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Fetal raphe neurons grafted into the hippocampus develop normal adult physiological properties
162
Brain Research, 364 (1986) 162-166 Elsevier
BRE 21319
Fetal raphe neurons grafted into the hippocampus develop normal adult physiological properties MENAHEM SEGAL 1and EFRAIN C. AZMITIA2 tCenter for Neuroscience, The Weizmann Institute of Science, Rehovot 76100 (Israel) and 2New York University, Department of Biology, New York, NY IO003 (U.S.A.)
(Accepted September 18th, 1985) Key words: raphe - - serotonin (5-HT) - - transplant
Embryonic midbrain raphe was grafted into ser0tonin-deficient adult rat hippocampus. Serotonin-containing neurons in the graft survive for at least 6 months after grafting. Grafted neurons develop physiological properties, not present on the day of grafting, identical to those of normal adult serotonin-containing neurons. These include (a) high input resistance and slow membrane time constant, (b) lack of inward rectification in response to hyperpolarizing current pulses and (c) a potent, 4-aminopyridine-sensitive transient outward rectification. The grafted neurons innervate the host tissue with axons that have a slow conduction velocity, and refractoriness. It is suggested that grafted CNS neurons may possess normal physiological properties. It has been clearly established that immature central neurons, when transplanted from a fetal donor into an adult brain, develop extensive fiber connections with the host tissue 3.4,t5. This finding has exciting clinical relevance and has led to several studies which have shown that neural transplants can lead to restoration of impaired functions 3.10. Despite the relative success in demonstrating some recovery of functions following transplantation of a n u m b e r of chemically specific neurons, it has not been established whether the transplanted neurons possess the same biophysical properties as normal cells, nor has a physiological analysis of synaptic connections between the grafted and host cells been undertaken. We now report that neurons taken from fetal brainstem raphe nuclei develop physiological properties virtually identical to those observed in raphe neurons of adult rats. Furthermore, the transplanted raphe cells innervate the host tissue with axons that share same properties with normal serotonergic axons, and may make viable synaptic connections with the host brain. Host animals were 2 - 3 month old Wistar rats which had received a prior injection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT).
Rat embryos, 14-15 days in utero, served as the source of immature neurons. They were surgically taken from the uterus under sterile conditions, their brains removed and a slice containing the mesencephalic midline dissected out as previously described 3. The slices were minced and microinjected in a volume of 1/A stereotaxically into the host dorsal hippocampus. Intracellular recording was made with KC1- or potassium acetate-filled micropipettes from neurons in coronal 300-ktm slices of (a) the adult midbrain containing the dorsal raphe ( D R ) nucleus, (b) the embryonic mesencephalic raphe (MR) slices or (c) CA1, dentate gyrus and grafted M R neurons in the host hippocampus. The slices were interfaced between normal Krebs m e d i u m and warm humidified 95% 0 2 - 5 % CO 2 gas mixture t2. Following termination of the experiments the slices were preincubated with 5-HTP (0.1 m M ) in the presence of pargyline (0.1 mM) and processed for serotonin immunocytochemistry 2 to verify the presence of viable 5-HTcontaining M R cells. The characteristics of the raphe cells in the normal brainstem slice were very similar to those which had been reported previously 1,6,13A8. The resting potential of these cells was rather consistant (-61 + 2 mV,
Correspondence: M. Segal, Center for Neuroscience, The Weizmann Institute of Science, Rehovot 76100, Israel.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
163
=
n 7 ) Their input resistance, measured with series of hyperpolarizing current pulses, was high (115 +_ 25 MQ, n = 7). The current-voltage relations were linear down to -140 inV. Unlike many other neuron types 9, hyperpolarization did not activate inward rectification in raphe cells. An analysis of the kinetics of the voltage response to hyperpolarizing current pulses indicates that the potential changes exponentially with a single, major, rather slow (20-30 ms) time constant. A faster component of the membrane time constant could not be peeled reliably. Action potentials occurred spontaneously in 3 of 12 of the cells analyzed, while the remaining neurons could fire action potentials in response to depolarizing current pulses. The duration of the action potential was relatively long (3-5 ms) and contained a slow compo-
nent that may reflect a calcium-dependent potential Is. Each individual spike was followed by a large afterhyperpolarization (Fig. IA) reflecting probably a calcium-dependent potassium current ~. We observed a striking property in all raphe ceils studied which has only recently been described 1.1~ This was an extremely potent transient outward rectification evoked in response to a depolarizing current pulse which virtually clamped the membrane below firing level for up to 50 ms. During this interval, action potentials could not be discharged (Fig. 1B). To fully activate this rectification the cell had to be primed-hyperpolarized for 30-50 ms by about 3(J mV from rest. At resting potential only a partial activation of the transient rectification could be detected. The kinetics and voltage dependence of this rectifica-
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Fig. 1. A I : electrical properties of a dorsal raphe (DR) neuron. Intracellular recording was made from a putative 5-HT containing D R neuron. Hyperpolarizing and depolarizing current pulses were applied to the neuron using a bridge circuit. A2: an analysis of thc hvperpolarizing charging curve indicates the presence of a single exponential curve with a time constant of 27 ms. A3: the c u r r e n t voltage curve is linear down to 60 m V below resting potential ( - 6 5 mV) and indicates an input resistance of 180 Mr2. No sag in the voltage response can be seen at any of the potentials. B: a 50-ms depolarizing current pulse produces a train of action potentials. A brief hyperpolarization prevents the cell from discharging action potentials to the same current pulse for about 45 ms. C: a conditioning hyperpolarization delays the discharge of action potentials in response to a depolarization pulse (top). This delay is blockcd after topical application of a microdrop of 5 m M 4-AP (bottom). 5 - 6 traces overlapped. This effect of 4-AP is not reproduced by 10 mM T E A (not shown).
164 lion indicate that it is likely to be p r o d u c e d by activation of a transient outward conductance ( F O C ) as seen elsewhere 5,s.ll,la. Since 4-aminopyridine (4-AP) has been shown to selectively bh)ck T O C in central neuronsL~S,~4.~% its effects were tested on D R cells. Indeed, the transient rectification was a t t e n u a t e d by 4-AP (5 cells tested, Fig. 1C) but not by 5 - 1 0 m M of t e t r a e t h y l a m m o n i u m ion ( T E A ) (3 cells, data not shown). These p r o p e r t i e s distinguish D R neurons from o t h e r neurons in the brain including hippocampal cells and facilitate their identification. None of these p r o p e r t i e s were found in 6 i m m a t u r e raphe neurons r e c o r d e d from 6 brainstem slices of 14-16-day-old embryos. These neurons had normal resting potential ( - 5 7 + 3 mV) and input resistance (41 + 3 M~2, n = 6) and slow m e m b r a n e time constant (20-30 s), yet had no action potential of the size and shape seen in the adult or any obvious transient rectification. In fact, the only p r o p e r t y of the embryonic neurons was a delayed rectification (unpublished observations). Action potentials were seen only at e m b r y o n i c day 17-18 (6 cells). Thus, when transplanted, D R neurons have no identifying physiological properties. Intracellular recording was m a d e from neurons in 8 midbrain grafts which were sliced together with the host hippocampus 2 - 5 months after grafting. The grafts, measured about 1 mms contained clusters of serotonin-positive immunoreactive neurons (Fig, 2A). These clusters were easily identified in the graft with the use of a stereomicroscope. Recording was made from cells located within the boundaries of the graft, away from any h i p p o c a m p a l neurons. Of 20 neurons r e c o r d e d from these clusters 12 exhibited physiological properties which were r e m a r k a b l y similar to those r e c o r d e d from serotonin-containing D R neurons in a midbrain slice. The o t h e r 8 cells did not exhibit these p r o p e r t i e s clearly and were not analyzed. Of the 12 neurons 3 fired action potentials spontaneously at a rate of 1 - 2 spikes per second (Fig. 2B). The others could fire spikes upon depolarization. The spikes were broad and had a typical 'slow c o m p o n e n t ' similar to that seen in D R cells r e c o r d e d in a brainstem slice. The spikes were followed by large, up to 10 mV, afterhyperpolarizations (Fig. 2B). The D R - l i k e neurons had an input resistance of up to 240 Mff2 (X = 115 _ 25 M Q ) , derived from a linear current-voltage relationship (Fig. 2C). Their
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Fig. 2. A: immunocytochemical staining of 5-HT-contaming neurons implanted into the hippocampus. Left low-power view of the graft. Small arrow-heads mark the boundaries of the graft. DG. dentate gyrus: sub, subiculum. Right highpower view of the grafted neurons. Somatals) and dendrites (d) are stained with 5-HT-immunoreactive material, Calibration 100,um for left and 10ktm for right. B: spontaneous activity of a grafted neuron. The action potentials are truncated by the recorder but a large, slow afterhyperpolarization is evident after each action potential. Calibration 10 mV. 0.5 s: resting membrane potential. -65 inV. C: a series of hyperpolarizing current pulses applied to a grafted neuron results in downward voltage deflections. The hyperpolarizing current pulses are followed by a depolarizing command (C). The current voltage relation in the hyperpolarizing direction is linear and indicates an input resistance of 150 Mr2 fC2). The voltage reaches its maximal value in an exponential manner with a time constant of 24 ms (CI). Calibration 10 ms. 20 inV. 1.0 nA.
mean resting potential was - 6 1 ___ 2 mV. These cells had no "sag' in the voltage responses to hyperpotarizing current pulses indicating the lack of inward rectification. Their m e m b r a n e time constant was slow (20-30 ms. Fig. 2C). A b o v e all, the cells possessed
165 c o n d u c t i o n v e l o c i t y of 5 - H T n e r v e fibers in situ 7. T h e
the characteristically s t r o n g t r a n s i e n t o u t w a r d rectification (Fig. 2C), which was partially i n a c t i v a t e d at
cells had a relatively slow r e f r a c t o r y p e r i o d t e s t e d
rest. T h e inactivation c o u l d be r e m o v e d by a brief
with high f r e q u e n c y s t i m u l a t i o n trains (Fig. 3C).
conditioning hyperpolarization.
T h e p r e s e n t results d e m o n s t r a t e that e m b r y o n i c
T h e t r a n s i e n t out-
ward rectification c o u l d t h e n be a c t i v a t e d by a d e p o -
raphe neurons transplanted
into the h i p p o c a m p u s
larizing c u r r e n t pulse. It was m a r k e d l y a t t e n u a t e d in
possess similar physiological p r o p e r t i e s to t h o s e seen
the p r e s e n c e of low c o n c e n t r a t i o n s of 4 - A P (Fig.
in n o r m a l r a p h e cells. T h u s , the natural input to the
3A). T h e s e p r o p e r t i e s w e r e not seen in n e u r o n s re-
r a p h e nuclei as well as the h o r m o n a l c o n d i t i o n s typic-
c o r d e d in the host h i p p o c a m p u s a d j a c e n t to the graft. P u t a t i v e M R n e u r o n s r e c o r d e d in the graft could
al of a n e w b o r n , do not s e e m to h a v e a crucial role in
be i n v a d e d a n t i d r o m i c a l l y by s t i m u l a t i n g s o m e sites
neurons.
d e t e r m i n i n g the physiological p r o p e r t i e s
of these
in the host tissue. T h e best results w e r e o b t a i n e d
T h e physiological d e t e r m i n a n t s of the firing r e p e r -
w h e n the d e n t a t e gyrus a d j a c e n t to the graft was
toire of the t r a n s p l a n t e d n e u r o n s are not yet clear. It
s t i m u l a t e d . T h e a n t i d r o m i c r e s p o n s e c o u l d be col-
is unlikely that these n e u r o n s r e c e i v e the s a m e inputs
lided with an action p o t e n t i a l e v o k e d by a d e p o l a r i -
as their in-situ c o u n t e r p a r t s do. In s o m e of the trans-
zing current pulse. T h e c o n d u c t i o n v e l o c i t y of t h e s e
planted cells spontaneous
axons was slow~ a b o u t 0.3 m - 0 . 5 m/s, similar to the
c o u l d be r e c o r d e d ( u n p u b l i s h e d o b s e r v a t i o n s ) that
postsynaptic
potentials
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Fig. 3. A: conditioning hyperpolarization removes inactivation of a transient outward rectifier. AI: depolarizing current pulses of various magnitudes clamp the membrane at about 10 mV positive to resting potential. A2: the transient rectification is blocked in the presence of 5 mM 4-AP and a depolarizing current pulse can now trigger an action potential discharge. Calibration 20 mV, 0.5 nA, 10 ms. B: action potentials can be discharged in a grafted neuron by low current stimulation of the host hippocampal slice some 2 mm away from the recorded cell. A collision test indicates that the response seen is an antidromic response to stimulation of the axon which grows out of the transplant and innervates the host hippocampus. An action potential triggered by passage of a depolarizing current pulse into the recorded neuron collides with the spike triggered by the stimulation of the axon. If, however, the intracellularly evoked spike discharges more than 6 ms before the antidromic stimulation, it does not collide and a spike is driven by the antidromic stimulation. Each trace is composed of 5-10 repetitions. C: estimates of refractoriness of a transplanted raphe neuron. A train of 3 antidromic stimuli is applied at a rate of 166 Hz (top) or 142 Hz (bottom). The cell can follow the slower rate but only the axon spike can follow rcliably the faster rate. The soma-dendritic spike fails intermittently. Calibration 50 mV, 5 ms for C, 2 ms for B.
166 w e r e similar to PSPs r e c o r d e d in D R cells l~. It is yet
In c o n c l u s i o n , these e x p e r i m e n t s d e m o n s t r a t e that
to be d e t e r m i n e d if t h e y m a i n t a i n firing p a t t e r n s that
e m b r y o n i c m i d b r a i n raphe n e u r o n s can d e v e l o p nor-
reflect b e h a v i o r a l states (i.e. s l e e p - w a k e
cycles):
real p r o p e r t i e s w h e n t r a n s p l a n t e d into a host hippo-
and thus m a y m o d u l a t e excitability of host h i p p o c a m -
c a m p u s . T h e s e results e n c o u r a g e r e s e a r c h into the
pal n e u r o n s in the s a m e way that r a p h e cells m o d -
possible use of t r a n s p l a n t a t i o n of neural tissue for the
ulate h i p p o c a m p a l f u n c t i o n s in n o r m a l brain. F u t u r e
facilitation of r e c o v e r y of f u n c t i o n s f o l l o w i n g specific
e x p e r i m e n t s are d e s i g n e d to e x a m i n e these possibili-
brain d a m a g e .
ties. A t any rate, the p r e s e n c e of intrinsic m e c h a activity in at least
This r e s e a r c h was s u p p o r t e d by G r a n t 84-245 f r o m
s o m e of t h e s e cells indicates that the transplanted cells
nisms for regular s p o n t a n e o u s
the U n i t e d S t a t e d - l s r a e l B i n a t i o n a l Science F o u n d a -
do not n e e d n o r m a l a f f e r e n t inputs to trigger cellular
tion, J e r u s a l e m , lsrael.
discharges.
4-AP CAt CNS DG 5,7-DHT DR
4-aminopyridine cornu amonis 1 central nervous system dentate gyrus 5,7-dihydroxytryptamine dorsal raphe
l Aghajanian, G,K., Modulation of a transient outward current in serotonergic neurons by alpha-1 adrenoreceptors, Nature (London), 315 (1985) 501-502. 2 Azmitia, E.C. and Gannon, P.J., The ultrastructural localization of serotonin immunoreactivity in myelinated axons within the medial forebrain bundle of the rat, J. Neurosci., 3 (1983) 2083-2090. 3 Azmitia, E.C. and Whitaker, P.M., Formation of a glial scar following microinjection of fetal neurons into the hippocampus or midbrain of the adult rat: an immunocytochemical study, Neurosci. Lett., 38 (1983) 145-150. 4 Bjorklund, A., Gage, F.H., Stenevi, U. and Dunnett, S.B., Survival and growth of intrahippocampal implants of septal cell suspensions, Acta Physiol. Scand. Suppl., 522 (1983) 49-58. 5 Connor, J.A. and Stevens, C.F., Voltage clamp studies of a transient outward membrane current in gastropod neural somata, J. Physiol. (London), 213 (1971) 21-30. 6 Crunelli, V., Forda, S., Brooks, P.A., Wilson, K.C.P., Wise, J.C.M. and Kelly, J.S., Passive membrane properties of neurones in the dorsal raphe and periaqueductal grey recorded in vitro, Neurosci. Lett., 40 (1983) 263-268. 7 Crunelli, V. and Segal, M., Electrophysiological study of the rat nucleus raphe medianus. J. Physiol (London), 328 (1982) 43-44. 8 Dekin, M.S. and Getting, P.A., Firing pattern of neurons in the nucleus tractus solitarius: modulation by membrane hyperpolarization, Brain Research, 324 (1984) 180-184. 9 Halliwell, J.V. and Adams, P . R , Voltage clamp analysis of muscarinic excitation in hippocampal neurons, Brain Research, 250 (1982) 71-92. 10 Low, W.C., Lewis, P.R., Bunch, T.S., Dunnett, S.B., Thomas, S.R., Iversen, S.D., Bjorklund. A. and Stenevi,
5-HT 5-HTP MR PSP Sub TEA
11
12
13 14
15
16 17
18
19
5-hydroxytryptamine (serotonin) 5-hydroxytryptophan mesencephalic raphe postsynaptic potentials subiculum tetraethylammonium
U.. Functional recovery following neural transplantation of embryonic septal nuclei in adult rats with septohippocampal lesions. Nature (London), 300 (1982J 260-262. Neher. E.. Two fast transient current components during voltage clamp on snail neurons. J. Gen. Phvsiol. 58 (197l) 36-53. Segal, M.. The action of serotonin in the rat hippocampal slice preparation. J. Physiol. :London,. 303 (198(1) 423-439. Segal, M., A potent transtent outward current regulates excitability of dorsal raphe neurons, Brain Research. in press. Segal, M.. Rogawski. M.A. and Barker. J.L.. A transient potassium conductance regulates the excitability of cultured hippocampal and spinal neurons. J Neurosci. 4 ( 1984~ 604-609. Stenevi. U. Bjorklund. A. and Svendgaard. N,A.. Transplantations of centra] and peripheral monoamine neurons to the adult rat brain: techniques and conditions for survival. Brain Research. 114 (1976) 1-20. Thompson. S.. Aminopyridine block of transient potassmm current. J. Gen. Physiol. 8011982] 1-18. Trulson. M.E. and Jacobs B.L. Raphe unit activity in freely moving cats: correlation with level ol behavioral arousal. Brain Research. 163 I 19791 135-150 Vandermaelen. C.P. and Aghajanian. G.K.. Electrophysiological and pharmacological characterizauon of serotonergic dorsal raphe neurons recorded extracellutarly and intraeellularly in rat brain slices. Brain Research. 289 (1983) 109-119. Yoshimura. M. and Higashi. H.. 5-Hydroxytryptamme mediates inhibitory postsynapnc potentials in rat dorsal raphe neurons. Neurosci. Lett.. 53 (1985) 69-74.
Brain Research, 364 (1986) 162-166 Elsevier
BRE 21319
Fetal raphe neurons grafted into the hippocampus develop normal adult physiological properties MENAHEM SEGAL 1and EFRAIN C. AZMITIA2 tCenter for Neuroscience, The Weizmann Institute of Science, Rehovot 76100 (Israel) and 2New York University, Department of Biology, New York, NY IO003 (U.S.A.)
(Accepted September 18th, 1985) Key words: raphe - - serotonin (5-HT) - - transplant
Embryonic midbrain raphe was grafted into ser0tonin-deficient adult rat hippocampus. Serotonin-containing neurons in the graft survive for at least 6 months after grafting. Grafted neurons develop physiological properties, not present on the day of grafting, identical to those of normal adult serotonin-containing neurons. These include (a) high input resistance and slow membrane time constant, (b) lack of inward rectification in response to hyperpolarizing current pulses and (c) a potent, 4-aminopyridine-sensitive transient outward rectification. The grafted neurons innervate the host tissue with axons that have a slow conduction velocity, and refractoriness. It is suggested that grafted CNS neurons may possess normal physiological properties. It has been clearly established that immature central neurons, when transplanted from a fetal donor into an adult brain, develop extensive fiber connections with the host tissue 3.4,t5. This finding has exciting clinical relevance and has led to several studies which have shown that neural transplants can lead to restoration of impaired functions 3.10. Despite the relative success in demonstrating some recovery of functions following transplantation of a n u m b e r of chemically specific neurons, it has not been established whether the transplanted neurons possess the same biophysical properties as normal cells, nor has a physiological analysis of synaptic connections between the grafted and host cells been undertaken. We now report that neurons taken from fetal brainstem raphe nuclei develop physiological properties virtually identical to those observed in raphe neurons of adult rats. Furthermore, the transplanted raphe cells innervate the host tissue with axons that share same properties with normal serotonergic axons, and may make viable synaptic connections with the host brain. Host animals were 2 - 3 month old Wistar rats which had received a prior injection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT).
Rat embryos, 14-15 days in utero, served as the source of immature neurons. They were surgically taken from the uterus under sterile conditions, their brains removed and a slice containing the mesencephalic midline dissected out as previously described 3. The slices were minced and microinjected in a volume of 1/A stereotaxically into the host dorsal hippocampus. Intracellular recording was made with KC1- or potassium acetate-filled micropipettes from neurons in coronal 300-ktm slices of (a) the adult midbrain containing the dorsal raphe ( D R ) nucleus, (b) the embryonic mesencephalic raphe (MR) slices or (c) CA1, dentate gyrus and grafted M R neurons in the host hippocampus. The slices were interfaced between normal Krebs m e d i u m and warm humidified 95% 0 2 - 5 % CO 2 gas mixture t2. Following termination of the experiments the slices were preincubated with 5-HTP (0.1 m M ) in the presence of pargyline (0.1 mM) and processed for serotonin immunocytochemistry 2 to verify the presence of viable 5-HTcontaining M R cells. The characteristics of the raphe cells in the normal brainstem slice were very similar to those which had been reported previously 1,6,13A8. The resting potential of these cells was rather consistant (-61 + 2 mV,
Correspondence: M. Segal, Center for Neuroscience, The Weizmann Institute of Science, Rehovot 76100, Israel.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
163
=
n 7 ) Their input resistance, measured with series of hyperpolarizing current pulses, was high (115 +_ 25 MQ, n = 7). The current-voltage relations were linear down to -140 inV. Unlike many other neuron types 9, hyperpolarization did not activate inward rectification in raphe cells. An analysis of the kinetics of the voltage response to hyperpolarizing current pulses indicates that the potential changes exponentially with a single, major, rather slow (20-30 ms) time constant. A faster component of the membrane time constant could not be peeled reliably. Action potentials occurred spontaneously in 3 of 12 of the cells analyzed, while the remaining neurons could fire action potentials in response to depolarizing current pulses. The duration of the action potential was relatively long (3-5 ms) and contained a slow compo-
nent that may reflect a calcium-dependent potential Is. Each individual spike was followed by a large afterhyperpolarization (Fig. IA) reflecting probably a calcium-dependent potassium current ~. We observed a striking property in all raphe ceils studied which has only recently been described 1.1~ This was an extremely potent transient outward rectification evoked in response to a depolarizing current pulse which virtually clamped the membrane below firing level for up to 50 ms. During this interval, action potentials could not be discharged (Fig. 1B). To fully activate this rectification the cell had to be primed-hyperpolarized for 30-50 ms by about 3(J mV from rest. At resting potential only a partial activation of the transient rectification could be detected. The kinetics and voltage dependence of this rectifica-
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Fig. 1. A I : electrical properties of a dorsal raphe (DR) neuron. Intracellular recording was made from a putative 5-HT containing D R neuron. Hyperpolarizing and depolarizing current pulses were applied to the neuron using a bridge circuit. A2: an analysis of thc hvperpolarizing charging curve indicates the presence of a single exponential curve with a time constant of 27 ms. A3: the c u r r e n t voltage curve is linear down to 60 m V below resting potential ( - 6 5 mV) and indicates an input resistance of 180 Mr2. No sag in the voltage response can be seen at any of the potentials. B: a 50-ms depolarizing current pulse produces a train of action potentials. A brief hyperpolarization prevents the cell from discharging action potentials to the same current pulse for about 45 ms. C: a conditioning hyperpolarization delays the discharge of action potentials in response to a depolarization pulse (top). This delay is blockcd after topical application of a microdrop of 5 m M 4-AP (bottom). 5 - 6 traces overlapped. This effect of 4-AP is not reproduced by 10 mM T E A (not shown).
164 lion indicate that it is likely to be p r o d u c e d by activation of a transient outward conductance ( F O C ) as seen elsewhere 5,s.ll,la. Since 4-aminopyridine (4-AP) has been shown to selectively bh)ck T O C in central neuronsL~S,~4.~% its effects were tested on D R cells. Indeed, the transient rectification was a t t e n u a t e d by 4-AP (5 cells tested, Fig. 1C) but not by 5 - 1 0 m M of t e t r a e t h y l a m m o n i u m ion ( T E A ) (3 cells, data not shown). These p r o p e r t i e s distinguish D R neurons from o t h e r neurons in the brain including hippocampal cells and facilitate their identification. None of these p r o p e r t i e s were found in 6 i m m a t u r e raphe neurons r e c o r d e d from 6 brainstem slices of 14-16-day-old embryos. These neurons had normal resting potential ( - 5 7 + 3 mV) and input resistance (41 + 3 M~2, n = 6) and slow m e m b r a n e time constant (20-30 s), yet had no action potential of the size and shape seen in the adult or any obvious transient rectification. In fact, the only p r o p e r t y of the embryonic neurons was a delayed rectification (unpublished observations). Action potentials were seen only at e m b r y o n i c day 17-18 (6 cells). Thus, when transplanted, D R neurons have no identifying physiological properties. Intracellular recording was m a d e from neurons in 8 midbrain grafts which were sliced together with the host hippocampus 2 - 5 months after grafting. The grafts, measured about 1 mms contained clusters of serotonin-positive immunoreactive neurons (Fig, 2A). These clusters were easily identified in the graft with the use of a stereomicroscope. Recording was made from cells located within the boundaries of the graft, away from any h i p p o c a m p a l neurons. Of 20 neurons r e c o r d e d from these clusters 12 exhibited physiological properties which were r e m a r k a b l y similar to those r e c o r d e d from serotonin-containing D R neurons in a midbrain slice. The o t h e r 8 cells did not exhibit these p r o p e r t i e s clearly and were not analyzed. Of the 12 neurons 3 fired action potentials spontaneously at a rate of 1 - 2 spikes per second (Fig. 2B). The others could fire spikes upon depolarization. The spikes were broad and had a typical 'slow c o m p o n e n t ' similar to that seen in D R cells r e c o r d e d in a brainstem slice. The spikes were followed by large, up to 10 mV, afterhyperpolarizations (Fig. 2B). The D R - l i k e neurons had an input resistance of up to 240 Mff2 (X = 115 _ 25 M Q ) , derived from a linear current-voltage relationship (Fig. 2C). Their
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Fig. 2. A: immunocytochemical staining of 5-HT-contaming neurons implanted into the hippocampus. Left low-power view of the graft. Small arrow-heads mark the boundaries of the graft. DG. dentate gyrus: sub, subiculum. Right highpower view of the grafted neurons. Somatals) and dendrites (d) are stained with 5-HT-immunoreactive material, Calibration 100,um for left and 10ktm for right. B: spontaneous activity of a grafted neuron. The action potentials are truncated by the recorder but a large, slow afterhyperpolarization is evident after each action potential. Calibration 10 mV. 0.5 s: resting membrane potential. -65 inV. C: a series of hyperpolarizing current pulses applied to a grafted neuron results in downward voltage deflections. The hyperpolarizing current pulses are followed by a depolarizing command (C). The current voltage relation in the hyperpolarizing direction is linear and indicates an input resistance of 150 Mr2 fC2). The voltage reaches its maximal value in an exponential manner with a time constant of 24 ms (CI). Calibration 10 ms. 20 inV. 1.0 nA.
mean resting potential was - 6 1 ___ 2 mV. These cells had no "sag' in the voltage responses to hyperpotarizing current pulses indicating the lack of inward rectification. Their m e m b r a n e time constant was slow (20-30 ms. Fig. 2C). A b o v e all, the cells possessed
165 c o n d u c t i o n v e l o c i t y of 5 - H T n e r v e fibers in situ 7. T h e
the characteristically s t r o n g t r a n s i e n t o u t w a r d rectification (Fig. 2C), which was partially i n a c t i v a t e d at
cells had a relatively slow r e f r a c t o r y p e r i o d t e s t e d
rest. T h e inactivation c o u l d be r e m o v e d by a brief
with high f r e q u e n c y s t i m u l a t i o n trains (Fig. 3C).
conditioning hyperpolarization.
T h e p r e s e n t results d e m o n s t r a t e that e m b r y o n i c
T h e t r a n s i e n t out-
ward rectification c o u l d t h e n be a c t i v a t e d by a d e p o -
raphe neurons transplanted
into the h i p p o c a m p u s
larizing c u r r e n t pulse. It was m a r k e d l y a t t e n u a t e d in
possess similar physiological p r o p e r t i e s to t h o s e seen
the p r e s e n c e of low c o n c e n t r a t i o n s of 4 - A P (Fig.
in n o r m a l r a p h e cells. T h u s , the natural input to the
3A). T h e s e p r o p e r t i e s w e r e not seen in n e u r o n s re-
r a p h e nuclei as well as the h o r m o n a l c o n d i t i o n s typic-
c o r d e d in the host h i p p o c a m p u s a d j a c e n t to the graft. P u t a t i v e M R n e u r o n s r e c o r d e d in the graft could
al of a n e w b o r n , do not s e e m to h a v e a crucial role in
be i n v a d e d a n t i d r o m i c a l l y by s t i m u l a t i n g s o m e sites
neurons.
d e t e r m i n i n g the physiological p r o p e r t i e s
of these
in the host tissue. T h e best results w e r e o b t a i n e d
T h e physiological d e t e r m i n a n t s of the firing r e p e r -
w h e n the d e n t a t e gyrus a d j a c e n t to the graft was
toire of the t r a n s p l a n t e d n e u r o n s are not yet clear. It
s t i m u l a t e d . T h e a n t i d r o m i c r e s p o n s e c o u l d be col-
is unlikely that these n e u r o n s r e c e i v e the s a m e inputs
lided with an action p o t e n t i a l e v o k e d by a d e p o l a r i -
as their in-situ c o u n t e r p a r t s do. In s o m e of the trans-
zing current pulse. T h e c o n d u c t i o n v e l o c i t y of t h e s e
planted cells spontaneous
axons was slow~ a b o u t 0.3 m - 0 . 5 m/s, similar to the
c o u l d be r e c o r d e d ( u n p u b l i s h e d o b s e r v a t i o n s ) that
postsynaptic
potentials
B
A1 i :
m
2-
V
I
I
2
A2 I --
I-
j I
V~
/
C
!
A
v I
~/~
-
I
Fig. 3. A: conditioning hyperpolarization removes inactivation of a transient outward rectifier. AI: depolarizing current pulses of various magnitudes clamp the membrane at about 10 mV positive to resting potential. A2: the transient rectification is blocked in the presence of 5 mM 4-AP and a depolarizing current pulse can now trigger an action potential discharge. Calibration 20 mV, 0.5 nA, 10 ms. B: action potentials can be discharged in a grafted neuron by low current stimulation of the host hippocampal slice some 2 mm away from the recorded cell. A collision test indicates that the response seen is an antidromic response to stimulation of the axon which grows out of the transplant and innervates the host hippocampus. An action potential triggered by passage of a depolarizing current pulse into the recorded neuron collides with the spike triggered by the stimulation of the axon. If, however, the intracellularly evoked spike discharges more than 6 ms before the antidromic stimulation, it does not collide and a spike is driven by the antidromic stimulation. Each trace is composed of 5-10 repetitions. C: estimates of refractoriness of a transplanted raphe neuron. A train of 3 antidromic stimuli is applied at a rate of 166 Hz (top) or 142 Hz (bottom). The cell can follow the slower rate but only the axon spike can follow rcliably the faster rate. The soma-dendritic spike fails intermittently. Calibration 50 mV, 5 ms for C, 2 ms for B.
166 w e r e similar to PSPs r e c o r d e d in D R cells l~. It is yet
In c o n c l u s i o n , these e x p e r i m e n t s d e m o n s t r a t e that
to be d e t e r m i n e d if t h e y m a i n t a i n firing p a t t e r n s that
e m b r y o n i c m i d b r a i n raphe n e u r o n s can d e v e l o p nor-
reflect b e h a v i o r a l states (i.e. s l e e p - w a k e
cycles):
real p r o p e r t i e s w h e n t r a n s p l a n t e d into a host hippo-
and thus m a y m o d u l a t e excitability of host h i p p o c a m -
c a m p u s . T h e s e results e n c o u r a g e r e s e a r c h into the
pal n e u r o n s in the s a m e way that r a p h e cells m o d -
possible use of t r a n s p l a n t a t i o n of neural tissue for the
ulate h i p p o c a m p a l f u n c t i o n s in n o r m a l brain. F u t u r e
facilitation of r e c o v e r y of f u n c t i o n s f o l l o w i n g specific
e x p e r i m e n t s are d e s i g n e d to e x a m i n e these possibili-
brain d a m a g e .
ties. A t any rate, the p r e s e n c e of intrinsic m e c h a activity in at least
This r e s e a r c h was s u p p o r t e d by G r a n t 84-245 f r o m
s o m e of t h e s e cells indicates that the transplanted cells
nisms for regular s p o n t a n e o u s
the U n i t e d S t a t e d - l s r a e l B i n a t i o n a l Science F o u n d a -
do not n e e d n o r m a l a f f e r e n t inputs to trigger cellular
tion, J e r u s a l e m , lsrael.
discharges.
4-AP CAt CNS DG 5,7-DHT DR
4-aminopyridine cornu amonis 1 central nervous system dentate gyrus 5,7-dihydroxytryptamine dorsal raphe
l Aghajanian, G,K., Modulation of a transient outward current in serotonergic neurons by alpha-1 adrenoreceptors, Nature (London), 315 (1985) 501-502. 2 Azmitia, E.C. and Gannon, P.J., The ultrastructural localization of serotonin immunoreactivity in myelinated axons within the medial forebrain bundle of the rat, J. Neurosci., 3 (1983) 2083-2090. 3 Azmitia, E.C. and Whitaker, P.M., Formation of a glial scar following microinjection of fetal neurons into the hippocampus or midbrain of the adult rat: an immunocytochemical study, Neurosci. Lett., 38 (1983) 145-150. 4 Bjorklund, A., Gage, F.H., Stenevi, U. and Dunnett, S.B., Survival and growth of intrahippocampal implants of septal cell suspensions, Acta Physiol. Scand. Suppl., 522 (1983) 49-58. 5 Connor, J.A. and Stevens, C.F., Voltage clamp studies of a transient outward membrane current in gastropod neural somata, J. Physiol. (London), 213 (1971) 21-30. 6 Crunelli, V., Forda, S., Brooks, P.A., Wilson, K.C.P., Wise, J.C.M. and Kelly, J.S., Passive membrane properties of neurones in the dorsal raphe and periaqueductal grey recorded in vitro, Neurosci. Lett., 40 (1983) 263-268. 7 Crunelli, V. and Segal, M., Electrophysiological study of the rat nucleus raphe medianus. J. Physiol (London), 328 (1982) 43-44. 8 Dekin, M.S. and Getting, P.A., Firing pattern of neurons in the nucleus tractus solitarius: modulation by membrane hyperpolarization, Brain Research, 324 (1984) 180-184. 9 Halliwell, J.V. and Adams, P . R , Voltage clamp analysis of muscarinic excitation in hippocampal neurons, Brain Research, 250 (1982) 71-92. 10 Low, W.C., Lewis, P.R., Bunch, T.S., Dunnett, S.B., Thomas, S.R., Iversen, S.D., Bjorklund. A. and Stenevi,
5-HT 5-HTP MR PSP Sub TEA
11
12
13 14
15
16 17
18
19
5-hydroxytryptamine (serotonin) 5-hydroxytryptophan mesencephalic raphe postsynaptic potentials subiculum tetraethylammonium
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