In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line

DEVELOPMENTAL BRAIN

~ ELSEVIER

H

DevelopmentalBrain Research 84 (1995) 130-141

Research report

In vivo electrophysiological maturation of neurons derived from a multil~tent precursor (embryonal carcinoma) cell line D a v i d S . K . M a g n u s o n a , . , D a n t e J. M o r a s s u t t i b, W i l l i a m A . S t a i n e s c, M i c h a e l W . M c B u m e y Kenneth C. Marshall "

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• Deoartment of Physiology, Uni~sisy of Ottawa, Ottawa, Ont., KIH 8M5 Canada De~nmmt of Medicine, Universiayof Ottaw, Ottawa, Ont., KIH 8M5 Canada ©Department of Anatomy and Neumbiology, University of Ottawa, Ottawa, Ont., K1H 8M5 Canada

Accepted 13 September 1994

k The multipotent embryonal carcinoma (EC) P19 ~eil line differentiates into neurons, gila and smooth muscle following exposure to retinoic acid (RA). RA-induced differentiation is irreversible and the neurons that develop are abundant, post-mitotic, and survive for prolonged periods in culture or when grafted into the CNS of adult rats. Striatal slices containing arafted PI9 cells were studied with intraceilular recording and labelling techniques to examine the development of electrophysinand morphological properties of Pl9-derived neurons over a period of 6 to 120 days after grafting into ibotenic acid lesioned strlatum. Cells from 1-week-old grafts had a range of immature e l e c t r o p h y s ~ characteristics including unstable resting membrane potentials (RMP's) and very high membrane input resistances (Rin's). Many were not able to produce action potentials (AP's). In coatrast, the majority of cells wAx}friedfrom 2- and 3-week-old grafts had stable RMP's, moderate Rin's, and were able to produce regenerative AP's. In grafts over 4 weeks of ase, the majority of P19-derived neurons had mature neuronal elcctrophyslological characteristics including R.MP's of - 6 0 mV, Rin's of 100-300 M.O, and overshooting AP's. 1 ~ , P19 derived neurons increase in soma size from 12-15 p. in diameter in 7-14-day-old grafts, to 25-35 p. in diameter in grafts 50-120 days old. Developing neurons exhibited a variety of m o r p ~ with increasingly complex processes and lengths of process extension. Our results demonstrate a developmental progression of the e l e c ~ of Pig-derived neurons, culminating in mature characteristics ctor,ely resembling those of adult rodent hippocampal or conical pyramidal neurons. The ability to easily alter these cells genetically provides a powerful model for addressing issues specific to neuronal development. geywords: Precursor cell; Embryonal carcinoma; Neuronal differentiation; Elnctrophysiololly; Deveh3ptnent; Transplant

1. Introduction Our understanding of the development of neuronal eiectmphysiological properties is derived primarily from studies of cultured cells isolated from rodent and chick embryos. Studies on such model systems as motoneutons [25,26] mammalian spinal cord [33], cervical ganglion [27] and septal neurons [13], have demonstrated that acquisition of neuronal electrophysiological properties continues throughout the middle and late embry-

• Cor~ins author. Spinal Cord Research Centre, University of Manitoba, 770 Bannstyne Arc, Winnipeg Man., R3E 0W3, Canada. Fax: (I) (204) 786-0932. Email: ([email protected]}. 0165-3806/95/f,09..¢0 © 1995Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00166-9

ouic stages. In each of these studies, however, the most immature neurons studied were already, capable of producing regenerative action potentials. Hockberger et al. [11] and Yool et al. [53], followed the late developing rat cerebellar Purkinie cells from E20 when the Purkinje neurons were largely inexcitable. In these studies, the timed arrival of four different K + conductances, large drops in Rin and a gradual maturation of action potentials were observed from E20 to postnatal days 10 to ~4. In several recent studies, embryonal carcinoma (EC) cells have been used to investigate early embryonic neuronal developmental processes because, these cells appear to differentiate via a patterned sequence of events similar to that followed in the embryo. Using

D.S.K Magnuson et aL / Decelopmental Brain Research 84 (1995) 130-141

whole-cell p a t c h c l a m p tecnnique% Simonr+oau a n d c o l l e a g u e s [41,42] e x a m i n e d the m o u s e 1003 E C cell line a n d f o u n d t h a t the electrophysiological c h a r a c t e r istics o f n e u r o n s c o u l d be d e m o n s t r a t e d by 11 days post-differentiation. C h e u n a n d Y e h [5] a n d K u b o [141 d o c u m e n t e d t h e a p p e a r a n c e o f the various ionic cond u c t a n c e s r e s p o n s i b l e f o r n e u r o n a l activity d u r i n g the d i f f e r e n t i a t i o n a n d e a r l y d e v e l o p m e n t o f P19 a n d P C C 4 - A z a l - F E C A 2 cells. T h e s e tissue c u l t u r e studies w e r e limited to a 7 - 1 4 d a y p e r i o d largely b e c a u s e o f progressive cell loss [47,5], a n d fully m a t u r e n e u r o n a l e l e c t r o p h y s i o l o g i c a l c h a r a c t e r i s t i c s w e r e not d e m o n strated. B r a i n slice p r e p a r a t i o n s of g r a f t e d fetal tissue have b e e n u s e d to s t u d y f u n c t i o n a l h o s t - g r a f t interactions, as well as n e u r o n a l electrophysiological d e v e l o p m e n t [46,31]. W e r e a s o n e d t h a t b r a i n slice p r e p a r a t i o n s o f n e u r o n s derived f r o m g r a f t e d m u l t i p o t e n t i a l p r e c u r s o r cells m i g h t provide a useful m o d e l for s t u d y i n g their electrophysiologieal d e v e l o p m e n t a n d plasticity. F o r o u r studies we u s e d P19 E C cells, which are i m m o r t a l a n d g r o w exponentially. T h e s e cells a r e diploid [21] a n d c o n t r i b u t e to the f o r m a t i o n o f a norreal c h i m e r i c m o u s e following injection into the i n n e r cell m a s s o f a b l a s t u l a [36]. Following a t h r e e d a y t r e a t m e n t with 10 - 6 M retinoic acid ( R A ) , they differe n t i a t e into a p o p u l a t i o n o f cells consisting o f n e u r o n s [15,20,21], mieroglia [2], astroglia [20], o l i g o d e n d r o g l i a [19] a n d v a s c u l a r s m o o t h m u s c l e [38]. D i f f e r e n t i a t e d cells c e a s e t h e i r e x p o n e n t i a l g r o w t h , a n d r e t a i n t h e i r d i f f e r e n t i a t e d c h a r a c t e r indefinitely [201. T h e n e u r o n a l p o p u l a t i o n wht~..h d e v e l o p s in c u l t u r e following R A t r e a t m e n t h a s b e e n f o u n d to express a variety o f n e u r o t r a n s m i t t e r s i n c l u d i n g g l u t a m a t e [17] G A B A , s o m a t o statin, n e u r o p e p t i d e Y, e n k e p h a l i n a n d c a t e c h o l a m i n e s [45]. R A - t r e a t e d P 1 9 cells h a v e b e e n g r a f t e d into the a d u l t r a t b r a i n w h e r e they survive, d i f f e r e n t i a t e a n d c o n t i n u e to express f o r e i g n g e n e p r o d u c t s [30]. In the p r e s e n t study, w e u s e d i n t r a c e l l u l a r r e c o r d i n g a n d labelling t e c h n i q u e s to e x a m i n e the d i f f e r e n t i a t i o n a n d e l e c t r o p h y s i o l o g i c a l m a t u r a t i o n o f retinoic acid ( R A ) t r e a t e d P19 cell~ g r a f t e d into ibotenic acid lesioned s t r i a t a o f a d u l t rats. T h i s h a s p e r m i t t e d s t u d y o f the development of electrophysiological and morphological c h a r a c t e r i s t i c s o f these m u l t i p o t e n t cells over a n ext e n d e d time c o u r s e [18].

2. M a t e r i a l s a n d m e t h o d s 2.1. Grafting

PI9 cells were cultured as previously de~rihed [37]. The clone used for the present experiments carries and expresses the E. coli lacZ gene product (,0-gaD [30]. prior to grahing, cells were treated with 10-e M RA for 3 d a d then aggregated overnight. Between 35.0110and 84,000 viable cells were stereotaxically injected, together

with 2fl ~g tboteni¢ acid, into the right striata of 280--300 gm Sprague-Dawley rats 130].l l ~ c a ~ PI9 cells are of mote~ raisin, the rat hosts were treated with the hlnmttnos~g,ln~..a.ant C y d o a l ~ A (Sandimmune. Sandoz). Without i m m a ~ xenografted PI9 cells were seldom f o ~ d surviving in the host mote than 2 weeks. However. with adequate imanun~,,~n~6ioa Pl9-derived survived and thrived for up to 120 days. Neurons, identified by the neuron-specific nadear antigen NeuN [32]. coeld he obsem~d as early as 4 days following ~ t a f i o n (7 days post-RA). Differentiated gila ceils (astroglia) were also apparent as ear~ as 7 days post-gsafting (10 days Ix~t-RA) using antibodics against GFAP (Ilia| fibrillary acidic protein). ZZ Slice preparation Striatal slices were prepared from the right forebraius of grafted animals between 6 da~ and 120 days after grafting Experiments were not done using gral~ less than 6 da~ old because of d ~ in obtaining good quality slices with young, poorly integrated graft tissue. Animals were injected with kctamine and exposed to 1.5% halothane in 100% e a t e n for 3 rain prior to decapitation. The dorsal aspects of the slmll were carefully removed and the dura reflected. The entire forebrain restrai to the cerebellum was lifted out of the skull and Placed in 4"C artificial cerebtospinal fluid (ACSF) ".quilibrated with 95% 0 2 / 5 % CO2. After 30-60 s the tissue was transferred to a chilled piafform cool,red with moist f'~er paper and cut by hand to leave a block of tissue a m t e l y 4 nmm mick. This block contained the b~k of the strintun~ incinding the graft, along with parts of the neocortex, septal nucleus and h~/pothalamus. The tissue block was then fixed to the cutting stain of a Campden Instruments vilnoslice with cyanoacr,Aate, submerged in chilled and bubbled ACSF, and 375 ~m thick slices were prepared. One slice was then transferred by wide-mouthed pipette onto the taut nylon mesh support tnylon stocking) of a submcrslon-style slice c~,,~]:~:r ,,~a cc.ntinuously perfiLsed at 1.3-1.7 ml/min, with ACSF bubbled with 9 5 % 0 2 / 5 % CO2. The ACSF was composed of (raM):. NaCI 118, KCI 3.0, N a H 2 P O + I.O, I M I ~ 4 0.81, C.aCI2 2.5, g l ~ 10, and N a H C O s 24. The p H of the A C S F in the chamber was 7.3-7.4. and the A C S F resen'oh" and the slice chamber were kept at 31-33°C using a recitculatingwater bath. Gold electron n ~

grids were placed onto the slice, and 1-1.5 mm long pieces of platinum wir, were used to hold the grids and slices in place. 2.3. Intracellular recording When trans-illuminated, unlesioued striatum appeared as mottied translucent ~ with bundles of opaque fibres passing through. Regions of lesioned striatum were opaque, and areas of grafted tissue were uaifonnl? translucent, apart from an obviously rich vasculafization. Under visual control, glass intrace!lular dectrodes (2 M KAc, 1% bloojtin) with DC tip resistances between 120 and 200 MJ~ were Iov~ered into the central region of the graft, well away from the lesioned host tissue, lntracelluinr recordings were made using an Axoa Instruments Axoclarap 2A ~nplificr ~ PClagnp data acquisition and analysis software on a 80386 perstmal o0mputer. Spike analysis, avernging, cun/e fitting and capacitance subtraction were all performed using the standard ¢L-'lamp(version 5.5) routines. Recordings were simultaneously monitored on a Gonld digital oscilloscope and chart recorder and final hard copy was produced using a Hewlen Packard Lmerjet liP printer. Voltage-current records were made using 200 o, 5(]0 ms rectangular current imlscs, injected intracellularly at 0.5 or 0.33 Hz with the cells at their resting membrane potential. In cells unable to maintain a resting potential of - 50 mV or less, current was injected to stabilize the potential at about - 55 mY to allow a voltage-current record to be made.

D.$.i~ Maouaon et nL~DevelopmentalBrain Research84 (1995) 130-141 2.4. lmracellularlabelling Bioeytin (1%) was routinely included in the recording electrodes and was injected into cells following electrop~siologkal enminalion. Hyperpoterizing current pulses of 800 ms duration and 1-4 nA in amplitude were delivered at 1 l-lz for 2-8 min. Membrane potentials were monitored continuously during the ejection of biocytht, and the ejection process was halted immediately if the membrane potential, resistance or capacitance changed. Slices were carefully rcumved from the bath and placed immediately in femtive consisting of 4% paraformaldebyde and 0.2% saturated picrlc acid in 0.16 M mdima phosphate buffer at a pH of 6.9 for I h at room temperature. Sh~ceswere then tr-_nsferred to 100 mM Na-phesphatc buffer contalaial 10% sucrose and 0.02% sodium azide for a period of no less tlum 48 h. Frozen serial sections (14 pm) through the entire slice were cut on a ctymtat and thaw mounted onto gelatin coated slides. B i o t i n filled cell* were visualized in conjunction with ~-salnctmidime immunorcuctivity. Slides were incubated with prinuny antiserum against O-galactmidese (Cappel, rabbit anti ~-gal, 1:2000) in 10 mM phosphate buffered ~atline (PBS) containing 0.3% Triton X-t00 for 18 h at 4"C. Slides were then rinsed in PEtS and incubated in fluorescent labelled secunda~ antibody solutions (FITC-labelled donkey anti-rabbit, 1:20, Amemham) for 45 rain at 37~C. After a another rime in PBS, bioojfin was detected by incubation with streptavidin-labelled Tens red (Amer~Qm, l:100) for 1 h at 37"C. After a final rinse in PBS, slides were coverslipped usins a mounting medium of PIELScontaining phenylamincdiaminc (0.1 raM) and 90% Ilyceroi and examined usi~ a Zelss Axioplan miamaWe equipped with hoth sinille and dual filter sets for the vi*.ualizationof FITC and Texas red. Biocytin labelled profiles were traced from serial sections ruing • drawing tube ~md lO0×ebjective and the~ were then compiled by manually superimpmins consecutive tracinss to reconstruct iDdividualfilled neur~mr...

3. Results T h e grafts into ibotenic acid lesioned striata all showed good survival if cyclosporin t r e a t m e n t was maintained. They t ~ i c a l l y h a d a d i a m e t e r of 1 to 1.5 m m and were s u r r o u n d e d by m y e l i n a t e d fiber b u n d l e s d e ~ : e n d i n g from the cortex a n d by a rim of non-lesioned striatal neurons. LesSoning o f striatal tissue at the t h n e of P19 cell i m p l a n t a t i o n was found to provide lot l a r g e r grafts with g r e a t e r survival p o t e n t i a l [29]. T h e ibotenic acid a d m i n i s t e r e d at the t i m e o f implantation did not lesion the R A - t r e a t e d P19 cells as they d o not express the excitotoxic classes of g l u t a m a t e receptors at this early time [18]. F o r this study, 71 cells w e r e r e c o r d e d in 35 grafts r a n g i n g from 6 to 120 days post-grafting ( D I G ) . T h r e e criteria were used to e n s u r e t h a t recordings were derived from graft r a t h e r t h a n host neurons. First, from intracellular biocytin injections we w e r e able to recover a n d examine the m o ~ h o l o g y of m o r e t h a n 40 P l 9 - d e rived neurons. Fifteen of these n e u r o n s w e r e phot o g r a p h e d a n d reconstructed from 14 / t i n serial sections as de-~'ribed in the methods. In every case, light microscopic e~amination using epifluorescence of double labelled sections ( F I T C labelling of 18-gal a n d streptavidin-Texas red labelling of biocytin), revealed

that the biocytin filled cells had n e u r o n a l morphologies and were c o n t a i n e d well within the graft boundaries. Secondly, on occasion, we r e c o r d e d and filled P19 derived neurons which c o n t i n u e d to express the / a c Z g e n e product, /3.gal. Hence, double labelled neurons w e r e observed [30], providing unequivocal e , : i d c n ~ that the recorded n e u r o n was P19 derived. Not all P19-derived neurons c o n t i n u e d to express /3-gal because the Pgk-1 p r o m o t e r that drives the / a c Z g e n e is d o w n - r e g u l a t e d in this cell type [22]. Finally, the electrophysiological characteristics of the grafted n e u r o n s w e r e very different from those of the surviving host striatal cells lying outside the area of the ibotenic acid lesion. Both active a n d passive m e m b r a n e characteristics of host and graft n e u r o n s were so different that these two cell types could b e u n a m b i g u o u s l y distinguished by these criteria.

3.1. Properties o f early (6-14 D I G ) neurons R e c o r d s m a d e from n i n e t e e n cells 6 - 1 4 days postgrafting ( D I G ) displayed a large r a n g e of electrophysiological states. T h e four most ' i m m a t u r e ' cells w e r e not able to m a i n t a i n a n e u r o n a l resting m e m b r a n e potently! ~.RMP), h a d very high m e m b r a n e input resist a n c e s ( R i n ) of 600 to 900 M f i , a n d h a d nearly l i n e a r (ohmic) v o l t a g e - c u r r e n t relations (Fig. ! ~ ) Tile =even most ' m a t u r e ' 6 - 1 4 D P G cells w e r e able to m a i n t a i n R M P s of - 5 5 t c --68 mV, h a d R i n ' s of 200 to 300 M n , a n d d e p o l a r i z i n g c*drrent injection elicited regenerative action p o t e n t i a l s of 15-65 m V in a m p l i t u d e a n d 3 - 1 5 m s in duration. Fig. I D shows the current-voltage r e c o r d for the least ' m a t u r e ' o f these seven. T h e morphology o f o n e of the most ' m a t u r e ' from this g r o u p is d e p i c t e d in Fig. 6A. F e w of the electrophysiologically ' i m m a t u r e ' n e u r o n s r e c o r d e d from 6 - 1 4 D P G slices

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Fill. I. Voltege-cun'ent responses of PI9 derived cells 6-14 DPG. Pl9-derived Irafts were prepared for recurdinil between 6 and 14 days pmt-sraftins, Cells were maintained under current clamp and the traces show the voltase responses to intracellular injections of rectangular current pulses. The current steps were :1:0.02 nA in amplitude. The recordings in panels A to D are from cells with prollressivcly more mature properties (A) n - 4, (n) n - 3, (C) n - 3 and (D) n - 4 out of the total of 19 cells recorded which were less than 11 DPG. The remaining 5 cells were more mature than that represented by D., and 4 out of these 5 were recurded from 14-day-old Starts.

D.S.K, Magnuson et aL / Derelopmental Brain Research 84 (1995) 130-141

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Fig. 2. Voltage-sensitive channels involved in Pig-derived neuron action potentials. These three voltage--current ~ ~ .~.'".".".".o' mgi'~ same Pl9-derived 6 DPG cell. Rectangular current steps of ±0.04 nA were used and the resting membrane potential of the neuron was -67 inV. Panel A was recorded under control conditions, panel B in the presence of 500 aM "lq'X and panel C in the presence of 500 nM TTX in a supeffusate containing 10 mM Mgz+ and 0.2 mM Caz" were successfully recovered following biocytin injection. This may be partially due to their relatively small size and lack of processes, m a k i n g them more likely to be pulled out of the slice by the intracellular electrode following the recording session. The morphology of the neuron-like cells most commonly seen with ~-gal immunoreactivity at this stage is shown in Fig. 7A. Note that this cell has a less ' m a t u r e ' morphotype than the cell from the same graft depicted in Fig. 6A. The other eight cells from 6 - 1 4 D P G displayed a variety of voltage-sensitive non-linearities to current injection. O f this group, two cells displayed rectification when depolarized without any sign ot spiking activity (Fig. 1C), suggesting that the voltage-sensitive channels responsible for this type of rectification (outward) develop prior to the TTX_-sen~itive Na + channels or the Ca z+ channels which are involved in action potential generation. The r e m a i n i n g six cells displayed both rectification and some r u d i m e n t a r y spikes (Fig. IB). Six of the cells exhibiting i m m a t u r e spikes were recorded in the presence of 300 to 600 n m T T X followed by T T X in a low C a Z + / h i g h M g 2+ superfusate containing 0.2 m M Ca 2 + (rather t h a n 2.5 raM) and 10 m M Mg z+ (rather than 0.81 raM). For each ceil tested, only the combination of both the "lq'X and low C a 2 + / h i g h Mg :+ completely blocked spiking activity indicating that both the "FIX sensitive N a + c h a n n e l s and the Ca 2+ channels participate in spike g e n e r a t i o n and that both a p p e a r to develop .¢imultaneol,~ly (Fig 2). Table I Summary of electmphysiolosical data Feature Graft age (days) 6-14 RMP(mV) 33.74. 16.5(16) a Rin(M.q) 493.1+208.8(19) ~ APAmp.(mV) 24.8+ 17.6(12) APDur.(ms) 15.1 4- 6.8(12) a AHP(mV) 3,04- 3.9(10) = Tau(ms) 32.4± 12.6(16)

15-28 56.4-t- 15.2(15) 220.34-102.6(17) 41.04. 19.8(15) 5,04- 4.0(15) 12.74- 7.5(15) 19.7± 10.5(15)

Cells within the same graft often had different prop. erties suggesting that these neurons m a t u r e in an asynchronous fashion. In transplants at 6 days of age the majority of cells visualized with biocytin o r / ] - g a l - i m munoreactivity were spheroid or slightly oblong with the soma measuring 15 to 20 t i m in d i a m e t e r (Fig. 7A). P19 cells in aggregates at the time of implantation are typically 8 - 1 2 t~m in d i a m e t e r and are devoid of pro. cesses (data not shcwn). Roughly 20% of the cells had a single thick process 30 to 40 /~m in length and a smaller 3ubpoptdation had four to five fine processes. Dye coupling of cells was seen on six different occasions, which is consistent with reports that P19 derived neurons express gap junction genes in vitro [3]. In these experiments single intraccllular bioolfin injections labelled ~vo adjacent cells and two such pairs of cells were recovered and reconstructed (Fig. 6A). A l t h o u g h the recordings were continuously m o n i t o ~ d d u r i n g biocytin injection, n o other steps were t a k e n to prevent or discount dye-coupling by m e a n s o t h e r than g a p junctions. By the e n d of this period (15 days) neurons with numerous, lengthy dendritic processes were the most commonly observed ~-gal immunoreactive cells in the transplant. 3.2. Maturation o f neurons between 2 and 4 weeks

By 15-28 DPG, the range of both Rin and R M P had decreased and the m e a n s were 220.3 + 102.6 M/~

29-59 62.8 4- 6.7 (15) a 216.6:1:72..1 (14) ~ 50.7 ± 10.8 (15) 1.624- 0.51(15) a 17.7 4- 2.5 (15) = 26.1 ±18.1 (11)

Data given as mean 3: standard deviation (n).

Lb Indicates significant differences determined by the Student's unpaired t-test, (p < 0.05).

> 60 67.5 :t: 7.5 (16) 202.8 -t-68.4 (16) b 46.6 ± 5.8 (16) 1.5121: 0.42(16) b 19.8:1:4.2 (16) b 19.5 ± 8.1 (14)

Imst striatal 73.5:t: 7.3(7) 116 +28.8(7) b 51.2+ 4.8(7) 2.6+ 0.6(7) b 6.5-1- 2.2(7) b 15.2± 6.5(7)

D.S.IC Magnttvon et aL / De~lopmental Brain Research 84 (1995) 130-141

(+S.D.) and - 5 6 . 4 ± 15.2 mV (+S.D.) respectively. The mean spike amplitude and duration for cells in grafts of this age was 41.0 ± 19.8 m V and 5.0 ± 4 ms respectively compared to 25 mV and 15 ms for the younger time period. Each spike was followed by an after-hyperpolarization (mean 12.7 ± 7.5 mV in amplitude), a characteristic not seen consistently in cells from younger grafts (Table 1). Fig. 3 shows voltage/ current records and action potential shapes for cells 15 (A, D), 30(B, E) and 90 (C, 17) DPG. The trend towards lower Rin's, higher RMP's and more mature spike characteristics can be seen in these records and graphically in Fig. 4. The development of robust after-byperpolarizations (AHP's) can also be seen following each spike elicited from these Pl9-d-rived cells.

3.3.Maturationof neurons between.4 and 9 weeks By 29-59 D I G , the ranges of all passive and active electrophysiological characteristics had narrowed substantially. This is evident in Tab:e 1, which shows the m e a n s ± s t a n d a r d deviations for all recordings ineluded in the data. All of the measured electrophysiological characteristics approached levels characteristic of several classes of adult CNS neurons, with RMP's averaging - 62.4 + 6.7 mV, Rin's averaging 216.6 + 72.1 MC/, action potentials which overshoot 0 m V and

robust AHP's averaging 17.7 ± 2.5 mV in size. The subtle changes in action potential shape involved primarily the duration and amplitude of the AHP, with little apparent change in threshold or amplitude. The findings for all the cells included in the study are summarized graphically in Fig. 4. Between 6 and 20 DPG, cells were heterogeneous for all properties but by 30 D P G most cells had electrical properties similar to those of mature neurons. The strongest relationship between two active properties of these cells can be seen in the A I t P amplitude and spike duration of the cells recorded in 15-28 D P G grafts. Of the fifteen cells in this group, three had A H P ' s less thar~ 3 mV, a fourth less than 10 mV, while the remaining cells had AHP's between 13 and 22 mV. T h e action potentials of the 3 cells with small A H P ' s (Fig. 4B and C) were each more than 9 ms in duration, while the average for the entire age group was 5 ms. Clearly, a rapid arrival of the K + channels responsible for the A H P in these cells occurs between 15 and 20 days post-grafting. By 9 weeks the values of the electrophysiological properties discussed above had reached a plateau. Also by this time period, the dendritic arbors of /~-gal labelled neurons were more elaborate, and extensive dendritic fields with secondary and tertiary branching were often seen. It is evident that P19 derived neurons exhibit a wide range of distinct morphologies

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Fig- 3. Voltage/current traces and action potential shapes from 3 diffnrem PI9 derived cells from 15 (A,D), 30 (B,E) and 90 (C,F) days

post-grafting.Voltage/current traces were made usinllrectangularcurrent steps of 5:0.05 nA for panels A and B, and 0.I nA for panel C. These neurons had Rin's of appro0cimately330 MQ (A), 175 MA (13)and 150 MKJ(C). The action potentials shownwere elicited during depolarizing current steps. These recordingsckn~nstraic ilm ~velopmenUd maturation of the action potentials from Pl9-dcrived neurons over an ll-week periQd. The records shown in panels A and D, B and E, and C and F were representativeof grafts 15-28, 29-59 and > 60 DPG. We recorded 17,15 and 16 cells in each age gxqoeprespcct~..ely.

135

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Fig. 4. Graphs of the development of Rin, RMP, AP height, AP duratio• ar.d AHP vs. time (DPG). Measurements from all file celh included in the study are shown versus their graft age in days. In A and B, the passive membrane properties are shown, as Rin and RMP vetoer ~ I f t I l e in days. I• C, D, and E, the properties of the action potentials are shown as spike amplitude (C) and duration (D) and afLer-hYl~Wal-rizatioa amplitude (E), also vs. graft age in days. The development of the active electrophysiological I~q0erties of Plq-derived cells appeared to occur along a very similar schedule to the passive properties outli•ed above.

136

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F'qg.5. Comparison of graft and striatal host action potential and rectification properties. The voltage responses to inhered current (A) and the idmp~ot an action potential elicited during a depolarizing current step (B), are shown for an adult host striatal neuron recorded from the same afice as the 80-day-old graft that the Pl9-derived neuron in parts C and F of Fig. 3 was recorded from. The current-voltage relationships for both the~ neurons are shown, providing a direct comparison of the characteristics of a host and a grafted cell (C). based on cell body size, shape and dendritic characteristics (Fig. 6~ Fig. 713). During this time period, fine axon-like processes bearing varicosities were common. 3. 4. Late development o f inward rectification

O n e electrophysiological characteristic, inward rectification during hyperpolarizing current injection, was observed in seven of nine neurons recorded in grafts 50-80 DPG. This characteristic was observed in only three out of seventeen cells recorded in 15-28 D P G grafts. Non-linear voltage-current plots throu.y,hout the voltage range of - 7 0 to - 1 2 0 m V are commonplace

for mammalian central neurons and the increased conductance at this level of membr.~ne polarization is due to increased potassium ot sodium and potassium permeability. Fig. 3C shows an example of rectification in a c~ll recorded from a 90*day-old graft. Fig. 5 provides for comparison, the voltage-current relationship and action potential shape of a host striatal neuron located in the unlesioned area outside the graft containing the cell recorded in Fig. 3C. The Rin, time constant, and inward rectification are very different for these two typical examples (Figs. 3C and Fig. 5). The linear voltage-current plot of the striatal cell response shows no inward rectification over the voltage range tested, in

F'ql. 6. Drawings of biocytin-filled neurons recorded from within PI9 cell intrastriatal ~afts. Each cc:i is reconstructed from drawings of serial scctior.s through the graft. Panel A depicts a neuron(s) recorded in a 6-day..nld graft. Panels B and C depict nem'ons recorded in two different 29-day-old grafts. Panels D and E depict neuruns record-.d in grafts 50 and 61 days old respectively.

D . S . ~ Magnuson et td. / Derelopmental Brain Research 84 (1995) 130-141

137

[12] The othei two sttiatal neurons and all three of the septal neurons rectified strongly through the - 6 5 to - 8 0 mV range. The morphology of grafted cells, beyond 9 weeks, was characterized by a dramatic increase in the number of biocytin or/3-gal innnunoreactive dendrites observed, in addition, many cells showed distinct dendritic spines which were not noted in younger transplants (Fig. 6E, Fig. 7C). Fig. 6 shows five examples from the fifteen bioojtin filled cells which were s::ccessfully, ecovered and reconstructed. These drawings illustrate that RA treated P19 EC cells, once grafted into a host brain, develop varied but distinctly neuronal morphologies with cell body diameters in the region of 25-35 #.m and multiple processes many hundreds of ~.m in length.

4. Discussion

Fill. 7. Photomicrographsof ~-galactcsidase immunoreactiveneurons in Pl9 cell intrastriatal grafts at 6 days (A), 29 days (B) and 68 days (C) Imst-grafling.

contrast to the graft cell which rectifies relatively strongly at - 9 0 to - 1 0 0 inV. in addition, the amplitude and duration of the action potentials and afterhyperpolarizations are dissimilar. A total of three host septal neurons and seven host striatal neurons were recorded. Five of the striatal neurons had linear current-voltage plots through the range - 65 to - 80 mV, and other electrophysiological characteristics, including Rin, RMP and action potential shape similar to those reported previously for large aspiny striatal neurons

Using intracellular recording and labelling techniques we have determined that RA-treated P19 cells grafted into ibotenic acid lesioned striata of adult rats differentiate into neurons which develop mature electrophysiological and morphological characteristics. During the first 4 weeks following RA-induced differentiation, Pl9-derived neurons mature from an undifferentiated electrophysiological state to a state consisting of stable and mature neuronal electrophysiological charactecistics. The changes observed ace similar to those reported for neurons from fetal and early postnatal mammalian nervous systems suggesting that differentiated EC cells provide a useful model of neuronal development. A number of P19 cells recorded in grafts less than 2 weeks old could not maintain a resting membrane potential once impaled, and showed no signs of voltage-dependent changes in membrane resistance. Even when manually clamped at - 60 mV and then depolarized via current injection, no spikes of any size were observed. Although this could be due to current shunt* ing around the intracellular electrode, this seems unlikely in light of their high membrane input resistances (see following paragraph). A few of these neurons did, however, show outward rectification during the depolarizing pulses suggesting that some voltage-dependent channels had developed. It is likely that the resistance decrease seen during depolarizing current steps is due to K + channel activation, and this is supported by the persistence of the decrease during superfusion with TI'X in a high Mg2+/low Ca 2+ solution, it appears, therefore, that these Pl9-derived neurons develop the K + channels which participate in the outward rectification described above before developing the ability to maintain a neuronal resting potential or to produce even rudimentary spikes.

D.S.I~ Magnuson et al. /Developmental Brain Research 84 (1995) 130-141

The very high membrane input resistances (Rin's) of 6-14 DPG neurons is probably due to a paucity of membrane ion channels. The graphs in Fig. 4 show that the passive membrane properties of these neurons develop together over a period of 12-20 days following retinoic acid treatme.n_t. It is plausible that the decrease in Rin with age is a direct result of the number of membrane ion channels necessary to achieve a normal neuronal resting membrane potential. The ability to maintain a RMP develops concomitantly with the drop in Rin. Similar findings have been reported for embry. ouic rat motoneurons by Ziskind-Couhaim [55] and neonatal rat cortical pyramidal neurons by McCormick and Prince [24]. Embryonic motoneurons were found to develop both a greater RMP ( - 5 2 to - 6 3 mV) and a lower Rin (271 to 65 M/I) over the period from E14/15 to postnatal day 0 [55]. Similarly, McCormick and Prince demonstrated that cortical pyramidal neurom have decreasing Rin's (from 125-150 M/I to less than 50 M/I) over the time period from P0 to P30 [24]. Similarly, neurons of the cat caudate nucleus develop more negative RMP's and lower Rin's over the period of fetal day 56 to postnatal day 10 [4]. The active properties of the P19 derived neurons developed rapidly following the establishment of the r ~ i n g membrane potential and decrease in input re. u ~ a c e . The earliest action potentials observed were small in amplitude, broad in duration and without any associated after-hyperpolarizations. A number of cells recorded at this stage were able to maintain a neuronal resting potential, but could not be made to display regenerative action potentials. Thus, the developmental sequence for neurons in this model involves the establishment of passive neuron-like properties before the appearance of functional voltage-dependent Na+channels capable of generating action potentials. This snggastion is supported by the observation that spontaneous post-synaptic potentials were infrequently observed in grafts less than 14 days old, but were routinely encountered in those 14-21 days old or older [18]. In another develpmental model, recordings from acutely isolated rat cerebeilar Purkinje neurons showed that E20 Purkinje neurons were largely inexeitable, had membrane input resistances four times greater than those at postnatal days 1-4 (stage 2), and had action potentials that did not consistently overshoot 0 mV until postnatal days 10-14 [11]. Action potential duration decreased gradually from postnatal dec 1 to 14 (stages 2, 3 and 4). This study also suggests that the neuronal mechanism for action potential generation develops after the passive properties of resting membrane potential. At least five neurons in 6-10 DPG grafts had action potentials 30 mV or more in amplitude with only one of these having an after-h~erpolarization over 5 mV in amplitude. Thus, the production of sizeable action

potentials is prerequisite to, or develops before, tile capability to elicit large after-hyperpolarizations (AHP's). The size and nature of ABe's are critical factors in determining the firing characteristics of most central neurons and are often used as defining characteristics of central neurcus. Developmental increases in the number of K + channels have been demol':strated to alter the firing characteristics but these changes do not necessarily alter the AHP. As illustrated by the example of cerebellar Purkinje neurons, neurons in early developmental stages express both Ca 2+.dependent and Ca2+-independent K + channels. As cerebellar Purkinje neurons mature they lose their Ca2+-independent K + channels, leading to a decrease in their AHP and the development of the familiar complex spiking pattern [531. By 2 to 3 weeks post-transplantation it was clear that the RA treated P19 cells developed into a number of different neuronal types based on morphology. This was most obvious from examination oi the 0-gal immunoreactivity but was clearly the case for the bioeytin-injected cells as well. Thus, the developmental rates and individual electrophysiological properties reported here represent the average across a heterogeneous population. This heterogeneity is in accord with the mo~hologieal and neuroeheimcal heterogeneity seen in P19 derived neurons developing in tissue culture [451. The P19 derived neurons recorded from the oldest of the grafts were similar in most respects to those recorded from 29-59-day-old grafts. AS an exception, a time-dependent inward rectification during hyperpolarizing current injection was infrequently observed in neurons from grafts less than 60 days old, but was routinely seen in neurons from older grafts. The timedependent inward rectLficution observed in most of the oldest category of implanted cells appears similar to those reported in various central neurons. A relativel3¢ fast inwardly rectifying current attributable to a potassium conductance has been reported in olfactory cortex neurons (Ifir) [6]. This current does not llive rise to a depolarization which overshoots the resting membrane potential on cessation of a hyperpolarizing pulse. Other inwardly rectifying currents which give rise to an overshoot seem generally to have a slow time course, are attributable to a combined sodium and potassium current, and are blocked by cesium, not barium. These have been referred to variously as iq (hippocampal pyramidal cells [10]), IAR (cortical neurons [44]) and lh (thalamic relay neurons [23]). Because the rectification of P19 derived neurons routinely exhibited time-dependent characteristics in the form of a sag in the hyperpolarizing voltage recurd and a depolarizing overshoot on the cessation of hyperpolarizing current pulses, it seems most likely that the inward rectifications we observed were of the second t ~ e (lq, IAR or

D.S.K. Magnuson et al. / DeL'elopmental Brain Research 84 (1995) l.YO-141

Ih). We did not, however, apply cesium or barium to distinguish between these possibilities. The presence of an inward rectification of this type would be consistent with the supposition that the P19-derived neurons resemble those from forebrain areas. The role played by this type of rectification for most mature mammalian CNS neurons is poorly understood, being observed only when cells are strongly hyperpolarized. Its appearance does indicate, however, a final stage of development during which P19-derived neurons reach a 'mature' and stable stage, appearing electrophysiologically similar to a number of adult forebrain neurons including cortical and hippocampai pyramidal neurons. It is conceivable that certain aspects of development could be influenced by factors arising from the transplantation process and associated methodology. Ibotenic acid was injected with the grafted cells, and their survival is attributable to the absence of glutamate receptors in the undifferentiated PI9 cells [17,18,47]. The excitotoxic death of host neurons rest:its, however, in a number of secondary events including activation of gila cells [28] and the release of neurotrophic substances [34,] which may have some effect on the developing P19-derived neurons. We can also not rule out the possibility that non-ionotropic glutamate receptors might he present in the cells at this stage. All of the host animals received cyclosporin A as inuuunosuppresive therapy, and it is known to act through various second messenger pathways [16]. Undifferentiated and differentiating P19 cells have been exposed to a wide range of concentrations of cyclosporin A in vitro, and no effect on differentiation was observed [29]. Results from studies of grafted fetal cells in brain slice preparations have suggested that the development of several electrophysiological parameters of transplanted neurons may he prolonged compared to normal developmental rates [49,52,31]. With multipotent precursor cell-derived neurons, only the present data are available for describing 'mature' cells, given the rapid progression of changes we observed, both dectrophysiologically and morphologically, it seems unlikely that the in vivo environment delays the development of differentiated EC cells. The morphological changes wc observed were similar to those seen in fetal striatal grafts into kainic acid-lesioned adult eat striatuna with re.~'~.ct to increases in soma size and process out growth with little or no process outgrowth beyond the graft boundary [51] (see Figs. 6, 7). There are now a number of multipotent neuron precursor cell lines available. Some were obtained using oncogene transformation of primary CNS tissue [9,39] while others were isolated using growth factors [48]. Some of these cells appear to diflerent]ate appropriately within the host brain following grafting into the neonatal hippocampns [35] or into neonatal cere-

bellum [43]. These cell lines are useful in studi¢~ concerning the isolation of factors or the elucidation of mechanisms important for neuronal cell commitment and differentiation. The electroph~.;o!ogieal properties of these cell lines have only been studied in a limited fashion in vitro. Some do not show ant'. evidence of Na + or Ca 2÷ channels [39], while others produce cells which display electrophysiological properties expected of immature neurons [48]. None have yet demonstrated mature neuronal electrophysiological responses. Therefore, the P19 EC multipotent precursor is, at present, the best characterized with respect to the development of mature neuronal electrophysiological characteristics and its cellular derivatives. We have also shown that, when given adequate protection from rejection, grafts of EC-derived neurons survive for up to 4 months in vivo, continuing to express E. coil ~-galactosidas¢ (/3-gal), a product of exogenous D N A with which the cells were transfeeted prior to differentiation. Grafted 1003 EC cells also continue transgene expression in vivo post-grafting [50]. The /3-gal can be used to identify the ceils following transplantation using histochemical or immunoc~ochemical techniques [40,7], even if they have been assimilated into the host or have migrated some distance from the graft site. Double-labelling of bioeytin injected neurons with anti-Ogal permitted the identification of some of the recorded cells as unequivocal~ Pl9-derived. Also, the presence of intracellular E. co// fl-gal did not appear to effect electroph~iological responsiveness or development. Using newly developed, non-leeching forms of FIX}, applied to ~-gal expressing grafts in a brain slice preparation, ~-gal expressing neurons should he identifiable in the slice using indirect fluorescence microscopy. This would be especially useful for targeting grafted neurons which become assimilated into the host and migrate away frorn the graft site [43,35]. In this study we have provided the first demonstration of the in vivo electrophysiological maturation of neurons derived from a multipotential precursor cell line. We have shown that P19 derived neurons are capable of developing mature electroph~iological properties normally associated with adult CNS neurons. The use of a grafting paradigm permitted extended neuronal survival times and the ability to observe electrophysiologieal development beyond that permitted by in vitro studies of these cells. Our data helps to establish the neuronal character of P19 derivatives, and serves as a baseline for future comparisons. P19 cells have been used to study the effects of the overexpression [54] or ablation [8] of ~ n e products on neuronal survival and differentiation. T h ~ and other studies may lead to the development of donor tissue sources which can he targeted for specific n e u r o k ~ c u l diseases.

D.S.K. Ma~uaon et aL /Developmental Brain Research 84 (1995) 130-141

~ m e n t s T h e a u t h o r s w o u l d like t o a c k n o w l e d g e t h e excellent t e c h n i c a l assistance given b y Dr. T o m a s z Woloszyn, Ms. B a b b e n T i n n e r - S t a i n e s a n d Ms. L u c y P i c k a v a n c e . T h a n k s also g o e s t o S a n d o z o f C a n a d a f o r p r o v i d i n g C y c l o s p o r i n A ( S a n d i m m u n e ) . W . A . S . is a n M R C Scholar, M . W . M . is a n N C I T e r r y F o x C a n c e r R e s e a r c h Scientist, D . S . K . M w a s a N C E F e l l o w a n d D . J . M . w a s a n M R C Fellow. S u p p o r t e d b y t h e C a n a d i a n N e t w o r k o f C e n t r e s o f Excellence in N e u r a l R e g e n e r a t i o n a n d F u n c t i o n a l R e c o v e r y . T h e P 1 9 cells u s e d f o r t h e s e e x p e r i m e n t s h a v e b e e n d e p o s i t e d in t h e A T C C w h e r e t h e y h a v e b e e n a s s i g n e d # C R L 1825.

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