- Email: [email protected]
Cultured hippocampal neurons from trisomy 16 mouse, a model for Down's syndrome, have an abnormal action potential due to a reduced inward sodium current
Brain Research, 604 (1993) 69-78
69
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 18530
Cultured hippocampal neurons from trisomy 16 mouse, a model for Down's syndrome, have an abnormal action potential due to a reduced inward sodium current Z. Galdzicki,
E. Coan
and S.I. Rapoport
Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD (USA)
(Accepted 8 September 1992)
Key words: Trisomy 16; Down's syndrome; Action potential; Current; Hippocampus; Primary culture; Mouse; Patch-clamp
Mouse trisomy 16 is an animal model for Down's syndrome (human trisomy 21). The whole-cell patch-clamp technique was used to compare passive and active electrical properties of trisomy 16 and diploid mouse 16 fetal hippocampal neurons maintained in culture for 2-5 weeks. There was no significant difference in any mean passive property, including resting potential, membrane resistance, capacitance and time constant. However, in trisomic neurons, the action potential had a 20% significantly slower rising phase and a 20% significantly smaller inward sodium current and inward sodium conductance than did control neurons. The outward conductance was not altered. The ratio of maximum inward conductance to maximum outward conductance was 30% less in the trisomy 16 cells. These results indicate that trisomy 16 hippocampal neurons have abnormal active electrical properties, most likely reflecting reduced sodium channel membrane density. Such subtle differences may influence elaboration of the hippocampus during development.
INTRODUCTION D o w n ' s s y n d r o m e (DS) is t h e result o f trisomy for c h r o m o s o m e 2133. D u p l i c a t i o n of g e n e d o s a g e o f t h e b a n d q22 r e g i o n o f this c h r o m o s o m e c o n t r i b u t e s to t h e D S p h e n o t y p e , which i n c l u d e s m e n t a l r e t a r d a t i o n 16. D S subjects also d e v e l o p t h e n e u r o p a t h o l o g i c a l a n d neurochemical defects of Alzheimer's disease after the age o f 35 y e a r s 51. It is n o t c l e a r if t h e s e a b n o r m a l i t i e s result f r o m o v e r - e x p r e s s i o n o f o n e specific g e n e on c h r o m o s o m e 21, o r of m a n y g e n e s on this chrom o s o m e 4'31. Clinical studies a r e u n d e r w a y to r e l a t e v a r i o u s a s p e c t s of the D S p h e n o t y p e to d u p l i c a t i o n of specific g e n e s o n d i f f e r e n t p a r t s of c h r o m o s o m e 2118. A n a n i m a l m o d e l w o u l d facilitate t h e s e studies. M u r i n e t r i s o m y 16 ( T s l 6 ) is c o n s i d e r e d such a m o d e l 17'29'5°, b e c a u s e m o u s e c h r o m o s o m e 16 is genetically h o m o l o g o u s to p a r t of t h e long a r m o f h u m a n c h r o m o s o m e 21. E a c h c h r o m o s o m e c o n t a i n s g e n e s for s u p e r o x i d e d i s m u t a s e ( S O D 1 ) , p r o t o - o n c o g e n e ETS2, /3-amyloid p r e c u r s o r p r o t e i n (APP), p h o s p h o r i b o s y l -
g l y c i n a m i d e s y n t h e t a s e ( P R G S ) , i n t e r f e r o n - a a n d -/3 cell surface r e c e p t o r s ( I F N R C ) , a n d c h r o m o s o m a l p r o tein HMG-1414'43 45. U n f o r t u n a t e l y trisomy 16 mice die in utero. T h e action p o t e n t i a l plays an essential role in comm u n i c a t i o n b e t w e e n n e u r o n s . D e p o l a r i z a t i o n o f presyn a p t i c n e u r o n a l m e m b r a n e s causes t h e r e l e a s e o f specific n e u r o t r a n s m i t t e r s a n d n e u r o m o d u l a t o r s . It has b e e n shown t h a t t h e d u r a t i o n o f t h e action p o t e n t i a l is s h o r t e r a n d its m a x i m u m r a t e s of d e p o l a r i z a t i o n a n d r e p o l a r i z a t i o n a r e l a r g e r in h u m a n trisomy 2138 a n d m u r i n e t r i s o m y 16 fetal d o r s a l r o o t g a n g l i a ( D R G ) n e u r o n s in c u l t u r e 2, as c o m p a r e d to c o n t r o l D R G n e u r o n s . T h e s e d i f f e r e n c e s a r e specific for h u m a n trisomy 21 a n d m o u s e trisomy 16, a n d do not occur in o t h e r h u m a n c h r o m o s o m a l a b n o r m a l i t i e s , such as K l i n e f e l t e r ' s , T u r n e r ' s or ' s u p e r f e m a l e ' s y n d r o m e s 38, in t r i s o m y 19 mice 1°, o r in s u p e r o x i d e d i s m u t a s e - 1 t r a n s g e n i c mice a. W e t h o u g h t it i m p o r t a n t to find o u t if t h e action p o t e n t i a l a n d its ionic c u r r e n t s a r e a b n o r m a l in cul-
Correspondence: Z. Galdzicki, Laboratory of Neurosciences, Building 10, Room 6C103, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892-010, USA. Fax: (1) (301) 402 0074.
70
tured non-DRG Hippocampal
n e u r o n s f r o m t h e t r i s o m y 16 m o u s e .
neurons were chosen for the study, be-
cause the hippocampus neous population
provides a relatively homoge-
of central nervous system neurons
with well-characterized properties,
and
because
the
h i p p o c a m p u s is p a r t i c u l a r l y v u l n e r a b l e t o A l z h e i m e r ' s d i s e a s e n e u r o p a t h o l o g y in a d u l t s w i t h O S 32. F u r t h e r m o r e , m o r p h o l o g i c a l d i f f e r e n c e s in t h e h i p p o c a m p a l f o r m a t i o n h a v e b e e n r e p o r t e d b e t w e e n t r i s o m y 16 a n d c o n t r o l m o u s e f e t u s e s a t e m b r y o n i c d a y 18 42 . S o m e o f t h e s e r e s u l t s h a v e b e e n p r e s e n t e d in a b s t r a c t f o r m t2. MATERIALS AND METHODS
Animals Trisomy 16 (Tsl6) and normal mouse fetuses were obtained by breeding males, doubly heterozygous for Robertsonian translocations of chromosome 16 (Rb(16.17)32Lub/Rb(ll.16)2H), with normal C57BL/6 females. After 15-16 days of gestation, "the fetuses were removed. Trisomy 16 fetuses were identified by their characteristic anasarca and retarded development36. To check the accuracy of the identification, chromosome spreads were prepared by amodification of the procedure of Crowe et al. 15.
A
Chromosome analysis Individual fetal livers were collected in dissection medium (calcium- and magnesium- free Puck's saline solution), minced with a sterile scalpel, and then incubated in a 0.25% (w/v) trypsin solution for 10 min at 37°C. Pieces of tissue were allowed to settle out. Individual cells which remained in suspension were collected by aspiration. These pieces of tissue were digested again and the single cell suspension was collected and added to the previous suspension. Cell suspensions were centrifuged for 5 rain at 460 X g and the cell pellet was resuspended in 10 ml Dulbecco's minimal essential medium (DMEM) supplemented by 10% fetal calf serum (v/v) and 0.1 ml 10 /zg/ml colcemid (Sigma, St. Louis, MO). Following incubation in Petri dishes for 90-120 rain at 37°C, the cells were harvested, collected by centrifugation and resuspended in 75 mM KCI at 37°C. After 15-20 min at 37°C, the cells were re-collected and resuspended in the residual volume of KC1 and fixed by incubation in a solution of methanol and glacial acetic acid (3:1) (v/v). This step was repeated twice. The fixed cells were dropped onto inclined slides and the chromosomes were stained with Giemsa solution. A magnification of x 400 was used to examine the spreads. Cell culture preparations Brains were removed from trisomy 16 and littermate control fetuses at day E15-E17 and hippocampal tissue was identified and dissected free as described by Banker and Cowan 3. This tissue was then cut into small pieces, transferred to a solution containing 0.05% trypsin (w/v), and incubated at 37°C for 15 min. The tissue was dissociated into a cell suspension by trituration with a fire-polished
B
J
Fig. 1. Metaphase chromosome spreads of liver cells stained with Giemsa solution from 16-day-old mouse embryos. Control cells have 39 separate chromosomes including one which is metacentric (A, arrow), giving a total of 40 chromosome arms. Trisomy 16 cells have 39 separate chromosomes including two metacentric chromosomes (B, arrows), giving a total of 41 chromosome arms.
71 Pasteur pipette in plating medium consisting of minimal essential m e d i u m (MEM), 10% fetal calf serum, 5% horse serum and 1% glutamine (all v/v). Neurons were maintained in low density culture 3 by plating t h e m on a confluent feeder layer of glial cells of the same ploidy type prepared 3 - 4 weeks previously. Dispersed cells survived for 5 or more weeks. After 24 h, and then every 3 - 4 days, m e d i u m was replaced with MEM, 5% horse serum and 1% glutamine (all v/v). Cytosine/3-D-arabinofuranoside (araC, 10/xM) was added as necessary to reduce growth of non-neuronal cells 2°. Cell diameter was m e a s u r e d with an eye-piece graticule.
Electrophysiology Electrophysiological recordings were performed at room temperature (22°C) on isolated neurons which had been cultured for at least 14 days. The culture medium in dishes was replaced by a solution containing (in mM): NaC1 140; KCI 5; CaC12 1; MgC12 2; dextrose 10; H E P E S / N a O H 10, pH 7.4. Currents and action potentials were recorded in a whole-cell configuration using a patch-clamp techrrique 23. Electrodes were pulled from borosilicate glass and backfilled with 'intracellular' solution containing (in mM): KCI 130; NaC1 3; CaC12 1; MgC12 2; E G T A 10; H E P E S / K O H 10; pH 7.4. T h e pipette resistance usually was less than 2 - 3 MgL Current and voltage recording and stimulation were controlled by a Labmaster board using the pClamp program (Axon Instruments, Burlingame, CA). Currents were filtered at 5 kHz (8-pole Bessel) and digitized at 50 kHz.
The resting potential of a cell was measured at the beginning of each recording and at intervals throughout an experiment to assess cell viability. A 5 m V hyperpolarizing step from a holding potential of - 6 0 mV was applied to estimate m e m b r a n e capacitance and resistance. Capacitance was calculated as the integral of the current response between the onset of the pulse and 5-fold of its initial decay time. A steady-state current level was used to calculate m e m b r a n e resistance. Action potentials were evoked in current-clamp mode by 5 ms depolarizing current steps of 0.1-0.8 nA. Analysis of voltage responses under these conditions was as described by Ault et al. 2. In voltage-clamp experiments, total current was recorded using 12 depolarization pulses of 10 ms duration, in 10 mV steps from a holding potential of - 6 0 mV. A typical current sweep included outward and inward components. Ionic currents were corrected on-line for linear leakage and capacitive currents using a P / 8 method 6, setting the control voltage of the P / 8 pulses to - 90 inV. A peak inward current, Ip, was estimated from a least-squares fit to experimental data in a short interval around the peak, using a third order polynomial. The same fitting procedure to the late component was used to estimate its steady-state value, /stst. From the peak current vs. m e m b r a n e potential, Vm, relation (Ip-Vm), the m a x i m u m peak inward current, i~nax, and the potential, Vpmax, that yielded i~nax, were estimated. Linear fitting of the first points after the peak value gave the m a x i m u m inward conductance, Gin, and then the m a x i m u m outward conductance, Gout , was defined as the m a x i m u m slope of the IStSt-gm relation. The ratio G i n / G o u t was calculated as a p a r a m e t e r independent of m e m b r a n e surface area. Likewise all current values were divided by m e m b r a n e capacitance (C).
Fig. 2. Phase contrast micrographs of two hippocampal neurons after 3 weeks in culture. A: control. B: trisomy 16. No morphological difference could be distinguished between the two neuron types at any stage in culture. Cells were triangular or had an oval/pear-like shape. Confluent glial cells are in the background. Bar = 5/xm.
72
-
-
CONTROL
A [SOMY
o 2 ms
.~1~
CONTROL
B TRISOMY
mosome arms are fused at their centromeres and appear metacentric. Cells from the diploid embryos had one Robertsonian chromosome, either the 11.16 or the 16.17 Robertsonian, giving 39 separate chromosomes with a normal complement of 40 chromosome arms (Fig. 1A). Ceils from trisomic embryos had one 11.16 Robertsonian and one 16.17 Robertsonian, and thus had 39 separate chromosomes but 41 chromosome arms (Fig. 1B). The control spread presented in Fig. 1A shows 39 chromosomes including the one Robertsonian translocation chromosome. The trisomy 16 cell in Fig. 1B shows 39 chromosomes including two Robertsonian translocation chromosomes. This indicates that the cell is aneuploid. We investigated 25 different fetuses that have been identified as trisomic according to phenotype. Twenty four of these were confirmed to have 39 separate chromosomes including two Robertsonian translocations (4% error), consistent with the report of Miyabara et al. 36. Therefore determination of a trisomy by its phenotypic characteristics was highly accurate.
2 ms
Cell culture
Fig. 3. A: comparison of action potentials obtained under currentclamp conditions from trisomy 16 and control hippocampal neurons after 25 days in culture. The holding potential was - 60 mV for both cells; the depolarizing current, Istim, was 0.3 hA. M e m b r a n e capacitance and m e m b r a n e resistance were, respectively, 22 pF and 270 MS2 for the control, 21 pF and 250 MS2 for the trisomy 16 neuron. B: comparison of the time derivative of the action potentials illustrated in A after subtraction of the contribution ( - Istim/C) due to the stimulating current. The slower rising phase of the action potential in the trisomic neuron is apparent only when looking at the derivative course in B.
Fig. 2 illustrates control diploid and trisomy 16 neurons which were used for recording. There was no difference in the size of the two cell types at any time. One day after plating minor processes were observed. After 4 - 5 days some cells created small networks, whereas others remained isolated. At the time of recording (between 2 - 4 weeks), there was approximately a 3-fold increase in the surface area of the cell body and increases in the length and thickness of the dendritic processes. Cell diameter was between 4 and 7 /xm.
Passiue electrical properties Differences between trisomy 16 and control neurons were evaluated using a two-tailed Student's t-test and a repetitive m e a s u r e s analysis of variance 22. Significance was assigned for P < 0.05.
Table I summarizes m e a n passive m e m b r a n e properties of the cultured hippocampal neurons. Capacitance, m e m b r a n e resistance, m e m b r a n e time constant
RESULTS TABLE I
Examination of chromosome spreads Metaphase spreads of fetal liver cells were visualized by the procedure described in Materials and Methods as illustrated in Fig. 1. In a control diploid mouse there were 19 pairs of autosomes plus one pair of sex chromosomes to give a total number of 40 chromosome arms. Mus musculus chromosomes are acrocentric (i.e. the centromere is near the end of the chromosome), so that they appear as single-armed chromosomes with a 'hinge' between the arms. In a Robertsonian translocation, two non-homologous chro-
Mean passi~,e membrane properties of trisomy 16 and control hippocampal neurons in culture N u m b e r s are means_+S.E.M. No m e a n s differed significantly between cell type. Sampling time was 20 /zs, and analog filter 10 kHz. N u m b e r of cells are given in parentheses.
Parameter Capacitance (mV) M e m b r a n e resistance (Mg2) M e m b r a n e time constant (ms) Resting potential(mV) Days in culture
Control 27 440
+ 1 +30
Trisomy 16 (53) (53)
11.1 + 1.0 (53) - 4 2 _+ 1 (54) 22 + 1 (54)
25 480
+ 1 +40
(56) (56)
10.8+ 1.0 (56) - 4 1 + 1 (53) 21 + 1 (56)
73
8
H
c~ V
~ v
~z
74 a n d resting m e m b r a n e p o t e n t i a l w e r e analyzed. T h e r e was no significant d i f f e r e n c e b e t w e e n trisomy 16 a n d c o n t r o l n e u r o n s for any of t h e s e p a r a m e t e r s .
A
KCl in pipette
~
120
)
~
~ - ~ - NORMAL
A
100
T
>
E 6O
<
.J 40
V
CONTROL ~-
20
1
ms
TRISOMY
B CsCI in pipette ,
i
i
i
200
400
600
800
CURRENT
1000
(pA) f
200
~-
+ TEA
/
•
B 160 •
120 • I-
Fig 5. A: tetrodotoxin (TTX) blocks inward currents. These currents were generated by a depolarizing 90 mV pulse from a holding potential of - 7 0 mV. In the presence of 1 /xM TTX, the inward component disappeared. B: in a different neuron, replacing KCI with CsCI in the pipette solution and application of 20 mM TEA into the bath completely abolished the delayed outward current as compared to A, when the same depolarizing step was applied.
N n," O~
80"
I$
40
"---"0---
CONTROL
:$
ISOMY
x
0 0
i
i
i
i
200
400
600
800
CURRENT
1000
(pA)
0.
> C
-10" ,,~ Z O I,-.-
-20 ' OMY
N
n" n~)
-30 '
LIJ ¢r
;IE -40 x
-50
,
i
i
,
200
400
600
800
CURRENT
1000
(pA)
Fig. 4. Action potential parameters in control and trisomy 16 neurons, with increasing current stimuli in current-clamp conditions. The holding potential equaled - 60 mV. A: amplitude increased with stimulus size. There was no difference between contro! and trisomy 16 neurons. B: the maximum depolarization rate increased with stimulus size and was smaller in trisomic neurons. C: the maximum repolarization rate decreased with stimulus size and was larger in trisomic cells.
Action potentials A c t i o n p o t e n t i a l s c o u l d n o t b e e v o k e d o n e day a f t e r plating. A f t e r two w e e k s in culture, a l m o s t 95% o f c o n t r o l n e u r o n s a n d 80% of t r i s o m i c n e u r o n s w e r e a b l e to g e n e r a t e an action p o t e n t i a l . Fig. 3 illustrates typical action p o t e n t i a l s in c o n t r o l a n d t r i s o m y 16 h i p p o c a m p a l n e u r o n s , which h a d b e e n p l a t e d for 25 a n d 27 days, respectively. T h e m a x i m u m r a t e of d e p o larization, t i m e to m a x i m u m d e p o l a r i z a t i o n , m a x i m u m r e p o l a r i z a t i o n rate, t i m e to m a x i m u m r e p o l a r i z a t i o n , action p o t e n t i a l a m p l i t u d e a n d w i d t h at 50% a m p l i t u d e w e r e analyzed. R e s u l t s a r e shown in T a b l e II. With increasing current steps from a suprathreshold stimulus o f 200 p A to a m a x i m u m stimulus o f 800 p A , trisomy a n d c o n t r o l cells fired p r o g r e s s i v e l y e a r l i e r a n d t h e action p o t e n t i a l s w e r e g r a d e d ( T a b l e II). A c t i o n p o t e n t i a l a m p l i t u d e i n c r e a s e d by a b o u t 40% a n d t h e m a x i m u m d e p o l a r i z a t i o n a n d r e p o l a r i z a t i o n r a t e s inc r e a s e d by 30% (Fig. 4A,B). T h e t i m e to p e a k a m p l i t u d e a n d the t i m e to m a x i m u m d e p o l a r i z a t i o n r a t e d e c r e a s e d a b o u t 50%, t h e t i m e to m a x i m u m r e p o l a r ization r a t e d e c r e a s e d a l m o s t 40%, a n d t h e a c t i o n p o t e n t i a l w i d t h d e c r e a s e d by 2 0 - 3 0 % .
75 fetal hippocampal neurons, maintained in primary culture for 2-5 weeks. This is consistent with previous results for mouse trisomy 16 and human trisomy 21 D R G neurons 38'2'1°. Similar ranges of values for membrane resistance, capacitance, membrane time constant and resting potential have been reported for hippocampal neurons in primary and acute culture, slices, and in vivo from mice and other mammalian species19,27,28,39,46.47.
The one statistically significant difference ( P < 0.05) between trisomy 16 and control hippocampal neurons was in the mean maximum depolarization rate (Table II and Fig. 4B), which was 20% slower in trisomic cells (118 V / s ) than in diploid controls (148 V / s ) at a current of 800 pA. Total currents
Whole-cell currents were analyzed in 49 control neurons and 40 trisomic neurons (Table III). By far the most predominant components of voltage-clamp currents were attributable to tetrodotoxin (TTX)-sensitive sodium channels and to delayed rectifier potassium channels. The early component of the current was blocked in a partially reversible way by 1 / z M T T X (see Fig. 5) but was not significantly affected by 2 mM CoC12 or 2 mM CdC12 (e.g. ref. 46). Replacing KC1 with CsC1 in the recording electrode (see Fig. 5), and putting 20 mM tetraethylammonium (TEA) in the bath virtually abolished the outward current 4s. Fig. 6 shows whole-cell currents in a trisomic cell and a control cell, evoked by a series of 10 ms depolarizing pulses, from a holding potential - 6 0 mV to the levels indicated. The mean maximum peak inward current / F ax was significantly smaller in trisomic than for control neurons ( - 1 2 8 p A / p F to - 1 6 9 p A / p F , P < 0.05). Fitting of the linear part of the I p - V m plot provided an estimation of the TTX-sensitive maximum inward conductance, Gin. For trisomic neurons Gin was smaller than for control neurons (942 p S / p F to 1192 p S / p F , P < 0.05). Both of these values for our fetal neurons are about 10-times smaller than reported for acutely dissociated adult guinea pig hippocampal neurons 46. Consistently, the ratio, (maximum inward conductance)/(maximum outward conductance), was significantly ( P < 0.01) smaller in trisomy 16 than control cells (0.45 to 0.67).
The only statistically significant difference in the action potential between trisomy 16 and control cells was in the mean rate of depolarization, which was 20% less in the trisomic neurons. This result is consistent with voltage clamp data which indicate a significantly smaller (25%) inward sodium current in trisomy 16 than in control cells, as the depolarization rate is directly proportional to voltage-dependent sodium current density 24. A lower rate of depolarization in cultured hippocampal trisomy 16 neurons differs from findings in cultured murine trisomy 16 and human trisomy 21 D R G neurons. The maximum depolarization rate of the action potential in D R G neurons is about 30% larger than in controls for both trisomy 16 and 21, and the maximum repolarization rate is about 30% greater for trisomy 16 and 50% greater for trisomy 21, whereas spike duration is reduced by about 20% for trisomy 16 and by about 40% for trisomy 212'38'4°. The differences between D R G and hippocampal neurons may be due to cell-specific regulation of ion channel expression or function 41. In addition, it has been reported that nerve growth factor (NGF) can influence action potential characteristics in D R G neurons 9, so a differential sensitivity to regulating factors like N G F could account for the differences in action potential properties u. D R G neurons in primary culture initially require N G F for survival 9, whereas hippocampal neurons are N G F independent 35. Two different types of sodium current have been reported in adult hippocampal neurons: a fast sodium current with a half decay time of 2 m s 46, and a slowly
DISCUSSION There was no difference in any mean passive electrical property between mouse trisomy 16 and control T A B L E III Voltage-clamp data from trisomy 16 and control hippocampal neurons
Parameters were evaluated from I - V m relations. N u m b e r of cells are given in parentheses. Parameter
Control
Trisomy 16
Maximum outward conductance ( p S / p F ) M a x i m u m inward conductance ( p S / p F ) Ratio of conductances (Gin / G o u t ) Maximum peak of inward current I v ( p A / p F ) Potential at m a x i m u m peak of inward current Vpma× (mV)
2,074 ± 149 (47) 1,187 ± 77 (47) 0.67± 0.05 (47)
2,033 + 161 (38) 931 ± 99 * (39) 0.45± 0.03 ** (38) -129 ± 12 ** (40)
- 162
±
11
(49)
-27
±
2
(49)
* Significantly different at P < 0.05; ** Significantly different at P < 0.01.
- 23
+
2
(40)
76 inactivating sodium current 2~. The first determines excitability threshold, whereas the second acts as a pacemaker current for modulating firing frequency. The 30% reduction in sodium inward current in trisomy 16 hippocampal neurons could be due to lower single sodium channel conductance or to a reduced density of sodium channels. As trisomy 16 is due to excessive gene expression rather than mutated genes, a change in conductance of a single channel is less likely.
30 mV
A
/
///
10 mV
-20 mV -40 mV
\/
o 2 ms
30 mV
B
f _ ~
S
j.7 ~ /
f"
10 mV
-20 mV ~ J~ -
~
-40 mV
fs
%
2 ms
If channel density is reduced this could occur in soma or dendrites, or in both regions. A reduced somatic sodium channel density would affect the action potential amplitude. A reduction in dendritic channel density also could produce important physiological differences. Sodium channels in dendrites have been demonstrated indirectly in guinea pig hippocampal slices 5, and directly using patch-clamp whole-cell recordings in isolated rat cortical pyramidal neurons 26, where the amplitudes of the dendritic and somatic sodium currents were comparable. A reduced current due to reduced sodium channel density in the dendritic tree could influence coupling between dendritic excitation and somatic activation, especially during development 25,26,3°,34. It is difficult to directly relate abnormalities in the active electrical properties of mouse trisomy 16 hippocampal neurons to the pathophysiology of Down's syndrome, as our experiments were not performed using trisomy 21 tissue. However, if the similarities found between D R G human trisomy 21 and murine trisomy 16 could be extended to hippocampal tissue, trisomy 16 as a model of Down's syndrome would be further validated. As neural development is related to integrated electrical activity 8, abnormalities such as we found might affect differentiation and morphogenesis and ultimately network wiring in the developing central nervous system 49. An implication of this study is that two neuron cell types, hippocampal neurons in the central nervous system, and D R G neurons in the peripheral nervous system, have abnormal action potential parameters, but that the abnormalities differ. The neurophysiological defects in Down's syndrome thus might be multiple and neuron specific. The abnormality in the hippocampus may be relevant to the participation of this region in learning and memory 13,37. Acknowledgments. We
thank A n d r e a Balbo for help in the maintenance of the cell cultures, Dr. Dan Brady for photographic assis-
C control-peak trisomy-peak
0
200 0
control-stst
0
trisomy-stst
100
•
0
IJ_ O Q. C
O
--
-20
40
[]
u
• o
2'0
"00J
o
Membrane Potential [ mV ]
'
6'0
Fig 6. Comparison of voltage-clamp responses to depolarizing steps from control (A) and trisomy 16 (B) hippocampal neurons. In both cases currents were generated by steps to - 40, - 20, 10, 30 m V from a holding potential of - 6 0 mV. A more complete series of records obtained from the same cells was used to plot the data in C, showing current-voltage relations for the peak inward (squares) and the steady state components (circles). The open symbols represent the control and the filled symbols represent the trisomy 16 neuron. The amplitude of the inward current component in the trisomy 16 neuron was 30% smaller than in the control cell. The ratio Gin~Gout, between the maximum slope conductances of the inward and outward components, was 0.66 in the control and 0.41 in the trisomy neuron. M e m b r a n e capacitance and m e m b r a n e resistance were 23 pF and 340 M~Q for the control and 21 pF and 280 MJ~ for the trisomy 16 neuron.
77 tance, Dr. Jim Stoll for providing us with the karyotyping procedure and discussions, and Dr. Franco Conti for valuable suggestions and discussions and for reading the manuscript. REFERENCES 1 Ault, B., Caviedes, P., Hidalgo, J., Epstein, C.J. and Rapoport, S.I., Electrophysiological analysis of cultured fetal mouse dorsal root ganglion neurons transgenic for human superoxide dismutase-l., a gene in the Down's syndrome region of chromosome 21, Brain Res., 497 (1989) 191-194. 2 Ault, B., Caviedes, P. and Rapoport, S.I., Neurophysiological abnormalities in cultured dorsal root ganglion neurons from the trisomy-16 mouse fetus, a model for Down's syndrome, Brain Res., 485 (1989) 165-170. 3 Banker, G.A. and Cowan, W.M., Rat hippocampal neurons in dispersed cell culture, Brain Res., 126 (1977) 397-325. 4 Berger, C.N. and Epstein, C.J., Genomic imprinting: normal complementation of murine chromosome 16, Genet. Res. Camb., 54 (1989) 227-230. 5 Bernardo, L.S., Masukawa, L.M. and Prince, D.A., Electrophysiology of isolated hippocampal dendrites, J. Neurosci., 2 (1982) 1614-1622. 6 Bezanilla, F. and Armstrong, C.M., Inactivation of the sodium channel. I. Sodium current experiments, J. Gen. Physiol., 70 (1977) 549-566. 7 Brown, T.H., Fricke, R.A. and Perkel, D.H., Passive electrical constants in three classes of hippocampal neurons, J. Neurophysiol., 46 (1981) 812-827. 8 Brown, T.H., Kairiss, E.W. and Keenan, C.L., Hebbian synapses: biophysical mechanisms and algorithms, Annu. Rev. Neurosci., 13 (1990) 475-511. 9 Chalazonitis, A., Peterson, E.R. and Crain, S.M., Nerve growth factor regulates the action potential duration of mature sensory neurons, Proc. Natl. Acad. Sci. USA, 84 (1987) 289-293. 10 Caviedes, P., Ault, B. and Rapoport, S.I., Electrical membrane properties of cultured dorsal root ganglion neurons from trisomy 19 mouse fetuses: a comparison with the trisomy 16 mouse fetus, a model for Down syndrome, Brain Res., 511 (1990) 169-172. 11 Caviedes, P., Koistinaho, J., Ault, B. and Rapoport, S.I., Effects of nerve growth factor on electrical membrane properties of cultured dorsal root ganglia neurons from normal and trisomy 21 human fetuses, Brain Res., 556 (1991) 285-291. 12 Coan, E.J., Galdzicki, Z. and Rapoport, S.I., Action potentials in cultured fetal hippocampal neurons from mouse trisomy 16, a model of Down's Syndrome, Soc. Neurosci. Abstr., 17 (1991) 1130. 13 Collingridge, G.L. and Davies, S.N., NMDA receptors and longterm potentiation in the hippocampus. In J.C. Watkins and G.L. Collingridge (Eds.), The NMDA Receptor, Oxford University Press, Oxford, 1989, pp. 123-135. 14 Cox, D.R., Epstein, L.B. and Epstein, C.J., Genes coding for sensitivity to interferon (IFREC) and soluble superoxide dismutase (SOD-l) are linked in mouse and man and map to mouse chromosome 16, Proe. Natl. Acad. Sci. USA, 77 (1980) 2168-2172. 15 Crowe, R., Ozer, H. and Rifkin, D., Experiments with Normal and Transformed Cells, Cold Spring Harbor, New York, 1978, pp. 96. 16 Epstein, C.J., The consequences of chromosome imbalance, Am. J. Med. Genet. Suppl., 7 (1990) 31-37. 17 Epstein, C.J., Berger, C.N., Carlson, E.J., Chan, P.H. and Huang, T.T., Models for Down's syndrome: chromosome 21-specific genes in mice, Prog. Clin. Biol. Res., 360 (1990) 215-232. 18 Epstein, C.J., Korenberg, J.R., Anneren, G., Antonarakis, S.E., Ayme, S., Courchesne, E., Epstein, L.B., Fowler, A., Groner, Y., Huret, J.L., Kemper, T.L., Lott, I.T., Lubin, B.H., Magenis, E., Opitz, J.M., Patterson, D., Priest, J.H., Pueschel, S.M., Rapoport, S.I., Sinet, P-M., Tanzi, R.E. and de la Cruz, F., Protocols to establish genotype-phenotype correlations in Down's syndrome, Am. J. Hum. Genet., 49 (1991) 207-235. 19 Finch, D.M., Fisher, R.S. and Jackson, M.B., Miniature excitatory synaptic currents in cultured hippocampal neurons, Brain Res., 518 (1990) 257-268.
20 Forsythe, I.D. and Westbrook, G.L., Slow excitatory postsynaptic currents mediated by N-methyl-D-aspartate receptors on cultured mouse central neurons, J. Physiol., 396 (1988) 515-533. 21 French, C.R. and Gage, P.W., A threshold sodium current in pyramidal cells in rat hippocampus, Neurosci. Lett., 56 (1985) 289-293. 22 Glantz, S.A., Primer to biostatistics (2nd edn.), McGraw-Hill, New York, 1987. 23 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F., Improved patch clamp techniques for high-resolution current recordings from cell and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 24 Hodgkin, A.L. and Huxley, A.F., A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol., 117 (1952) 500-544. 25 Hounsgaard, J. and Midtgaard, J., Dendrite processing in more ways than one, Trends Neurosci., 12 (1989) 313-315. 26 Huguenard, J.R., Hamill, O.P. and Prince, D.A., Sodium channels in dendrites of rat cortical pyramidal neurons, Proc. Natl. Acad. Sci. USA, 86 (1989) 2473-2477. 27 Johansson, S. and ,~rhem, P., Graded action potentials in small cultured hippocampal neurones, Neurosci. Len., 118 (1990) 155158. 28 Kay, A.R., Wong, R.K.S., Calcium currents activation kinetics in isolated pyramidal neurons of the CA1 region of the mature guinea-pig hippocampus, J. Physiol., 392 (1987) 603-616. 29 Kiss, J., Schlumpf, M. and Balazs, R., Selective retardation of the development of the basal forebrain cholinergic and pontine catecholaminergic nuclei in the brain of trisomy 16 mouse, an animal model of Down's syndrome, Dev. Brain Res. 50 (1989) 251-264. 30 Koch, C. and Poggio, T., Electrical properties of dendritic spines, Trends Neurosci., 3 (1983) 80-83. 31 Korenberg, J.R., Kawashima, H., Pulst, S.M., Allen, L., Magenis, E. and Epstein, C.J., Down syndrome: toward a molecular definition of the phenotype, Am. J. Med. Genet. Suppl., 7 (1990) 91-97. 32 Kumar, A., Schapiro, M.B., Grady, C.L., Matocha, M.F., Haxby, J.V., Moore, A.M., Luxenberg, J.S., St.George-Hyslop, P.H., Robinette, C.D. and Ball, M.J., Anatomic, metabolic, neurophysiological, and molecular genetic studies of three pair of identical twins discordant for dementia of the Alzheimer's type, Arch. Neurol., 48 (1991) 160-168. 33 Lejeune, J., Turpin, R. and Gautier, M., Le mogolisme, premier exemple d'aberration autosomique humaine, Ann. Genet., 1 (1959) 41-49. 34 Llinas, R.R., The intrinsic electrophysiological properties of mammalian neurons: insight into central nervous system function, Science, 242 (1988) 1654-1664. 35 Matsuda, S., Saito, H. and Nishiyama, N., Effect of basic fibroblast growth factor on neurones cultured from various regions of postnatal rat brain, Brain Res., 520 (1990) 310-316. 36 Miyabara, S., Gropp, A. and Winking, H., Trisomy 16 in the mouse fetus associated with generalized edema and cardiovascular and urinary tract anomalies, Teratology, 25 (1982) 369-380. 37 Morris, R.G.M., Davis, S., Butcher, S.P., The role of NMDA receptors in learning and memory. In J.C. Watkins and G.L. Collingridge (Eds.), The NMDA Receptor, Oxford University Press, Oxford, 1989, pp. 137-151. 38 Nieminen, K., Suarez-Isla, B.A. and Rapoport, S.I., Electrical properties of cultured dorsal root ganglion neurons from normal and trisomy 21 human fetal tissue, Brain Res., 474 (1988) 246-254. 39 Numann, R.E., Wadman, W.J. and Wong, R.K.S., Outward currents of single hippocampal cells obtained from the adult guineapig, J. Physiol., 393 (1987) 331-353. 40 Orozco, C.B., Smith, S.A., Epstein, C.J. and Rapoport, S.I., Electrophysiologica[ properties of cultured dorsal root ganglion and spinal cord neurons of normal and trisomy 16 fetal mice, Dev. Brain Res., 32 (1987) 111-122. 41 Orozco, C.B., Epstein, C.J. and Rapoport, S.I., Voltage-activated sodium conductances in cultured normal and trisomy 16 dorsal root ganglion neurons from the fetal mouse, Dev. Brain Res., 38 (1988) 265-274. 42 Oster-Granite, M.L. and Hzidimitriou, G., Development of the
78
43
44
45
46
hippocampal formation in trisomy 16 mice, Pediatr. Res. 19, (1985) 328A. Pash, J., Popescu, N., Matocha, M., Rapoport, S.I. and Bustin, M., Chromosomal protein HMG-14 gene maps to the Down's syndrome region of human chromosome 21 and is overexpressed in mouse trisomy 16, Proc. Natl. Acad. Sci. USA, 87 (1990) 3836-3840. Reeves, R.H., Gearhart, J.D. and Littlefield, J.W. Genetics basis for a mouse model of Down Syndrome, Brain Res. Bull., 16 (1986) 803-814. Reeves, R.H., Crowley, M.R., Lorenzon, N., Pavan, W.J., Smeyne, R.J. and Goldowitz, D., The mouse neurological mutant weaver maps within the region of chromosome 16 that is homologous to human chromosome 21, Genomics, 5 (1989) 522-526. Sah, P., Gibb, A.J. and Gage, P.W., The sodium current underlying action potentials in guinea pig hippocampal CA1 neurons, J. Gen. Physiol., 91 (1988) 373-398.
47 Spencer, W.A. and Kandel, E.R., Electrophysiolgy of hippocampal neurons II and IV, J. Neurophysiol., 24 (1961) 260-285. 48 Storm, J.F., Potassium currents in hippocampal pyramidal cells, Progr. Brain Res., 83 (1990) 161-187. 49 Suetsugu, M. and Mehraein, P., Spine distribution along the apical dendrites of the pyramidal neurons in Down's syndrome, Acta Neuropathol., 50 (1980) 207-210. 50 Sweeney, J.E., H6hmann, C.F., Oster-Granite, M.L. and Coyle, J.T., Neurogenesis of the basal forebrain in euploid and trisomy 16 mice: an animal model for developmental disorders in Down's syndrome, Neuroscience, 31 (1989) 413-425. 51 Wisniewski, K.E., Wisniewski, H.M. and Wen, G.Y., Occurrence of neuropathological changes and dementia of Alzheimer's disease in Down's syndrome, Ann. Neurol., 17 (1985) 278-282.
69
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 18530
Cultured hippocampal neurons from trisomy 16 mouse, a model for Down's syndrome, have an abnormal action potential due to a reduced inward sodium current Z. Galdzicki,
E. Coan
and S.I. Rapoport
Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD (USA)
(Accepted 8 September 1992)
Key words: Trisomy 16; Down's syndrome; Action potential; Current; Hippocampus; Primary culture; Mouse; Patch-clamp
Mouse trisomy 16 is an animal model for Down's syndrome (human trisomy 21). The whole-cell patch-clamp technique was used to compare passive and active electrical properties of trisomy 16 and diploid mouse 16 fetal hippocampal neurons maintained in culture for 2-5 weeks. There was no significant difference in any mean passive property, including resting potential, membrane resistance, capacitance and time constant. However, in trisomic neurons, the action potential had a 20% significantly slower rising phase and a 20% significantly smaller inward sodium current and inward sodium conductance than did control neurons. The outward conductance was not altered. The ratio of maximum inward conductance to maximum outward conductance was 30% less in the trisomy 16 cells. These results indicate that trisomy 16 hippocampal neurons have abnormal active electrical properties, most likely reflecting reduced sodium channel membrane density. Such subtle differences may influence elaboration of the hippocampus during development.
INTRODUCTION D o w n ' s s y n d r o m e (DS) is t h e result o f trisomy for c h r o m o s o m e 2133. D u p l i c a t i o n of g e n e d o s a g e o f t h e b a n d q22 r e g i o n o f this c h r o m o s o m e c o n t r i b u t e s to t h e D S p h e n o t y p e , which i n c l u d e s m e n t a l r e t a r d a t i o n 16. D S subjects also d e v e l o p t h e n e u r o p a t h o l o g i c a l a n d neurochemical defects of Alzheimer's disease after the age o f 35 y e a r s 51. It is n o t c l e a r if t h e s e a b n o r m a l i t i e s result f r o m o v e r - e x p r e s s i o n o f o n e specific g e n e on c h r o m o s o m e 21, o r of m a n y g e n e s on this chrom o s o m e 4'31. Clinical studies a r e u n d e r w a y to r e l a t e v a r i o u s a s p e c t s of the D S p h e n o t y p e to d u p l i c a t i o n of specific g e n e s o n d i f f e r e n t p a r t s of c h r o m o s o m e 2118. A n a n i m a l m o d e l w o u l d facilitate t h e s e studies. M u r i n e t r i s o m y 16 ( T s l 6 ) is c o n s i d e r e d such a m o d e l 17'29'5°, b e c a u s e m o u s e c h r o m o s o m e 16 is genetically h o m o l o g o u s to p a r t of t h e long a r m o f h u m a n c h r o m o s o m e 21. E a c h c h r o m o s o m e c o n t a i n s g e n e s for s u p e r o x i d e d i s m u t a s e ( S O D 1 ) , p r o t o - o n c o g e n e ETS2, /3-amyloid p r e c u r s o r p r o t e i n (APP), p h o s p h o r i b o s y l -
g l y c i n a m i d e s y n t h e t a s e ( P R G S ) , i n t e r f e r o n - a a n d -/3 cell surface r e c e p t o r s ( I F N R C ) , a n d c h r o m o s o m a l p r o tein HMG-1414'43 45. U n f o r t u n a t e l y trisomy 16 mice die in utero. T h e action p o t e n t i a l plays an essential role in comm u n i c a t i o n b e t w e e n n e u r o n s . D e p o l a r i z a t i o n o f presyn a p t i c n e u r o n a l m e m b r a n e s causes t h e r e l e a s e o f specific n e u r o t r a n s m i t t e r s a n d n e u r o m o d u l a t o r s . It has b e e n shown t h a t t h e d u r a t i o n o f t h e action p o t e n t i a l is s h o r t e r a n d its m a x i m u m r a t e s of d e p o l a r i z a t i o n a n d r e p o l a r i z a t i o n a r e l a r g e r in h u m a n trisomy 2138 a n d m u r i n e t r i s o m y 16 fetal d o r s a l r o o t g a n g l i a ( D R G ) n e u r o n s in c u l t u r e 2, as c o m p a r e d to c o n t r o l D R G n e u r o n s . T h e s e d i f f e r e n c e s a r e specific for h u m a n trisomy 21 a n d m o u s e trisomy 16, a n d do not occur in o t h e r h u m a n c h r o m o s o m a l a b n o r m a l i t i e s , such as K l i n e f e l t e r ' s , T u r n e r ' s or ' s u p e r f e m a l e ' s y n d r o m e s 38, in t r i s o m y 19 mice 1°, o r in s u p e r o x i d e d i s m u t a s e - 1 t r a n s g e n i c mice a. W e t h o u g h t it i m p o r t a n t to find o u t if t h e action p o t e n t i a l a n d its ionic c u r r e n t s a r e a b n o r m a l in cul-
Correspondence: Z. Galdzicki, Laboratory of Neurosciences, Building 10, Room 6C103, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892-010, USA. Fax: (1) (301) 402 0074.
70
tured non-DRG Hippocampal
n e u r o n s f r o m t h e t r i s o m y 16 m o u s e .
neurons were chosen for the study, be-
cause the hippocampus neous population
provides a relatively homoge-
of central nervous system neurons
with well-characterized properties,
and
because
the
h i p p o c a m p u s is p a r t i c u l a r l y v u l n e r a b l e t o A l z h e i m e r ' s d i s e a s e n e u r o p a t h o l o g y in a d u l t s w i t h O S 32. F u r t h e r m o r e , m o r p h o l o g i c a l d i f f e r e n c e s in t h e h i p p o c a m p a l f o r m a t i o n h a v e b e e n r e p o r t e d b e t w e e n t r i s o m y 16 a n d c o n t r o l m o u s e f e t u s e s a t e m b r y o n i c d a y 18 42 . S o m e o f t h e s e r e s u l t s h a v e b e e n p r e s e n t e d in a b s t r a c t f o r m t2. MATERIALS AND METHODS
Animals Trisomy 16 (Tsl6) and normal mouse fetuses were obtained by breeding males, doubly heterozygous for Robertsonian translocations of chromosome 16 (Rb(16.17)32Lub/Rb(ll.16)2H), with normal C57BL/6 females. After 15-16 days of gestation, "the fetuses were removed. Trisomy 16 fetuses were identified by their characteristic anasarca and retarded development36. To check the accuracy of the identification, chromosome spreads were prepared by amodification of the procedure of Crowe et al. 15.
A
Chromosome analysis Individual fetal livers were collected in dissection medium (calcium- and magnesium- free Puck's saline solution), minced with a sterile scalpel, and then incubated in a 0.25% (w/v) trypsin solution for 10 min at 37°C. Pieces of tissue were allowed to settle out. Individual cells which remained in suspension were collected by aspiration. These pieces of tissue were digested again and the single cell suspension was collected and added to the previous suspension. Cell suspensions were centrifuged for 5 rain at 460 X g and the cell pellet was resuspended in 10 ml Dulbecco's minimal essential medium (DMEM) supplemented by 10% fetal calf serum (v/v) and 0.1 ml 10 /zg/ml colcemid (Sigma, St. Louis, MO). Following incubation in Petri dishes for 90-120 rain at 37°C, the cells were harvested, collected by centrifugation and resuspended in 75 mM KCI at 37°C. After 15-20 min at 37°C, the cells were re-collected and resuspended in the residual volume of KC1 and fixed by incubation in a solution of methanol and glacial acetic acid (3:1) (v/v). This step was repeated twice. The fixed cells were dropped onto inclined slides and the chromosomes were stained with Giemsa solution. A magnification of x 400 was used to examine the spreads. Cell culture preparations Brains were removed from trisomy 16 and littermate control fetuses at day E15-E17 and hippocampal tissue was identified and dissected free as described by Banker and Cowan 3. This tissue was then cut into small pieces, transferred to a solution containing 0.05% trypsin (w/v), and incubated at 37°C for 15 min. The tissue was dissociated into a cell suspension by trituration with a fire-polished
B
J
Fig. 1. Metaphase chromosome spreads of liver cells stained with Giemsa solution from 16-day-old mouse embryos. Control cells have 39 separate chromosomes including one which is metacentric (A, arrow), giving a total of 40 chromosome arms. Trisomy 16 cells have 39 separate chromosomes including two metacentric chromosomes (B, arrows), giving a total of 41 chromosome arms.
71 Pasteur pipette in plating medium consisting of minimal essential m e d i u m (MEM), 10% fetal calf serum, 5% horse serum and 1% glutamine (all v/v). Neurons were maintained in low density culture 3 by plating t h e m on a confluent feeder layer of glial cells of the same ploidy type prepared 3 - 4 weeks previously. Dispersed cells survived for 5 or more weeks. After 24 h, and then every 3 - 4 days, m e d i u m was replaced with MEM, 5% horse serum and 1% glutamine (all v/v). Cytosine/3-D-arabinofuranoside (araC, 10/xM) was added as necessary to reduce growth of non-neuronal cells 2°. Cell diameter was m e a s u r e d with an eye-piece graticule.
Electrophysiology Electrophysiological recordings were performed at room temperature (22°C) on isolated neurons which had been cultured for at least 14 days. The culture medium in dishes was replaced by a solution containing (in mM): NaC1 140; KCI 5; CaC12 1; MgC12 2; dextrose 10; H E P E S / N a O H 10, pH 7.4. Currents and action potentials were recorded in a whole-cell configuration using a patch-clamp techrrique 23. Electrodes were pulled from borosilicate glass and backfilled with 'intracellular' solution containing (in mM): KCI 130; NaC1 3; CaC12 1; MgC12 2; E G T A 10; H E P E S / K O H 10; pH 7.4. T h e pipette resistance usually was less than 2 - 3 MgL Current and voltage recording and stimulation were controlled by a Labmaster board using the pClamp program (Axon Instruments, Burlingame, CA). Currents were filtered at 5 kHz (8-pole Bessel) and digitized at 50 kHz.
The resting potential of a cell was measured at the beginning of each recording and at intervals throughout an experiment to assess cell viability. A 5 m V hyperpolarizing step from a holding potential of - 6 0 mV was applied to estimate m e m b r a n e capacitance and resistance. Capacitance was calculated as the integral of the current response between the onset of the pulse and 5-fold of its initial decay time. A steady-state current level was used to calculate m e m b r a n e resistance. Action potentials were evoked in current-clamp mode by 5 ms depolarizing current steps of 0.1-0.8 nA. Analysis of voltage responses under these conditions was as described by Ault et al. 2. In voltage-clamp experiments, total current was recorded using 12 depolarization pulses of 10 ms duration, in 10 mV steps from a holding potential of - 6 0 mV. A typical current sweep included outward and inward components. Ionic currents were corrected on-line for linear leakage and capacitive currents using a P / 8 method 6, setting the control voltage of the P / 8 pulses to - 90 inV. A peak inward current, Ip, was estimated from a least-squares fit to experimental data in a short interval around the peak, using a third order polynomial. The same fitting procedure to the late component was used to estimate its steady-state value, /stst. From the peak current vs. m e m b r a n e potential, Vm, relation (Ip-Vm), the m a x i m u m peak inward current, i~nax, and the potential, Vpmax, that yielded i~nax, were estimated. Linear fitting of the first points after the peak value gave the m a x i m u m inward conductance, Gin, and then the m a x i m u m outward conductance, Gout , was defined as the m a x i m u m slope of the IStSt-gm relation. The ratio G i n / G o u t was calculated as a p a r a m e t e r independent of m e m b r a n e surface area. Likewise all current values were divided by m e m b r a n e capacitance (C).
Fig. 2. Phase contrast micrographs of two hippocampal neurons after 3 weeks in culture. A: control. B: trisomy 16. No morphological difference could be distinguished between the two neuron types at any stage in culture. Cells were triangular or had an oval/pear-like shape. Confluent glial cells are in the background. Bar = 5/xm.
72
-
-
CONTROL
A [SOMY
o 2 ms
.~1~
CONTROL
B TRISOMY
mosome arms are fused at their centromeres and appear metacentric. Cells from the diploid embryos had one Robertsonian chromosome, either the 11.16 or the 16.17 Robertsonian, giving 39 separate chromosomes with a normal complement of 40 chromosome arms (Fig. 1A). Ceils from trisomic embryos had one 11.16 Robertsonian and one 16.17 Robertsonian, and thus had 39 separate chromosomes but 41 chromosome arms (Fig. 1B). The control spread presented in Fig. 1A shows 39 chromosomes including the one Robertsonian translocation chromosome. The trisomy 16 cell in Fig. 1B shows 39 chromosomes including two Robertsonian translocation chromosomes. This indicates that the cell is aneuploid. We investigated 25 different fetuses that have been identified as trisomic according to phenotype. Twenty four of these were confirmed to have 39 separate chromosomes including two Robertsonian translocations (4% error), consistent with the report of Miyabara et al. 36. Therefore determination of a trisomy by its phenotypic characteristics was highly accurate.
2 ms
Cell culture
Fig. 3. A: comparison of action potentials obtained under currentclamp conditions from trisomy 16 and control hippocampal neurons after 25 days in culture. The holding potential was - 60 mV for both cells; the depolarizing current, Istim, was 0.3 hA. M e m b r a n e capacitance and m e m b r a n e resistance were, respectively, 22 pF and 270 MS2 for the control, 21 pF and 250 MS2 for the trisomy 16 neuron. B: comparison of the time derivative of the action potentials illustrated in A after subtraction of the contribution ( - Istim/C) due to the stimulating current. The slower rising phase of the action potential in the trisomic neuron is apparent only when looking at the derivative course in B.
Fig. 2 illustrates control diploid and trisomy 16 neurons which were used for recording. There was no difference in the size of the two cell types at any time. One day after plating minor processes were observed. After 4 - 5 days some cells created small networks, whereas others remained isolated. At the time of recording (between 2 - 4 weeks), there was approximately a 3-fold increase in the surface area of the cell body and increases in the length and thickness of the dendritic processes. Cell diameter was between 4 and 7 /xm.
Passiue electrical properties Differences between trisomy 16 and control neurons were evaluated using a two-tailed Student's t-test and a repetitive m e a s u r e s analysis of variance 22. Significance was assigned for P < 0.05.
Table I summarizes m e a n passive m e m b r a n e properties of the cultured hippocampal neurons. Capacitance, m e m b r a n e resistance, m e m b r a n e time constant
RESULTS TABLE I
Examination of chromosome spreads Metaphase spreads of fetal liver cells were visualized by the procedure described in Materials and Methods as illustrated in Fig. 1. In a control diploid mouse there were 19 pairs of autosomes plus one pair of sex chromosomes to give a total number of 40 chromosome arms. Mus musculus chromosomes are acrocentric (i.e. the centromere is near the end of the chromosome), so that they appear as single-armed chromosomes with a 'hinge' between the arms. In a Robertsonian translocation, two non-homologous chro-
Mean passi~,e membrane properties of trisomy 16 and control hippocampal neurons in culture N u m b e r s are means_+S.E.M. No m e a n s differed significantly between cell type. Sampling time was 20 /zs, and analog filter 10 kHz. N u m b e r of cells are given in parentheses.
Parameter Capacitance (mV) M e m b r a n e resistance (Mg2) M e m b r a n e time constant (ms) Resting potential(mV) Days in culture
Control 27 440
+ 1 +30
Trisomy 16 (53) (53)
11.1 + 1.0 (53) - 4 2 _+ 1 (54) 22 + 1 (54)
25 480
+ 1 +40
(56) (56)
10.8+ 1.0 (56) - 4 1 + 1 (53) 21 + 1 (56)
73
8
H
c~ V
~ v
~z
74 a n d resting m e m b r a n e p o t e n t i a l w e r e analyzed. T h e r e was no significant d i f f e r e n c e b e t w e e n trisomy 16 a n d c o n t r o l n e u r o n s for any of t h e s e p a r a m e t e r s .
A
KCl in pipette
~
120
)
~
~ - ~ - NORMAL
A
100
T
>
E 6O
<
.J 40
V
CONTROL ~-
20
1
ms
TRISOMY
B CsCI in pipette ,
i
i
i
200
400
600
800
CURRENT
1000
(pA) f
200
~-
+ TEA
/
•
B 160 •
120 • I-
Fig 5. A: tetrodotoxin (TTX) blocks inward currents. These currents were generated by a depolarizing 90 mV pulse from a holding potential of - 7 0 mV. In the presence of 1 /xM TTX, the inward component disappeared. B: in a different neuron, replacing KCI with CsCI in the pipette solution and application of 20 mM TEA into the bath completely abolished the delayed outward current as compared to A, when the same depolarizing step was applied.
N n," O~
80"
I$
40
"---"0---
CONTROL
:$
ISOMY
x
0 0
i
i
i
i
200
400
600
800
CURRENT
1000
(pA)
0.
> C
-10" ,,~ Z O I,-.-
-20 ' OMY
N
n" n~)
-30 '
LIJ ¢r
;IE -40 x
-50
,
i
i
,
200
400
600
800
CURRENT
1000
(pA)
Fig. 4. Action potential parameters in control and trisomy 16 neurons, with increasing current stimuli in current-clamp conditions. The holding potential equaled - 60 mV. A: amplitude increased with stimulus size. There was no difference between contro! and trisomy 16 neurons. B: the maximum depolarization rate increased with stimulus size and was smaller in trisomic neurons. C: the maximum repolarization rate decreased with stimulus size and was larger in trisomic cells.
Action potentials A c t i o n p o t e n t i a l s c o u l d n o t b e e v o k e d o n e day a f t e r plating. A f t e r two w e e k s in culture, a l m o s t 95% o f c o n t r o l n e u r o n s a n d 80% of t r i s o m i c n e u r o n s w e r e a b l e to g e n e r a t e an action p o t e n t i a l . Fig. 3 illustrates typical action p o t e n t i a l s in c o n t r o l a n d t r i s o m y 16 h i p p o c a m p a l n e u r o n s , which h a d b e e n p l a t e d for 25 a n d 27 days, respectively. T h e m a x i m u m r a t e of d e p o larization, t i m e to m a x i m u m d e p o l a r i z a t i o n , m a x i m u m r e p o l a r i z a t i o n rate, t i m e to m a x i m u m r e p o l a r i z a t i o n , action p o t e n t i a l a m p l i t u d e a n d w i d t h at 50% a m p l i t u d e w e r e analyzed. R e s u l t s a r e shown in T a b l e II. With increasing current steps from a suprathreshold stimulus o f 200 p A to a m a x i m u m stimulus o f 800 p A , trisomy a n d c o n t r o l cells fired p r o g r e s s i v e l y e a r l i e r a n d t h e action p o t e n t i a l s w e r e g r a d e d ( T a b l e II). A c t i o n p o t e n t i a l a m p l i t u d e i n c r e a s e d by a b o u t 40% a n d t h e m a x i m u m d e p o l a r i z a t i o n a n d r e p o l a r i z a t i o n r a t e s inc r e a s e d by 30% (Fig. 4A,B). T h e t i m e to p e a k a m p l i t u d e a n d the t i m e to m a x i m u m d e p o l a r i z a t i o n r a t e d e c r e a s e d a b o u t 50%, t h e t i m e to m a x i m u m r e p o l a r ization r a t e d e c r e a s e d a l m o s t 40%, a n d t h e a c t i o n p o t e n t i a l w i d t h d e c r e a s e d by 2 0 - 3 0 % .
75 fetal hippocampal neurons, maintained in primary culture for 2-5 weeks. This is consistent with previous results for mouse trisomy 16 and human trisomy 21 D R G neurons 38'2'1°. Similar ranges of values for membrane resistance, capacitance, membrane time constant and resting potential have been reported for hippocampal neurons in primary and acute culture, slices, and in vivo from mice and other mammalian species19,27,28,39,46.47.
The one statistically significant difference ( P < 0.05) between trisomy 16 and control hippocampal neurons was in the mean maximum depolarization rate (Table II and Fig. 4B), which was 20% slower in trisomic cells (118 V / s ) than in diploid controls (148 V / s ) at a current of 800 pA. Total currents
Whole-cell currents were analyzed in 49 control neurons and 40 trisomic neurons (Table III). By far the most predominant components of voltage-clamp currents were attributable to tetrodotoxin (TTX)-sensitive sodium channels and to delayed rectifier potassium channels. The early component of the current was blocked in a partially reversible way by 1 / z M T T X (see Fig. 5) but was not significantly affected by 2 mM CoC12 or 2 mM CdC12 (e.g. ref. 46). Replacing KC1 with CsC1 in the recording electrode (see Fig. 5), and putting 20 mM tetraethylammonium (TEA) in the bath virtually abolished the outward current 4s. Fig. 6 shows whole-cell currents in a trisomic cell and a control cell, evoked by a series of 10 ms depolarizing pulses, from a holding potential - 6 0 mV to the levels indicated. The mean maximum peak inward current / F ax was significantly smaller in trisomic than for control neurons ( - 1 2 8 p A / p F to - 1 6 9 p A / p F , P < 0.05). Fitting of the linear part of the I p - V m plot provided an estimation of the TTX-sensitive maximum inward conductance, Gin. For trisomic neurons Gin was smaller than for control neurons (942 p S / p F to 1192 p S / p F , P < 0.05). Both of these values for our fetal neurons are about 10-times smaller than reported for acutely dissociated adult guinea pig hippocampal neurons 46. Consistently, the ratio, (maximum inward conductance)/(maximum outward conductance), was significantly ( P < 0.01) smaller in trisomy 16 than control cells (0.45 to 0.67).
The only statistically significant difference in the action potential between trisomy 16 and control cells was in the mean rate of depolarization, which was 20% less in the trisomic neurons. This result is consistent with voltage clamp data which indicate a significantly smaller (25%) inward sodium current in trisomy 16 than in control cells, as the depolarization rate is directly proportional to voltage-dependent sodium current density 24. A lower rate of depolarization in cultured hippocampal trisomy 16 neurons differs from findings in cultured murine trisomy 16 and human trisomy 21 D R G neurons. The maximum depolarization rate of the action potential in D R G neurons is about 30% larger than in controls for both trisomy 16 and 21, and the maximum repolarization rate is about 30% greater for trisomy 16 and 50% greater for trisomy 21, whereas spike duration is reduced by about 20% for trisomy 16 and by about 40% for trisomy 212'38'4°. The differences between D R G and hippocampal neurons may be due to cell-specific regulation of ion channel expression or function 41. In addition, it has been reported that nerve growth factor (NGF) can influence action potential characteristics in D R G neurons 9, so a differential sensitivity to regulating factors like N G F could account for the differences in action potential properties u. D R G neurons in primary culture initially require N G F for survival 9, whereas hippocampal neurons are N G F independent 35. Two different types of sodium current have been reported in adult hippocampal neurons: a fast sodium current with a half decay time of 2 m s 46, and a slowly
DISCUSSION There was no difference in any mean passive electrical property between mouse trisomy 16 and control T A B L E III Voltage-clamp data from trisomy 16 and control hippocampal neurons
Parameters were evaluated from I - V m relations. N u m b e r of cells are given in parentheses. Parameter
Control
Trisomy 16
Maximum outward conductance ( p S / p F ) M a x i m u m inward conductance ( p S / p F ) Ratio of conductances (Gin / G o u t ) Maximum peak of inward current I v ( p A / p F ) Potential at m a x i m u m peak of inward current Vpma× (mV)
2,074 ± 149 (47) 1,187 ± 77 (47) 0.67± 0.05 (47)
2,033 + 161 (38) 931 ± 99 * (39) 0.45± 0.03 ** (38) -129 ± 12 ** (40)
- 162
±
11
(49)
-27
±
2
(49)
* Significantly different at P < 0.05; ** Significantly different at P < 0.01.
- 23
+
2
(40)
76 inactivating sodium current 2~. The first determines excitability threshold, whereas the second acts as a pacemaker current for modulating firing frequency. The 30% reduction in sodium inward current in trisomy 16 hippocampal neurons could be due to lower single sodium channel conductance or to a reduced density of sodium channels. As trisomy 16 is due to excessive gene expression rather than mutated genes, a change in conductance of a single channel is less likely.
30 mV
A
/
///
10 mV
-20 mV -40 mV
\/
o 2 ms
30 mV
B
f _ ~
S
j.7 ~ /
f"
10 mV
-20 mV ~ J~ -
~
-40 mV
fs
%
2 ms
If channel density is reduced this could occur in soma or dendrites, or in both regions. A reduced somatic sodium channel density would affect the action potential amplitude. A reduction in dendritic channel density also could produce important physiological differences. Sodium channels in dendrites have been demonstrated indirectly in guinea pig hippocampal slices 5, and directly using patch-clamp whole-cell recordings in isolated rat cortical pyramidal neurons 26, where the amplitudes of the dendritic and somatic sodium currents were comparable. A reduced current due to reduced sodium channel density in the dendritic tree could influence coupling between dendritic excitation and somatic activation, especially during development 25,26,3°,34. It is difficult to directly relate abnormalities in the active electrical properties of mouse trisomy 16 hippocampal neurons to the pathophysiology of Down's syndrome, as our experiments were not performed using trisomy 21 tissue. However, if the similarities found between D R G human trisomy 21 and murine trisomy 16 could be extended to hippocampal tissue, trisomy 16 as a model of Down's syndrome would be further validated. As neural development is related to integrated electrical activity 8, abnormalities such as we found might affect differentiation and morphogenesis and ultimately network wiring in the developing central nervous system 49. An implication of this study is that two neuron cell types, hippocampal neurons in the central nervous system, and D R G neurons in the peripheral nervous system, have abnormal action potential parameters, but that the abnormalities differ. The neurophysiological defects in Down's syndrome thus might be multiple and neuron specific. The abnormality in the hippocampus may be relevant to the participation of this region in learning and memory 13,37. Acknowledgments. We
thank A n d r e a Balbo for help in the maintenance of the cell cultures, Dr. Dan Brady for photographic assis-
C control-peak trisomy-peak
0
200 0
control-stst
0
trisomy-stst
100
•
0
IJ_ O Q. C
O
--
-20
40
[]
u
• o
2'0
"00J
o
Membrane Potential [ mV ]
'
6'0
Fig 6. Comparison of voltage-clamp responses to depolarizing steps from control (A) and trisomy 16 (B) hippocampal neurons. In both cases currents were generated by steps to - 40, - 20, 10, 30 m V from a holding potential of - 6 0 mV. A more complete series of records obtained from the same cells was used to plot the data in C, showing current-voltage relations for the peak inward (squares) and the steady state components (circles). The open symbols represent the control and the filled symbols represent the trisomy 16 neuron. The amplitude of the inward current component in the trisomy 16 neuron was 30% smaller than in the control cell. The ratio Gin~Gout, between the maximum slope conductances of the inward and outward components, was 0.66 in the control and 0.41 in the trisomy neuron. M e m b r a n e capacitance and m e m b r a n e resistance were 23 pF and 340 M~Q for the control and 21 pF and 280 MJ~ for the trisomy 16 neuron.
77 tance, Dr. Jim Stoll for providing us with the karyotyping procedure and discussions, and Dr. Franco Conti for valuable suggestions and discussions and for reading the manuscript. REFERENCES 1 Ault, B., Caviedes, P., Hidalgo, J., Epstein, C.J. and Rapoport, S.I., Electrophysiological analysis of cultured fetal mouse dorsal root ganglion neurons transgenic for human superoxide dismutase-l., a gene in the Down's syndrome region of chromosome 21, Brain Res., 497 (1989) 191-194. 2 Ault, B., Caviedes, P. and Rapoport, S.I., Neurophysiological abnormalities in cultured dorsal root ganglion neurons from the trisomy-16 mouse fetus, a model for Down's syndrome, Brain Res., 485 (1989) 165-170. 3 Banker, G.A. and Cowan, W.M., Rat hippocampal neurons in dispersed cell culture, Brain Res., 126 (1977) 397-325. 4 Berger, C.N. and Epstein, C.J., Genomic imprinting: normal complementation of murine chromosome 16, Genet. Res. Camb., 54 (1989) 227-230. 5 Bernardo, L.S., Masukawa, L.M. and Prince, D.A., Electrophysiology of isolated hippocampal dendrites, J. Neurosci., 2 (1982) 1614-1622. 6 Bezanilla, F. and Armstrong, C.M., Inactivation of the sodium channel. I. Sodium current experiments, J. Gen. Physiol., 70 (1977) 549-566. 7 Brown, T.H., Fricke, R.A. and Perkel, D.H., Passive electrical constants in three classes of hippocampal neurons, J. Neurophysiol., 46 (1981) 812-827. 8 Brown, T.H., Kairiss, E.W. and Keenan, C.L., Hebbian synapses: biophysical mechanisms and algorithms, Annu. Rev. Neurosci., 13 (1990) 475-511. 9 Chalazonitis, A., Peterson, E.R. and Crain, S.M., Nerve growth factor regulates the action potential duration of mature sensory neurons, Proc. Natl. Acad. Sci. USA, 84 (1987) 289-293. 10 Caviedes, P., Ault, B. and Rapoport, S.I., Electrical membrane properties of cultured dorsal root ganglion neurons from trisomy 19 mouse fetuses: a comparison with the trisomy 16 mouse fetus, a model for Down syndrome, Brain Res., 511 (1990) 169-172. 11 Caviedes, P., Koistinaho, J., Ault, B. and Rapoport, S.I., Effects of nerve growth factor on electrical membrane properties of cultured dorsal root ganglia neurons from normal and trisomy 21 human fetuses, Brain Res., 556 (1991) 285-291. 12 Coan, E.J., Galdzicki, Z. and Rapoport, S.I., Action potentials in cultured fetal hippocampal neurons from mouse trisomy 16, a model of Down's Syndrome, Soc. Neurosci. Abstr., 17 (1991) 1130. 13 Collingridge, G.L. and Davies, S.N., NMDA receptors and longterm potentiation in the hippocampus. In J.C. Watkins and G.L. Collingridge (Eds.), The NMDA Receptor, Oxford University Press, Oxford, 1989, pp. 123-135. 14 Cox, D.R., Epstein, L.B. and Epstein, C.J., Genes coding for sensitivity to interferon (IFREC) and soluble superoxide dismutase (SOD-l) are linked in mouse and man and map to mouse chromosome 16, Proe. Natl. Acad. Sci. USA, 77 (1980) 2168-2172. 15 Crowe, R., Ozer, H. and Rifkin, D., Experiments with Normal and Transformed Cells, Cold Spring Harbor, New York, 1978, pp. 96. 16 Epstein, C.J., The consequences of chromosome imbalance, Am. J. Med. Genet. Suppl., 7 (1990) 31-37. 17 Epstein, C.J., Berger, C.N., Carlson, E.J., Chan, P.H. and Huang, T.T., Models for Down's syndrome: chromosome 21-specific genes in mice, Prog. Clin. Biol. Res., 360 (1990) 215-232. 18 Epstein, C.J., Korenberg, J.R., Anneren, G., Antonarakis, S.E., Ayme, S., Courchesne, E., Epstein, L.B., Fowler, A., Groner, Y., Huret, J.L., Kemper, T.L., Lott, I.T., Lubin, B.H., Magenis, E., Opitz, J.M., Patterson, D., Priest, J.H., Pueschel, S.M., Rapoport, S.I., Sinet, P-M., Tanzi, R.E. and de la Cruz, F., Protocols to establish genotype-phenotype correlations in Down's syndrome, Am. J. Hum. Genet., 49 (1991) 207-235. 19 Finch, D.M., Fisher, R.S. and Jackson, M.B., Miniature excitatory synaptic currents in cultured hippocampal neurons, Brain Res., 518 (1990) 257-268.
20 Forsythe, I.D. and Westbrook, G.L., Slow excitatory postsynaptic currents mediated by N-methyl-D-aspartate receptors on cultured mouse central neurons, J. Physiol., 396 (1988) 515-533. 21 French, C.R. and Gage, P.W., A threshold sodium current in pyramidal cells in rat hippocampus, Neurosci. Lett., 56 (1985) 289-293. 22 Glantz, S.A., Primer to biostatistics (2nd edn.), McGraw-Hill, New York, 1987. 23 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F., Improved patch clamp techniques for high-resolution current recordings from cell and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 24 Hodgkin, A.L. and Huxley, A.F., A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol., 117 (1952) 500-544. 25 Hounsgaard, J. and Midtgaard, J., Dendrite processing in more ways than one, Trends Neurosci., 12 (1989) 313-315. 26 Huguenard, J.R., Hamill, O.P. and Prince, D.A., Sodium channels in dendrites of rat cortical pyramidal neurons, Proc. Natl. Acad. Sci. USA, 86 (1989) 2473-2477. 27 Johansson, S. and ,~rhem, P., Graded action potentials in small cultured hippocampal neurones, Neurosci. Len., 118 (1990) 155158. 28 Kay, A.R., Wong, R.K.S., Calcium currents activation kinetics in isolated pyramidal neurons of the CA1 region of the mature guinea-pig hippocampus, J. Physiol., 392 (1987) 603-616. 29 Kiss, J., Schlumpf, M. and Balazs, R., Selective retardation of the development of the basal forebrain cholinergic and pontine catecholaminergic nuclei in the brain of trisomy 16 mouse, an animal model of Down's syndrome, Dev. Brain Res. 50 (1989) 251-264. 30 Koch, C. and Poggio, T., Electrical properties of dendritic spines, Trends Neurosci., 3 (1983) 80-83. 31 Korenberg, J.R., Kawashima, H., Pulst, S.M., Allen, L., Magenis, E. and Epstein, C.J., Down syndrome: toward a molecular definition of the phenotype, Am. J. Med. Genet. Suppl., 7 (1990) 91-97. 32 Kumar, A., Schapiro, M.B., Grady, C.L., Matocha, M.F., Haxby, J.V., Moore, A.M., Luxenberg, J.S., St.George-Hyslop, P.H., Robinette, C.D. and Ball, M.J., Anatomic, metabolic, neurophysiological, and molecular genetic studies of three pair of identical twins discordant for dementia of the Alzheimer's type, Arch. Neurol., 48 (1991) 160-168. 33 Lejeune, J., Turpin, R. and Gautier, M., Le mogolisme, premier exemple d'aberration autosomique humaine, Ann. Genet., 1 (1959) 41-49. 34 Llinas, R.R., The intrinsic electrophysiological properties of mammalian neurons: insight into central nervous system function, Science, 242 (1988) 1654-1664. 35 Matsuda, S., Saito, H. and Nishiyama, N., Effect of basic fibroblast growth factor on neurones cultured from various regions of postnatal rat brain, Brain Res., 520 (1990) 310-316. 36 Miyabara, S., Gropp, A. and Winking, H., Trisomy 16 in the mouse fetus associated with generalized edema and cardiovascular and urinary tract anomalies, Teratology, 25 (1982) 369-380. 37 Morris, R.G.M., Davis, S., Butcher, S.P., The role of NMDA receptors in learning and memory. In J.C. Watkins and G.L. Collingridge (Eds.), The NMDA Receptor, Oxford University Press, Oxford, 1989, pp. 137-151. 38 Nieminen, K., Suarez-Isla, B.A. and Rapoport, S.I., Electrical properties of cultured dorsal root ganglion neurons from normal and trisomy 21 human fetal tissue, Brain Res., 474 (1988) 246-254. 39 Numann, R.E., Wadman, W.J. and Wong, R.K.S., Outward currents of single hippocampal cells obtained from the adult guineapig, J. Physiol., 393 (1987) 331-353. 40 Orozco, C.B., Smith, S.A., Epstein, C.J. and Rapoport, S.I., Electrophysiologica[ properties of cultured dorsal root ganglion and spinal cord neurons of normal and trisomy 16 fetal mice, Dev. Brain Res., 32 (1987) 111-122. 41 Orozco, C.B., Epstein, C.J. and Rapoport, S.I., Voltage-activated sodium conductances in cultured normal and trisomy 16 dorsal root ganglion neurons from the fetal mouse, Dev. Brain Res., 38 (1988) 265-274. 42 Oster-Granite, M.L. and Hzidimitriou, G., Development of the
78
43
44
45
46
hippocampal formation in trisomy 16 mice, Pediatr. Res. 19, (1985) 328A. Pash, J., Popescu, N., Matocha, M., Rapoport, S.I. and Bustin, M., Chromosomal protein HMG-14 gene maps to the Down's syndrome region of human chromosome 21 and is overexpressed in mouse trisomy 16, Proc. Natl. Acad. Sci. USA, 87 (1990) 3836-3840. Reeves, R.H., Gearhart, J.D. and Littlefield, J.W. Genetics basis for a mouse model of Down Syndrome, Brain Res. Bull., 16 (1986) 803-814. Reeves, R.H., Crowley, M.R., Lorenzon, N., Pavan, W.J., Smeyne, R.J. and Goldowitz, D., The mouse neurological mutant weaver maps within the region of chromosome 16 that is homologous to human chromosome 21, Genomics, 5 (1989) 522-526. Sah, P., Gibb, A.J. and Gage, P.W., The sodium current underlying action potentials in guinea pig hippocampal CA1 neurons, J. Gen. Physiol., 91 (1988) 373-398.
47 Spencer, W.A. and Kandel, E.R., Electrophysiolgy of hippocampal neurons II and IV, J. Neurophysiol., 24 (1961) 260-285. 48 Storm, J.F., Potassium currents in hippocampal pyramidal cells, Progr. Brain Res., 83 (1990) 161-187. 49 Suetsugu, M. and Mehraein, P., Spine distribution along the apical dendrites of the pyramidal neurons in Down's syndrome, Acta Neuropathol., 50 (1980) 207-210. 50 Sweeney, J.E., H6hmann, C.F., Oster-Granite, M.L. and Coyle, J.T., Neurogenesis of the basal forebrain in euploid and trisomy 16 mice: an animal model for developmental disorders in Down's syndrome, Neuroscience, 31 (1989) 413-425. 51 Wisniewski, K.E., Wisniewski, H.M. and Wen, G.Y., Occurrence of neuropathological changes and dementia of Alzheimer's disease in Down's syndrome, Ann. Neurol., 17 (1985) 278-282.