ significantly from those of littermate controls; Tsl6, cerebral hemispheres 14.6 + 0.8 (mean + S.E.M.), diencepha-
TABLE II
Catecholaminergic markers in embryonic and adult cerebral hemispheres Tyrosine hydroxylase activity (TH), norepinephrine (NE) and dopamine (DA) were measured as described under Methods, with the following units: TH, pmol DOPA/mg protein/h; NE and DA, pmol/mg protein. Values are expressed as mean _+ S.E.M. for 4 animals in each group. Analysis of variance showed a significant gene dose effect for norepinephrine (P < 0.05) and dopamine (P < 0.05).
Age 15 Days gestation Normal Tsl6 16 Days gestation Normal Tsl6 17 Days gestation Normal Tsl6 18 Days gestation Normal Tsl6 Adult
Tyrosine hydroxylase activity
Norepinephrine
Dopamine
nd nd 178.9 ± 43.9 145.7 ± 18.9 269.2 ± 32.8 186.7 ± 37.6
6.6±0.8 5.3±0.5
10.2±0.9 7.7±1.6
274.8 _+ 59.5 240.9 ± 65.5 758.3 + 53.4
9.0±0.7 6.9±1.2 34.0±6.2
13.5±0.5 11.5±1.0 59.0±14.9
nd, not detected;-, not assayed.
TABLE III
Catecholaminergic markers in embryonic and adult diencephalon/brainstem Tyrosine hydroxylase acitivity (TH), norepinephrine (NE), and dopamine (DA) were measured as described under Methods, with the following units: TH, pmol DOPA/mg protein/h (embryonic) and nmol/mg protein/h (adult); NE and DA, pmol/mg protein. Values are expressed as mean ± S.E.M. for 4 animals in each group. Analysis of variance showed a significant gene dose effect for NE (e < 0.05).
Age 15 Days gestation Normal Ts16 16 Days gestation Normal Tsl6 17 Days gestation Normal Tsl6 18 Days gestation Normal Tsl6 Adult
Tyrosine hydroxylase activity nd nd
Norepinephrine
Dopamine
-
353.8 ± 41.0 342.8 ± 49.6
-
382.9 + 38.5 397.0 ± 68.9
15.4 + 1.0 13.3 + 1.2
6.1±1.3 5.8±1.0
471.8 + 121.1 394.5 ___82.3 20.5 ± 1.1
12.0 ± 1.5 9.2 ± 1.1 47.0 + 18.3
9.4±0.6 7.8+0.9 96.2±20.7
nd, not detected; -, not assayed.
161 lon/brainstem 15.8 + 2.3; control, cerebral hemi-
found in either embryonic brain region through D G
spheres 15.7 + 1.4, diencephalon/brainstem 17.3 + 1.7. Levels of other putative amino acid neurotransmitters were as follows (nmol/mg prot): T s l 6 cere-
18. Cholinergic markers. The cholinergic n e u r o n a l system was assessed by quantitating choline acetyl-
bral hemispheres aspartate 29.4 + 3.6, glutamate
transferase (CHAT) activity and [3H]quinuclidinyl
74.8 + 7.4; T s l 6 diencephalon/brainstem aspartate 33.1 + 6.7, glutamate 84.6 + 7.5; control cerebral
benzilate (QNB) binding. In the cerebral hemi-
hemispheres aspartate 39.5 ___ 4.5, glutamate 87.5 _+ 8.5; control diencephalon/brainstem aspartate 33.7
significant differences were found for C h A T activity
+ 3.4 and glutamate 78.4 + 4.9. Detectable specific
D G 18 C h A T activity was 3% and QNB specific
binding for [3H]muscimol or [3H]glutamate was not
binding was 12-14% of adult levels. In contrast, in
spheres of the fetal trisomic and normal mouse, no (Fig. 3) or QNB receptor binding (Table VI). O n
TABLE IV
Serotonin and 5-hydroxyindoleacetic acid in embryonic and adult cerebral hemispheres and diencephalon/brainstem regions Compounds were measured as described under Methods and are given as pmol/mg protein. Values are expressed as mean ± S.E.M. for 4 animals in each group. Analysis of variance showed a significant gene dose effect for 5-HT in the diencephalon/brainstem region (P < 0.01). Values for 5-HT and 5-HIAA in the diencephalon/brainstem on DG 17 were significantly reduced on two-sample t-test analysis.
Region and compound
Gestational day
Adult
17 ~mbralhemispher~ Serotonin (5-HT) Normal Tsl6 5-Hydroxyindoleaceticacid(5-HIAA) Normal Tsl6 Diencephalon/brainstem Serotonin (5-HT) Normal Tsl65-Hydroxyindoleacetic acid (5-HIAA) Normal Tsl6 * P<
0 . 0 5 , ** P <
18
3.8±0.1 3.0±0.5
9.6±0.4 12.3±1.7
67.8±18.9
2.5±0.1 2.0±0.2
8.5±1.2 7.0±1.2
54.5±19.2
11.7 + 1.1 6.8 + 0.5**
19.7 ± 1.3 17.4 + 1.6
97.9 + 13.4
6.7 ± 0.9 4.4 + 0.2*
26.4 ± 5.1 19.0 + 3.5
87.0 ± 8.4
0.01.
TABLE V
Glutamate decarboxylase activity in embryonic and adult mouse brain Enzyme activity, nmol GABA/mg protein/h, was measured as described in Methods. Values are expressed as mean + S.E.M. for 4 animals in each group. No significant group or interaction effect was shown on analysis of variance.
Region
Enzyme activity on gestational day 15
Adult
16
17
18
Cerebral hemispheres Normal 2.9 ± 0.5 Tsl6 2.1 + 0.2
2.5±0.5 2.7±0.6
3.3±0.9 3.1±0.7
4.1±1.6 4.4±1.6
21.8±0.6
Diencephalon/brainstem Normal 4.8 + 0.6 Tsl6 3.6 + 0.9
6.7 + 1.5 6.3 + 1.5
7.5 + 1.7 8,1 + 1.9
8.7 + 2.4 7.7 + 2.2
31.3 + 3.0
162 Cerebral Hemispheres
t-
Diencephalon/Brainstem
sJ/~
5
/t/t
C
P
cL
4
4
O~
/// "
3
3
~.~
>
<
~4,~
2
2
(J v,. tJ O.
u) p-
0 . . . . 0 Normal
1
1
0----0 Normal
H
Ts 16
e-
Ts16
C3 l
I
I
I
I
I
I
I
15
16
17
18
15
16
17
18
Gestational Day
Gestational Day
Fig. 3. Choline acetyltransferase (CHAT) activity in embryonic Ts16 and control mouse cerebral hemispheres and diencephalon/brainstem. Values shown are the means (nmol ACh/mg protein/h) + S.E.M. (bars) for 4 animals in each group. Analysis of variance showed a significant gene dose effect (P < 0.001) in the diencephalon/brainstem region. Paired t-test analysis was also significant (*) in this region on DG 16-18 (P < 0.01, P < 0.05 and P < 0.025 respectively).
the d i e n c e p h a l o n / b r a i n s t e m an analysis of v a r i a n c e of C h A T activity d e m o n s t r a t e d a significant difference ( P < 0.001) b e t w e e n the trisomic a n d control fetus; there was n o significant i n t e r a c t i o n with gestational days (Fig. 3). In a d d i t i o n , on D G 16, 17 and 18 the specific activities of C h A T were significantly r e d u c e d ( p a i r e d t-test) in the trisomic m o u s e c o m p a r e d to l i t t e r m a t e controls ( P < 0.01, < 0.05,
and < 0.025 respectively). Assays were p e r f o r m e d on the s a m e tissue samples used to m e a s u r e d G A D a n d T H activities, which were u n c h a n g e d from littermate controls. T h e specific activity of c o n t r o l C h A T activity in the d i e n c e p h a l o n / b r a i n s t e m o n D G 18 repr e s e n t e d only 3 . 6 % of that f o u n d in adult mice. T h e a p p a r e n t density of [3H]QNB b i n d i n g sites in the T s l 6 d i e n c e p h a l o n / b r a i n s t e m did n o t differ signifi-
TABLE VI Quinuclidinyl benzilate receptor-ligand binding in embryonic and adult mouse brain Quinuclidinylbenzilate receptor-ligand binding, fmol QNB bound/mg protein, was measured as described in Methods. Values are expressed as mean + S.E.M. for 4 animals in each group. Analysis of variance showed no significant gene dose or interaction effect. Region
Cerebral hemispheres Normal Ts16 Diencephalon/brainstem Normal Tsl6
Ligand binding on gestational day
Adult
16
17
18
164 + 42 106 + 13
205 + 30 202 + 24
235 + 43 266 + 31
1897 + 101
258 + 75 189 + 27
275 + 76 285 _+ 34
391 + 53 365 + 17
978 + 189
163 cantly from littermate controls (Table VI). The density of [3H]QNB specific binding in D G 18 controls was 40% of the level of adult mice. Thus, the significant reduction in ChAT activity observed in the trisomic mouse did not appear to affect the development of muscarinic cholinergic receptors. DISCUSSION This study was designated to determine possible selective effects of trisomy 16 on neuronal characteristics in the mouse. To date, general developmental retardation and organ hypoplasia are the most obvious abnormal features observed in all murine trisomic conditions 23. The statistically reduced brain weight and brain protein content between DG 15 and 18 in the Ts16 mouse embryo is consistent with previously reported reductions in the body weight of embryos and their placentas (Gearhart, unpublished observations) 32. In our morphometric analysis, the reduced brain weight appears to be primarily due to a delay in neuronal differentiation rather than neuroblast formation. Furthermore, the ratio of the thickness of the ventricular germinal zone as compared to the fetal cortex is elevated in the Tsl6 embryos such that the ratio at DG 18 in Tsl6 is comparable to that observed in unaffected controls at DG 17. Thus, maturational increases in brain weight in the Tsl6 embryo are delayed by approximately one day in Tsl6 embl-yos as compared to their littermate controls. In order to determine possible selective effects of the trisomic condition on identified populations of neurons, we have compared in Tsl6 mice embryos and their unaffected littermates several pre- and postsynaptic markers for neurotransmitter-defined neuronal systems implicated in the neuropathology of DS. Thus, we examined presynaptic markers for GABAergic neurons, which are local circuit stellate cells in the cerebral cortex, because of the reported reduction of cortical small cells in DS 15,21. Furthermore, studies on peripheral and central serotonin levelsS,40, platelet serotonin uptake 5, catecholamine metabolic enzymes4.7.19.25 and CSF biogenic amine metabolites 30 suggest that dysfunction of the central serotonergic/catecholaminergic neuronal systems may be a feature of DS. Finally, studies have demonstrated in middle-aged DS patients the presence of neuropathologic stigmata of Alzheimer's dementia,
reduction in cortical activity of ChAT and loss of cholinergic neuronal perikarya in the nucleus basalis of Meynert35,36,~. Unfortunately, since Ts16 mice generally die by DG 17-18, the present studies were limited to the assessment of the embryonic neurogenesis of these systems. Nevertheless, [3H]thymidine autoradiographic, immunocytochemical and histofluorescence studies in rodents indicate that the bulk of the catecholaminergic, serotonergic and cholinergic nuclei in the brainstem core have probably been formed by DG 17-1811,12,14,22,33. Neurogenesis of the cortical GABAergic neurons is likely to continue beyond this date13,37,41. In our dissection, dopamine is the predominant catecholamine in the telencephalic sample because it contains the developing striatum. TH activity was first detectable at DG 16 in both the hemispheres and the diencephalon/brainstem and exhibited a modest increase in specific activity in both controls and Ts16 embryos between DG 16 and 18. Furthermore, endogenous norepinephrine and dopamine were also detectable in both the hemispheres and diencephalon/brainstem at DG 17 and 18. Significant differences between the Ts16 and littermate controls were observed for norepinephrine in the two regions and for dopamine in the cerebral hemispheres. In addition, in 13 out of the 14 experimental determinations of catecholaminergic parameters the mean value in the Ts16 was less than that measured in the littermate controls, producing a significant Wilcoxon rank order. These data suggest an impairment in the development of the catecholaminergic systems in Ts16. The concentration of serotonin, a presynaptic marker for serotonergic neurons, was significantly reduced in the Ts16 diencephalon/brainstem at DG 17.5-Hydroxyindoleacetic acid (5-HIAA), the serotonin metabolite, was also significantly reduced in the diencephalon/brainstem on DG 17 (two sample t-test), although the analysis of variance did not achieve significance. Thus, reductions in the concentrations of serotonin and 5-HIAA within the embryonic brain region containing the median raphe, suggest an impairment in the ontogeny of this neuronal system. Glutamate decarboxylase (GAD), appears to be a highly specific presynaptic marker for GABAergic neurons 13. GABAergic neurons have been shown by immunocytochemical studies to be stellate type II in-
164 terneurons in the cerebral cortex, interneurons scattered through the brainstem core and Golgi type II neurons within the striatal efferent system. Neurogenesis of these neurons appears to occur over a longer period of time than noted for non-GABAergic neurons in the cortex and brainstem core. In the Tsl6 mouse, no significant differences in the specific activity of G A D were observed either in the hemispheres or the diencephalon/brainstem between DG 15 and 18. Furthermore, unlike the presynaptic markers for the catecholaminergic and serotonergic systems, no consistent pattern of reduction in G A D activity was observed in the Tsl6 versus the littermate controls. Similarly, in developmental studies of the GABAergic system in which neurogenesis was temporarily arrested by treatment with the alkylating agent methylazoxymethanol acetate, no significant depressions in the concentrations of presynaptic markers for GABAergic neurons were detected 27. However, in both the chemically lesioned and this chromosomally imbalanced model, the total content of the GABAergic markers is reduced within a hypoplastic cortex. Choline acetyltransferase (CHAT), is the most specific presynaptic marker for the cholinergic neuronal system 38. Whereas the specific activity of ChAT in the cerebral hemispheres did not differ in Tsl6 and littermate controls between D G 15 and 18, significant reductions in its specific activity were observed in the diencephalon/brainstem region. At this early stage of development, however, the specific activity of ChAT represents only 3 - 4 % of that occurring in the adult within these same regions. Approximately 60-80% of ChAT activity in the cerebral cortex is localized to afferents derived from the magnocellular complex which includes the nucleus basalis of Meynert located adjacent to the diencephalon. In addition, a small component of cortical cholinergic innervation appears to be derived from neurons intrinsic to the region 28,29. The reason for the differential reduction in specific activity of ChAT in the Tsl6 diencephalon/brainstem as compared to the normal concentration in the hemispheres remains unclear. One possibility is that the reduced activity reflects impaired development of the cholinergic nucleus basalis neurons, which would be included in the diencephalon/brainstem sample. Nevertheless, assessment of muscarinic receptor density by ligand binding did not disclose significant differences between
Tsl6 and littermate controls within the two major brain regions on the three gestational days examined. Thus, at these early stages, impairments in presynaptic cholinergic development, especially in the diencephalon/brainstem, are not mirrored by alterations in its receptor site. Our studies point to a delay in the neurogenesis and growth of the brain in the Tsl6 mouse embryo as compared to littermate controls. Neurochemical assessment of presynaptic markers for defined neuronal systems in the hemispheres and diencephalon/brainstem indicates that certain neuronal systems may be more severely affected than others. Thus, whereas the specific activity of the presynaptic marker for the GABAergic neurons in Tsl6 and controls did not differ at any gestational date, significant reductions in the presynaptic markers for catecholaminergic and serotonergic neurons were observed. In addition, the specific activity of ChAT was significantly reduced in the diencephalon/brainstem, suggesting that cholinergic systems in this region are more specifically affected by the genetic defect. Our neurochemical data also provide additional support for the hypothesis of delayed, but not aberrant neuronal development in trisomy 16. For example, the analysis of variance, even in regions with significant gene-dose effects, shows no evidence for a meaningful interaction between aneuploidy and the subsequent rate of accumulation of enzyme activity or neurotransmitter content. In the Ts16 embryo, when the reductions in weight of the hemispheres and the diencephalon/brainstem are taken into account, it is clear that the total complement of synaptic markers for the neuronal systems is significantly reduced. Thus, expression of data only in terms of concentration of the markers minimizes the more global deficit in neurogenesis and synaptic neurochemical differentiation in the fetal trisomic brain. Validity of this alternative form of presentation has been adequately demonstrated in previous studies in which telencephalic and cerebellar neuronal groups have been selectively eliminated by drugs that arrest neurogenesis 27,39. The mechanism by which an aneuploid condition results in abnormal neurogenesis with more selective effects on specifically defined neuronal systems remains unknown, although possible explanations include the effects of gene dose or secondary biochem-
165 ical abnormalities17, 23. R e s o l u t i o n of this m o r e fund-
tor D e v e l o p m e n t A w a r d 5 K07 NS 00506-04 f r o m the
a m e n t a l q u e s t i o n m a y be possible t h r o u g h the study
N I N C D S ( H . S . S . ) , by U . S . Public H e a l t h S e r v i c e
of T s l 6 c h i m e r a s , which survive to a d u l t h o o d .
Grant RSDA
ACKNOWLEDGEMENTS
n e d y , Jr. F o u n d a t i o n . J . G . is a K e n n e d y F o u n d a t i o n Scholar.
II K03-00125, a S u r d n a F o u n d a t i o n
G r a n t ( J . T . C . ) and a g r a n t f r o m the J o s e p h P. K e n -
This w o r k was s u p p o r t e d by a T e a c h e r - I n v e s t i g a -
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Developmental Brain Research, 15 (1984) 155-166
Elsevier BRD 50081
Morphologic and Neurochemical Studies of Embryonic Brain Development Murine Trisomy 16 HARVEY S. SINGER, MICHAEL TIEMEYER, Departments of Neurology,
Neuroscience,
JOHN C. HEDREEN,
JOHN GEARHART
in
and JOSEPH T. COYLE
Pathology, Pediatrics, Pharmacology and Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, MD (U.S.A.)
(Accepted April lOth, 1984) Key words: murine trisomy 16 -
chromosomal imbalance - brain development-synaptic
neurochemistry - neurotransmitters
Telencephalic and diencephalicbrainstem regions from embryonic trisomy-16 mice (Ts16) between gestational days 15-18 were analyzed for alterations of morphologic and neurochemical parameters and compared to phenotypically normal littermates. Mean trisomic wet weights from both regions were significantly diminished (> 20%) and total protein content was reduced. Ratios of the thickness of the ventricular (germinal) zone to the thickness of the whole cortex were increased, suggesting a delay in neuronal differentiation. Pre- and postsynaptic markers for GABAergic, cholinergic, catecholaminergic and serotonergic transmitter systems were compared. A significant impairment of the trisomic brain catecholaminergic and serotonergic system development was observed, based upon regional reductions in norepinephrine, dopamine and serotonin content. Choline acetyltransferase activity in the diencephalonbrainstem was reduced by 21-26% in contrast to normal levels within the cerebral hemispheres. Presynaptic GABAergic markers were not affected in the Ts16 embryos. It is concluded that although a genetic imbalance involving chromosome 16 in the mouse embryo produces a delay in neurogenesis, it has a more selective effect on the catecholaminergic, serotonergic and cholinergic systems than on GABAergic neurons. INTRODUCTION
Chromosomal imbalance or aneuploidy is a frequent outcome of conception in mammals. The genetic imbalance of autosomal trisomies, usually results in embryonic and fetal death or, in those who survive the perinatal period, mental retardation. Primarily because of the difficulty of obtaining brain tissue, little is known about morphologic and neurochemical consequences of autosomal trisomies within the central nervous system. Recently, selective breeding schemes have been utilized in mice to obtain a variety of autosomal trisomies with high frequency=.%. Among these trisomies, trisomy 16 (Ts16) is of particular interest since it may represent a model system for the study of neurobiologic alterations observed in Down’s syndrome (DS)i6JJJ. Several lines of evidence support the hypothesis that murine trisomy 16 may provide a useful animal model for understanding the pathophysiology of DS.
Correspondence:
Current evidence of homology between the two chromosomal disorders includes synteny of several gene loci, common morphogenic features and a high rate of fetal wastage. Genes for superoxide dismutase-1, phosphoribosyl glycinamide synthetase and the cell surface receptor for interferon have been mapped to the distal segment of both the mouse chromosome 16 and human chromosome 21*,93. The full extent of genetic homology between the murine chromosome 16 and human chromosome 21 remains to be determined. Morphologically, for example, the cardiovascular atrio-ventricular defects characteristic of DS occur frequently in murine Ts1632. In these studies, we have analyzed morphologic and synaptic neurochemical parameters of brain development in prenatal Ts16 mice and their unaffected littermates with the objective of determining possible selective effects related to a trisomic condition. The catecholaminergic, serotonergic, GABAergic, and cholinergic neuronal systems were evaluated since all
H. S. Singer, Pediatric Neurology, Meyer S-109, The Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore,
MD 21205, U.S.A. 0165-3806/84/$03.00 0 1984 Elsevier Science Publishers B.V.
have been implicated in the Down’s syndrome~.i.7.19,‘1.7.i(i.ih.~ll.~l,
neuropathology We
served that the effect of chromosomal
have
of ob_
aneuploidy,
ventricular (germinal) zone to the thickness whole cortex was determined. Measurements obtained
bilaterally
along a line located midway be-
represented by Ts16 in the mouse. is one of generalized retardation of neuronal development. Findings
tween the dorsolateral
from the analysis of neurochemical
drawn perpendicular
cephalic
markers
and diencephalonibrainstem
ever, indicate that certain neuronal severely affected than others. MATERIALS
in telen-
regions,
how-
systems are more
AND METHODS
Litters containing mating
normal
doubly
heterozygous
Ts16 progeny
C57BLi6J for
female
were obtained
by
mice with males
Robertsonian
transloca-
tions, Rb (16.17) 7BnriRb (9.16) YRma. Since we and others*3 have observed that Ts16 fetuses may remain viable only until the late prenatal period, the progeny of such matings were studied on gestational days (DG) 1.5-18 (plug day = DG 0). Pregnant females were killed by cervical dislocation. The conceptuses were removed quickly and placed in phosphate-buffered isotonic saline. pH 7.4. Placental membranes were removed with the aid of a dissecting microscope. The fetuses were then examined for the phenotypic characteristics of Ts16; those with some or all of the following characteristics were selected for study: developmental growth retardation, moderate hypoplasia, anasarca, hygroma of the neck and back, open eyelids, bifid sternum, persistent omphalocoele, and petechiae. No macerated or dead fetuses or deciduomas (moles) were colFor each trisomic fetus selected, a lected. phenotypically normal littermate was collected to serve as its paired control. Each animal was decapitated and the brain was carefully dissected and weighed. Histology Brains from selected trisomic (n = 3) and littermate control (n = 3) animals (DG 15-18) were fixed by immersion in Bouin’s solution for 6-18 h, passed through repeated changes of 70% ethanol until free of yellow color, and then dehydrated, cleared and embedded in paraffin. Tissue blocks were cut in the coronal plane at lo-pm intervals and every 20th section was stained with cresyl violet. As a measure of cortical development, the ratio of the thickness of the
edge of the ganglionic
nence and the dorsalmost
to the pial and ventricular
da1 section which contained
emi-
part of the lateral ventricle
faces. Four levels so analyzed connection
of the were
included
sur-
the most cau-
the septum with its dorsal
to cortex and corpus callosum.
three succeeding
more posterior
sections.
section contained
rostra1 hippocampus.
and the The third
Neurochemical analyses For
the
hemispheres
neurochemical (‘cerebral
analyses, hemispheres’)
telencephalic were
sepa-
rated and the brainstem was transected at the level of the rudimentary cerebellum (‘diencephalon/brainstem’). Occasionally, assays were performed on the entire brain, including cerebral hemispheres, diencephalon, and dissected brainstem (‘whole brain’). The tissues were dried by blotting, then frozen on dry ice and maintained at -70 “C until assay. Measurements of the content of neurotransmitter synthesizing enzymes and endogenous neurotransmitters, as well as receptor binding assays, were carried out on paired samples derived from Ts16 and littermate control animals. Neurotransmitter synthetic enzymes were analyzed after sonication in 10 ~01s. of ice-cold 50 mM Tris-HCl, pH 7.4, containing 0.2% (v/v) aton X-100. Homogenates were centrifuged at 12,000g at 4 “C for 15 min and portions of these supematant fractions were assayed. Activity of choline ace tyitransferase, the enzyme that catalyzes the synthesis of acetylcholine, (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) was measured by the method of Bull and Oderfeld-Nowake. Glutamate decarboxylase, the enzyme that synthesizes the inhibitory amino acid GABA, (GAD; L-glutamate l-carboxylase, EC 4.1.1.15) was assayed by a modification of the method of Wilson et al.42 with l-DL-[r4C]glutamic acid as the substrate. Tyrosine hydroxylase, the initial and rate-limiting enzyme in the synthesis of catecholamines, (TH; tyrosine 3-hydroxylase. EC 1.14.16.2) activity was determined by the method of Coylet” except that D,t_-methyl-5,6,7,8_tetrahydropterine was used as the cofactor. Tissue protein was measured by the method of Lowry et al.31 with bo-
157 tant fluid for amino acid determination
was treated
vine serum albumin as standard. The specific binding of [3H]quinuclidinyl
benzilate
with o-phthalaldehyde
(0.3 nM QNB; 29 Ci/mmol;
Nuclear)
od of Hill et al.26 before being separated
New England
to muscarinic cholinergic receptors, was measured by a modification of the method of Yamamura and Snydera.
Non-specific
binding is determined
by incu-
formance Associates;
liquid
chromatography
Waltham,
tos fluorescence
and ethanethiol
by the methby high per-
(HPLC;
MA) and measured
detector.
Biogenic
perchloric
acid supernatant
fraction
by HPLC
and quantified
Waters by a Kra-
amines
in the
were separated
bating membranes with [3H]QNB in the presence of the muscarinic agonist oxotremorine (0.1 mM). The gamma-amino butyric acid (GABA) receptor was
tion according to the method of Zaczek and Coyle45.
studied (6 nM;
Statisticalanalyses
by analyzing the binding of [3H]muscimol 10.3 Ci/mmol; New England Nuclear) to
membrane
preparationsz.
[3H]L-glutamic
The
specific
acid was measured
binding
by the method
Slevin et al.39 which was a modification
of of
of the method
Significance
by electrochemical
of differences
assays in the Ts16 embryos mate controls
were assessed
detec-
for the neurochemical and their matched by an analysis
litterof vari-
of Foster and Robertsls. Serotonin receptors were measured by utilizing the binding of [3H]D-lysergic acid diethylamide (4.25 nM; 58.7 Ci/mmol; New England Nuclear) according to the method of Bennett
ance. Synthesizing enzyme activity, neurotransmitter content and receptor-ligand binding were analyzed for a gene dose effect (Ts16 vs control) and a possible interaction of aneuploidy with rate of accu-
and SnyderJ. Beta adrenergic receptors were assayed by incubating crude membranes with [3H]dihydroalprenolol(18 nM; 49.4 Ci/mmol; New England Nuclear) by the method of Alexander et al.1. Biogenic amines and amino acids were measured in frozen tissues obtained from cerebral hemispheres and diencephalon/brainstem regions. For each determination, 4 paired samples (Ts16 and control litter-
mulation during maturation. Statistical comparison of values on individual gestational days was assessed by a two-sample and paired t-test.
mate) were collected for DG 17 and 18. Samples were assayed in duplicate. Tissues were homogenized in either 20 ~01s. of methanol (amino acid determination) or 10 ~01s. of ice-cold 0.1 N perchloric acid containing 0.5 ,uM dihydroxybenzylamine as an internal standard (monoamines). Homogenates were centrifuged at 10,000 g for 15 min at 5 “C. Superna-
RESULTS Morphologic observations Both the cerebral hemispheres and the diencephalon/brainstem regions of Ts16 mice were grossly smaller than those of littermate controls. An analysis of variance of brain weight showed a significant gene dose effect in both the cerebral hemispheres (P < 0.001) and diencephalon/brainstem (P < 0.001) regions. A gene dose-maturational rate of accumulation interaction (P < 0.02) was present in the cere-
TABLE I Mean wet weights of dissected embryonic and adult structures
Results are given as means f S.E.M. of measurements on several animals in each group (number in parentheses). Analysis of variance showed a significant gene dose effect in both the cerebral hemisphere (P < 0.001) and diencephalon/brainstem (P < 0.001). A significant interaction effect between gene dose and rate of weight change was noted in the hemisphere (P < 0.02). Two-sample t-test analyses were also significant. Days of gestation
Cerebral hemispheres Normal 21.6 + 2
15 (5)
16 (7) 17 (15) 18 (15) Adult (8) * p
25.4 + 32.4 f 46.8 + 190.9 + P
1.2 1.8 0.9 20.6
P
Diencephalonlbrainsrm Ts16
Normal
Ts16
17.2 f 1.5’
25.4 f. 1.3 29.4 + 0.8 36.4 + 0.8 43.3 f 1.0 195.2 ?I 33.8
17.3 23.4 26.6 31.2
19.6 f l.l** 24.4 f 0.9*** 34.3 + 0.9***
f f f +
1.3** 1.2** 0.5*** 2.2***
158 bral hemispheres. In addition, the mean wet weights of the trisomic cerebral hemispheres and diencephalon/brainstem were significantly reduced (P < 0.05 to < 0.001) on DG 15 through 18 (Table I). Total content of protein was also significantly reduced in the Tsl6 brain (Fig. 1). Analysis of variance showed a gene dose effect for protein content in cerebral hemispheres (P < 0.002) and diencephalon/brainstem (P < 0.001). There was no significant gene dose-accumulation rate interaction. Examination of Nissl-stained sections of the trisomic telencephalon disclosed a delay in neuronal development when compared to paired controls (Fig. 2). In the control embryo as the cerebral cortex matured, cortical thickness increased, concomitant with a reduction in the size of the germinal zone. The average ratio of the thickness of the germinal zone to that of the whole cortex in the normal embryo was 0.220 at D G 15, 0.149 at D G 17 and 0.141 at D G 18. In contrast, comparable ratios in the trisomic cerebral hemisphere, were 0.309, 0.211 and 0.206 on D G 15, 17 and 18, respectively. No qualitative differ-
ences in cell groups or their architectonic arrangement were observed in any of the brain sections, from the rostral end of the forebrain to the level of the locus coeruleus and cerebellum. The cholinergic neurons in the basal forebrain were not cytologically distinct from other cells in either the trisomic or littermate control animals at these ages.
Synaptic neurochemistry The development of afferent and intrinsic cortical neuronal systems was assayed by monitoring pre- and postsynaptic neurochemical markers in the cerebral hemispheres and diencephalon/brainstem. Catecholaminergic markers: tyrosine hydroxylase (TH) activity, a presynaptic marker for both noradrenergic and dopaminergic systems, was not detected on D G 15 in either the cerebral hemispheres (Table II) or diencephalon/brainstem (Table III). On D G 18 the specific activity for TH in trisomic and control hemispheres represented 34% of their adult activity. TH activity tended to be reduced in the brain regions of the trisomic embryo although a sta-
Cerebral Hemispheres
Dlencephalon/Brainstem
.o 3
,//'t
3
!.
tS e st
o
Q. v
o~ E
2
sss S
2i
fs ff"
C
o 0
$ C
o
n
s j s s s ' ~
1
j SS°
1
" ~
I
15
0 .... 0 Normal e----e Ts16 I 16
I 17
Gestational Day
I 18
t
,
~
Normal
~ Ts16
I 15
I 16
....
I 17
I 18
Gestational Day
Fig. 1. Total protein content in embryonic Ts16 and control mouse cerebral hemispheres and diencephalon/brainstem. Values shown are the means ± S.E.M. (bars) for 4-5 animals in each group. Analysis of variance showed a significantgene dose effect in both the cerebral hemispheres (P < 0.002) and diencephalon/brainstem (P < 0.00l). Two-sample t-test analyses were also significant (P < o.o5)*.
159
P
p
¢
V
v Fig. 2. Nissl-stained sections of cortex from Ts16 (A) and control (B) embryosat 17 days of gestation. Sectionswere obtained from an area midwaybetween the dorsolateral edge of the ganglioniceminence and the dorsalmost part of the lateral ventricle; p, pial; v, ventricular surface. Note, the reduced cortical thickness and larger germinal zone in the Tsl6 section. Magnificationin both x 260.
tistically significant difference was not achieved. Norepinephrine (NE) concentrations were higher in the diencephalon/brainstem region whereas dopamine (DA) was higher in the telencephalic region. Analysis of variance showed a significant gene dose effect for NE (P < 0.05) and dopamine (P < 0.05) in the hemispheres and for NE (P < 0.05) in the diencephalon/brainstem. There was no statistical interaction between gene dose and maturational rate of accumulation. In 13 out of the 14 experimental catecholaminergic points the mean values in the Tsl6 were lower than that measured in littermate controls
(P < 0.02 Wilcoxon rank order). Specific [3H]dihydroalprenoloi binding was not detected in the fetal mouse brains. Serotonergic markers. 5-Hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured on gestational days 17 and 18 (Table IV). In the cerebral hemispheres, neither compound was significantly reduced. However, in the diencephalon/brainstem region analysis of variance showed a gene dose effect for 5-HT (P < 0.01); no interaction with rate of accumulation was observed. In addition, values for 5-HT and 5-HIAA in
160
this region were significantly reduced on DG 17 (two sample t-test, P < 0.05). Specific binding for [3H]LSD was not detected in embryonic brains. Putative amino acid neurotransmitter markers. Presynaptic markers assayed for GABAergic neurons included glutamate decarboxylase (GAD) activity and the concentration of G A B A . GAD activity was detected on all gestational days studied and reached
19-20% of adult activity in the hemispheres on DG 18 (Table V). No significant differences were found between Tsl6 and littermate control embryos. On DG 18, levels of G A B A (nmol/mg prot) in the trisomic cerebral hemispheres (n = 4) and diencephalon/brainstem (n = 4) did not differ significantly from those of littermate controls; Tsl6, cerebral hemispheres 14.6 + 0.8 (mean + S.E.M.), diencepha-
TABLE II
Catecholaminergic markers in embryonic and adult cerebral hemispheres Tyrosine hydroxylase activity (TH), norepinephrine (NE) and dopamine (DA) were measured as described under Methods, with the following units: TH, pmol DOPA/mg protein/h; NE and DA, pmol/mg protein. Values are expressed as mean _+ S.E.M. for 4 animals in each group. Analysis of variance showed a significant gene dose effect for norepinephrine (P < 0.05) and dopamine (P < 0.05).
Age 15 Days gestation Normal Tsl6 16 Days gestation Normal Tsl6 17 Days gestation Normal Tsl6 18 Days gestation Normal Tsl6 Adult
Tyrosine hydroxylase activity
Norepinephrine
Dopamine
nd nd 178.9 ± 43.9 145.7 ± 18.9 269.2 ± 32.8 186.7 ± 37.6
6.6±0.8 5.3±0.5
10.2±0.9 7.7±1.6
274.8 _+ 59.5 240.9 ± 65.5 758.3 + 53.4
9.0±0.7 6.9±1.2 34.0±6.2
13.5±0.5 11.5±1.0 59.0±14.9
nd, not detected;-, not assayed.
TABLE III
Catecholaminergic markers in embryonic and adult diencephalon/brainstem Tyrosine hydroxylase acitivity (TH), norepinephrine (NE), and dopamine (DA) were measured as described under Methods, with the following units: TH, pmol DOPA/mg protein/h (embryonic) and nmol/mg protein/h (adult); NE and DA, pmol/mg protein. Values are expressed as mean ± S.E.M. for 4 animals in each group. Analysis of variance showed a significant gene dose effect for NE (e < 0.05).
Age 15 Days gestation Normal Ts16 16 Days gestation Normal Tsl6 17 Days gestation Normal Tsl6 18 Days gestation Normal Tsl6 Adult
Tyrosine hydroxylase activity nd nd
Norepinephrine
Dopamine
-
353.8 ± 41.0 342.8 ± 49.6
-
382.9 + 38.5 397.0 ± 68.9
15.4 + 1.0 13.3 + 1.2
6.1±1.3 5.8±1.0
471.8 + 121.1 394.5 ___82.3 20.5 ± 1.1
12.0 ± 1.5 9.2 ± 1.1 47.0 + 18.3
9.4±0.6 7.8+0.9 96.2±20.7
nd, not detected; -, not assayed.
161 lon/brainstem 15.8 + 2.3; control, cerebral hemi-
found in either embryonic brain region through D G
spheres 15.7 + 1.4, diencephalon/brainstem 17.3 + 1.7. Levels of other putative amino acid neurotransmitters were as follows (nmol/mg prot): T s l 6 cere-
18. Cholinergic markers. The cholinergic n e u r o n a l system was assessed by quantitating choline acetyl-
bral hemispheres aspartate 29.4 + 3.6, glutamate
transferase (CHAT) activity and [3H]quinuclidinyl
74.8 + 7.4; T s l 6 diencephalon/brainstem aspartate 33.1 + 6.7, glutamate 84.6 + 7.5; control cerebral
benzilate (QNB) binding. In the cerebral hemi-
hemispheres aspartate 39.5 ___ 4.5, glutamate 87.5 _+ 8.5; control diencephalon/brainstem aspartate 33.7
significant differences were found for C h A T activity
+ 3.4 and glutamate 78.4 + 4.9. Detectable specific
D G 18 C h A T activity was 3% and QNB specific
binding for [3H]muscimol or [3H]glutamate was not
binding was 12-14% of adult levels. In contrast, in
spheres of the fetal trisomic and normal mouse, no (Fig. 3) or QNB receptor binding (Table VI). O n
TABLE IV
Serotonin and 5-hydroxyindoleacetic acid in embryonic and adult cerebral hemispheres and diencephalon/brainstem regions Compounds were measured as described under Methods and are given as pmol/mg protein. Values are expressed as mean ± S.E.M. for 4 animals in each group. Analysis of variance showed a significant gene dose effect for 5-HT in the diencephalon/brainstem region (P < 0.01). Values for 5-HT and 5-HIAA in the diencephalon/brainstem on DG 17 were significantly reduced on two-sample t-test analysis.
Region and compound
Gestational day
Adult
17 ~mbralhemispher~ Serotonin (5-HT) Normal Tsl6 5-Hydroxyindoleaceticacid(5-HIAA) Normal Tsl6 Diencephalon/brainstem Serotonin (5-HT) Normal Tsl65-Hydroxyindoleacetic acid (5-HIAA) Normal Tsl6 * P<
0 . 0 5 , ** P <
18
3.8±0.1 3.0±0.5
9.6±0.4 12.3±1.7
67.8±18.9
2.5±0.1 2.0±0.2
8.5±1.2 7.0±1.2
54.5±19.2
11.7 + 1.1 6.8 + 0.5**
19.7 ± 1.3 17.4 + 1.6
97.9 + 13.4
6.7 ± 0.9 4.4 + 0.2*
26.4 ± 5.1 19.0 + 3.5
87.0 ± 8.4
0.01.
TABLE V
Glutamate decarboxylase activity in embryonic and adult mouse brain Enzyme activity, nmol GABA/mg protein/h, was measured as described in Methods. Values are expressed as mean + S.E.M. for 4 animals in each group. No significant group or interaction effect was shown on analysis of variance.
Region
Enzyme activity on gestational day 15
Adult
16
17
18
Cerebral hemispheres Normal 2.9 ± 0.5 Tsl6 2.1 + 0.2
2.5±0.5 2.7±0.6
3.3±0.9 3.1±0.7
4.1±1.6 4.4±1.6
21.8±0.6
Diencephalon/brainstem Normal 4.8 + 0.6 Tsl6 3.6 + 0.9
6.7 + 1.5 6.3 + 1.5
7.5 + 1.7 8,1 + 1.9
8.7 + 2.4 7.7 + 2.2
31.3 + 3.0
162 Cerebral Hemispheres
t-
Diencephalon/Brainstem
sJ/~
5
/t/t
C
P
cL
4
4
O~
/// "
3
3
~.~
>
<
~4,~
2
2
(J v,. tJ O.
u) p-
0 . . . . 0 Normal
1
1
0----0 Normal
H
Ts 16
e-
Ts16
C3 l
I
I
I
I
I
I
I
15
16
17
18
15
16
17
18
Gestational Day
Gestational Day
Fig. 3. Choline acetyltransferase (CHAT) activity in embryonic Ts16 and control mouse cerebral hemispheres and diencephalon/brainstem. Values shown are the means (nmol ACh/mg protein/h) + S.E.M. (bars) for 4 animals in each group. Analysis of variance showed a significant gene dose effect (P < 0.001) in the diencephalon/brainstem region. Paired t-test analysis was also significant (*) in this region on DG 16-18 (P < 0.01, P < 0.05 and P < 0.025 respectively).
the d i e n c e p h a l o n / b r a i n s t e m an analysis of v a r i a n c e of C h A T activity d e m o n s t r a t e d a significant difference ( P < 0.001) b e t w e e n the trisomic a n d control fetus; there was n o significant i n t e r a c t i o n with gestational days (Fig. 3). In a d d i t i o n , on D G 16, 17 and 18 the specific activities of C h A T were significantly r e d u c e d ( p a i r e d t-test) in the trisomic m o u s e c o m p a r e d to l i t t e r m a t e controls ( P < 0.01, < 0.05,
and < 0.025 respectively). Assays were p e r f o r m e d on the s a m e tissue samples used to m e a s u r e d G A D a n d T H activities, which were u n c h a n g e d from littermate controls. T h e specific activity of c o n t r o l C h A T activity in the d i e n c e p h a l o n / b r a i n s t e m o n D G 18 repr e s e n t e d only 3 . 6 % of that f o u n d in adult mice. T h e a p p a r e n t density of [3H]QNB b i n d i n g sites in the T s l 6 d i e n c e p h a l o n / b r a i n s t e m did n o t differ signifi-
TABLE VI Quinuclidinyl benzilate receptor-ligand binding in embryonic and adult mouse brain Quinuclidinylbenzilate receptor-ligand binding, fmol QNB bound/mg protein, was measured as described in Methods. Values are expressed as mean + S.E.M. for 4 animals in each group. Analysis of variance showed no significant gene dose or interaction effect. Region
Cerebral hemispheres Normal Ts16 Diencephalon/brainstem Normal Tsl6
Ligand binding on gestational day
Adult
16
17
18
164 + 42 106 + 13
205 + 30 202 + 24
235 + 43 266 + 31
1897 + 101
258 + 75 189 + 27
275 + 76 285 _+ 34
391 + 53 365 + 17
978 + 189
163 cantly from littermate controls (Table VI). The density of [3H]QNB specific binding in D G 18 controls was 40% of the level of adult mice. Thus, the significant reduction in ChAT activity observed in the trisomic mouse did not appear to affect the development of muscarinic cholinergic receptors. DISCUSSION This study was designated to determine possible selective effects of trisomy 16 on neuronal characteristics in the mouse. To date, general developmental retardation and organ hypoplasia are the most obvious abnormal features observed in all murine trisomic conditions 23. The statistically reduced brain weight and brain protein content between DG 15 and 18 in the Ts16 mouse embryo is consistent with previously reported reductions in the body weight of embryos and their placentas (Gearhart, unpublished observations) 32. In our morphometric analysis, the reduced brain weight appears to be primarily due to a delay in neuronal differentiation rather than neuroblast formation. Furthermore, the ratio of the thickness of the ventricular germinal zone as compared to the fetal cortex is elevated in the Tsl6 embryos such that the ratio at DG 18 in Tsl6 is comparable to that observed in unaffected controls at DG 17. Thus, maturational increases in brain weight in the Tsl6 embryo are delayed by approximately one day in Tsl6 embl-yos as compared to their littermate controls. In order to determine possible selective effects of the trisomic condition on identified populations of neurons, we have compared in Tsl6 mice embryos and their unaffected littermates several pre- and postsynaptic markers for neurotransmitter-defined neuronal systems implicated in the neuropathology of DS. Thus, we examined presynaptic markers for GABAergic neurons, which are local circuit stellate cells in the cerebral cortex, because of the reported reduction of cortical small cells in DS 15,21. Furthermore, studies on peripheral and central serotonin levelsS,40, platelet serotonin uptake 5, catecholamine metabolic enzymes4.7.19.25 and CSF biogenic amine metabolites 30 suggest that dysfunction of the central serotonergic/catecholaminergic neuronal systems may be a feature of DS. Finally, studies have demonstrated in middle-aged DS patients the presence of neuropathologic stigmata of Alzheimer's dementia,
reduction in cortical activity of ChAT and loss of cholinergic neuronal perikarya in the nucleus basalis of Meynert35,36,~. Unfortunately, since Ts16 mice generally die by DG 17-18, the present studies were limited to the assessment of the embryonic neurogenesis of these systems. Nevertheless, [3H]thymidine autoradiographic, immunocytochemical and histofluorescence studies in rodents indicate that the bulk of the catecholaminergic, serotonergic and cholinergic nuclei in the brainstem core have probably been formed by DG 17-1811,12,14,22,33. Neurogenesis of the cortical GABAergic neurons is likely to continue beyond this date13,37,41. In our dissection, dopamine is the predominant catecholamine in the telencephalic sample because it contains the developing striatum. TH activity was first detectable at DG 16 in both the hemispheres and the diencephalon/brainstem and exhibited a modest increase in specific activity in both controls and Ts16 embryos between DG 16 and 18. Furthermore, endogenous norepinephrine and dopamine were also detectable in both the hemispheres and diencephalon/brainstem at DG 17 and 18. Significant differences between the Ts16 and littermate controls were observed for norepinephrine in the two regions and for dopamine in the cerebral hemispheres. In addition, in 13 out of the 14 experimental determinations of catecholaminergic parameters the mean value in the Ts16 was less than that measured in the littermate controls, producing a significant Wilcoxon rank order. These data suggest an impairment in the development of the catecholaminergic systems in Ts16. The concentration of serotonin, a presynaptic marker for serotonergic neurons, was significantly reduced in the Ts16 diencephalon/brainstem at DG 17.5-Hydroxyindoleacetic acid (5-HIAA), the serotonin metabolite, was also significantly reduced in the diencephalon/brainstem on DG 17 (two sample t-test), although the analysis of variance did not achieve significance. Thus, reductions in the concentrations of serotonin and 5-HIAA within the embryonic brain region containing the median raphe, suggest an impairment in the ontogeny of this neuronal system. Glutamate decarboxylase (GAD), appears to be a highly specific presynaptic marker for GABAergic neurons 13. GABAergic neurons have been shown by immunocytochemical studies to be stellate type II in-
164 terneurons in the cerebral cortex, interneurons scattered through the brainstem core and Golgi type II neurons within the striatal efferent system. Neurogenesis of these neurons appears to occur over a longer period of time than noted for non-GABAergic neurons in the cortex and brainstem core. In the Tsl6 mouse, no significant differences in the specific activity of G A D were observed either in the hemispheres or the diencephalon/brainstem between DG 15 and 18. Furthermore, unlike the presynaptic markers for the catecholaminergic and serotonergic systems, no consistent pattern of reduction in G A D activity was observed in the Tsl6 versus the littermate controls. Similarly, in developmental studies of the GABAergic system in which neurogenesis was temporarily arrested by treatment with the alkylating agent methylazoxymethanol acetate, no significant depressions in the concentrations of presynaptic markers for GABAergic neurons were detected 27. However, in both the chemically lesioned and this chromosomally imbalanced model, the total content of the GABAergic markers is reduced within a hypoplastic cortex. Choline acetyltransferase (CHAT), is the most specific presynaptic marker for the cholinergic neuronal system 38. Whereas the specific activity of ChAT in the cerebral hemispheres did not differ in Tsl6 and littermate controls between D G 15 and 18, significant reductions in its specific activity were observed in the diencephalon/brainstem region. At this early stage of development, however, the specific activity of ChAT represents only 3 - 4 % of that occurring in the adult within these same regions. Approximately 60-80% of ChAT activity in the cerebral cortex is localized to afferents derived from the magnocellular complex which includes the nucleus basalis of Meynert located adjacent to the diencephalon. In addition, a small component of cortical cholinergic innervation appears to be derived from neurons intrinsic to the region 28,29. The reason for the differential reduction in specific activity of ChAT in the Tsl6 diencephalon/brainstem as compared to the normal concentration in the hemispheres remains unclear. One possibility is that the reduced activity reflects impaired development of the cholinergic nucleus basalis neurons, which would be included in the diencephalon/brainstem sample. Nevertheless, assessment of muscarinic receptor density by ligand binding did not disclose significant differences between
Tsl6 and littermate controls within the two major brain regions on the three gestational days examined. Thus, at these early stages, impairments in presynaptic cholinergic development, especially in the diencephalon/brainstem, are not mirrored by alterations in its receptor site. Our studies point to a delay in the neurogenesis and growth of the brain in the Tsl6 mouse embryo as compared to littermate controls. Neurochemical assessment of presynaptic markers for defined neuronal systems in the hemispheres and diencephalon/brainstem indicates that certain neuronal systems may be more severely affected than others. Thus, whereas the specific activity of the presynaptic marker for the GABAergic neurons in Tsl6 and controls did not differ at any gestational date, significant reductions in the presynaptic markers for catecholaminergic and serotonergic neurons were observed. In addition, the specific activity of ChAT was significantly reduced in the diencephalon/brainstem, suggesting that cholinergic systems in this region are more specifically affected by the genetic defect. Our neurochemical data also provide additional support for the hypothesis of delayed, but not aberrant neuronal development in trisomy 16. For example, the analysis of variance, even in regions with significant gene-dose effects, shows no evidence for a meaningful interaction between aneuploidy and the subsequent rate of accumulation of enzyme activity or neurotransmitter content. In the Ts16 embryo, when the reductions in weight of the hemispheres and the diencephalon/brainstem are taken into account, it is clear that the total complement of synaptic markers for the neuronal systems is significantly reduced. Thus, expression of data only in terms of concentration of the markers minimizes the more global deficit in neurogenesis and synaptic neurochemical differentiation in the fetal trisomic brain. Validity of this alternative form of presentation has been adequately demonstrated in previous studies in which telencephalic and cerebellar neuronal groups have been selectively eliminated by drugs that arrest neurogenesis 27,39. The mechanism by which an aneuploid condition results in abnormal neurogenesis with more selective effects on specifically defined neuronal systems remains unknown, although possible explanations include the effects of gene dose or secondary biochem-
165 ical abnormalities17, 23. R e s o l u t i o n of this m o r e fund-
tor D e v e l o p m e n t A w a r d 5 K07 NS 00506-04 f r o m the
a m e n t a l q u e s t i o n m a y be possible t h r o u g h the study
N I N C D S ( H . S . S . ) , by U . S . Public H e a l t h S e r v i c e
of T s l 6 c h i m e r a s , which survive to a d u l t h o o d .
Grant RSDA
ACKNOWLEDGEMENTS
n e d y , Jr. F o u n d a t i o n . J . G . is a K e n n e d y F o u n d a t i o n Scholar.
II K03-00125, a S u r d n a F o u n d a t i o n
G r a n t ( J . T . C . ) and a g r a n t f r o m the J o s e p h P. K e n -
This w o r k was s u p p o r t e d by a T e a c h e r - I n v e s t i g a -
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166
29
30 31
32
33
34
35
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