Electrophysiological study of mammalian neurons from ventral mesencephalon grown in primary dissociated cell culture

Brain Research, 310 (1984) 142-148 Elsevier

142 BRE 20363

Electrophysiological study of mammalian neurons from ventral mesencephalon grown in primary dissociated cell culture ERIC J. HEYER*

Department of Neurology, The Mount Sinai School of Medicine, New York, NY (U.S.A.) (Accepted May 8th, 1984)

Key words: ventral mesencephalon neurons - - cell culture - - action potential - - calcium

Mammalian neurons from ventral mesencephalon were grown in primary dissociated cell culture. While dopaminergic neurons were found in culture and used as a marker for this area of the nervous system, our study did not segregate the neurons in terms of their dopaminergic nature. Indeed all of the results were probably from nondopaminergic neurons. Action potentials dependent on sodium and potassium could be elicited from neurons placed in a balanced salt solution. Following blockage of sodium current by tetrodotoxin, and reduction of potassium current by tetraethylammonium and 3-aminopyridine, long-duration action potentials could be elicited by intracellular stimulation. These action potentials were most probably calcium-dependent since they could not be elicited in bathing solution containing manganese, a calcium-conductance blocker.

Mammalian neurons from ventral mesencephalon (VM) were grown in primary dissociated cell culture. Previous work has utilized these cultures for biochemical and morphological studies 3.34,35. Recent

dendrites 13,24,31,39 and acts on its own dendrites presumably in an autoregulatory capacity 1,5,7~16,17~25. Re-

work by Nowak 3e has used these cultures to look at

rons using slices of mesencephalon from guinea pig and presented data on calcium conductances underlying calcium dependent dendritic release. As well,

glutamate-activated responses. We have used these cultures to perform intracellular electrophysiological experiments. Electrophysioiogical investigation of neurons from this area has great neurophysiological and clinical interest. VM includes areas regulating a number of physiological functions, for example, substantia nigra is part of the 'basal ganglia system' concerned with modifying motor actions. In vivo, VM includes substantia nigra zona compacta where dopaminergic neurons are located 8.27. These dopaminergic neurons project primarily to striatum8,40. Loss of these dopaminergic neurons and their action in striaturn produces Parkinson's Disease. In addition, there is evidence that in vivo dopaminergic neurons act locally. These neurons extend processes within substantia nigra zona compacta 15 and to zona reticulata~. Dopamine is released from dopaminergic neuron

cently, Llinas and coworkers 26 characterized some electrophysiological properties of dopaminergic neu-

dopamine is presently thought to modulate input from striatum38,41. Because dopamine is synthesized, released and acts in substantia nigra specifically and VM generally, electrophysiological study of cultured neurons from VM is a useful model in which to investigate actions of dopamine. The first step in such an investigation is to demonstrate the electrophysiological characteristics of VM neurons. This study presents such evidence: cultured neurons o f V M can be impaled by micropipettes; spontaneous activity can be recorded from VM neurons; action potentials can be elicited to determine active m e m b r a n e properties and current pulses passed across the m e m b r a n e to determine passive m e m b r a n e properties. These action potentials depend on sodium and potassium cur-

• Supported in part by NINCDS Clinical Center for Research in Parkinson's and Allied Diseases (NS 11631-10), Research Grant from the American Parkinson Disease Association, NINCDS Teacher-Investigator Development Award (NS 00657), and NIH Grant RR-71, Division of Research Resources, General Clinical Research Centers Branch.

Correspondence: E. J. Heyer, The Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, U.S.A. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.

143 rents. Under special circumstances, in which sodium current is blocked by tetrodotoxin and potassium current is reduced by both tetraethylammonium and 3aminopyridine, action potentials dependent on calcium and potassium can be elicited. Since dopaminergic neurons represent less than ten percent of the neurons in culture, the data gathered can be assumed to be from nondopaminergic neurons in general. Cell cultures were prepared from VM dissected from 13-day old fetal mice. At this age dopaminergic neurons are already present in vivo as shown by fluorescent histochemistry 14. The dissection3, 34 was carried out using a dissecting microscope equipped with both direct and indirect illumination. The mesencephalon was located visually using the pontine flexure and diencephalon as landmarks for the caudal and rostral boundaries. The ventral surface was removed using the aqueduct as a horizontal landmark. Plating and maintenance of cultures 37 were as described previously. The VMs were minced as a batch, mechanically disrupted and plated onto collagen-coated 22mm coverships placed in 35-mm culture dishes (Falcon 3001) or directly onto 35-mm culture dishes (Falcon Primaria 3801); 1.5 to 2 VMs were placed in each culture. Cultures were maintained at 35 °C in 10% carbon dioxide and 90% room air in a growth medium containing Eagle's minimum essential medium (MEM). MEM was always supplemented with increased glucose (6 g/liter) and sodium bicarbonate (3.7 g/liter). The cultures were initially grown in a mixture of MEM plus 10% fetal calf serum (FCS) and 10% horse serum (HS) (MEM, FCS, and HS were obtained from Gibco). After one week 5-fluorodeoxyuridine (6.7 btg/ml/culture) and uridine (16.5/~g/ml/ culture) were added to MEM plus 10% HS for 3 days, and the cultures thereafter maintained in MEM plus 10% HS. They were fed twice weekly. Electrophysiological experiments were performed 4-8 weeks following plating. Cultures were prepared for catecholamine histofluorescence using a modified glyoxylic acid method in which the cultures were incubated in 10/~M alpha-methylnorepinephrine prior to exposure to glyoxylic acid 42. They were viewed using a'fluorescent Leitz microscope. Intracellular recordings were made with glass micropipettes (30-50 Mr2) filled with 4 M potassium acetate. A conventional bridge circuit (WPI M707) permitted simultaneous measurement of membrane

potential and injection of current through a single micropipette. Maximum rate of rise, duration and magnitude of action potential overshoot were measured from film of action potentials obtained from the screen of a storage oscilloscope. The maximum rate of rise was measured manually using a film enlarger of pictures of expanded oscilloscope sweeps, and the duration of the action potentials from half maximal amplitude. Statistical analyses were performed using a computer program (CLINFO). Recordings were made from cultures viewed through an inverted phase microscope (Leitz Diavert) where the stage was modified to maintain the temperature between 35 and 37 °C. For electrophysiological study, the cultures were maintained in 3 ml of Tris buffered saline solution (control bathing solution) (TBS) consisting of (in mM): NaCI 145; KC1 5.3; CaCI 2 5.0; MgC12 0.8; Tris-HC1 13; and glucose 5.6. Osmolarity was 300-310 mOsm and pH was 7.2-7.4. In solutions with reduced sodium concentration, Tris-HC1 was substituted for sodium on a one to one molar basis. In calcium-free solutions, Tris-HC1 was substituted for calcium chloride in the molar ratio of 3:2, and in increased magnesium bathing solution (12 mM) (Mg TBS), sodium chloride was reduced by the same ratio. Since we did not use calcium chelators, calcium-free solutions probably contained 20-100¢tM calcium 12. When tetraethylammonium (25 mM) and 3-aminopyridine (5 mM) were added to the bathing solution, sodium chloride concentration was lowered to maintain constant osmolarity. Tetrodotoxin was added from a 1 mM stock solution to obtain concentrations of 1-33/~M. All chemicals were reagent grade (Fisher or Sigma). A few experiments were performed in phosphate buffered saline (PBS) solution consisting of (in mM): NaC1 137; KC1 2.7; Na2HPO 4 15.7; KH2PO 4 1.2; CaCI 2 0.9; MgC12 0.5. Heavy mineral oil was applied to the surface of the bathing solution to retard evaporation. Primary dissociated cell cultures of VM contained neurons which ranged in size from 10-25 microns. Each culture batch was stained with glyoxylic acid to be certain that VM was indeed isolated. About 5-10% of the neurons were dopaminergic as evidenced by the number of catecholamine fluorescent cells. All of the fluorescent cells were dopaminergic because preincubation with a dopamine uptake blocker, benztropine (5 ~tM)3"23, eliminated cellular

144

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4 0 mV Fig. 1. A: spontaneously occurring action potentials were recorded from VM neurons in PBS solution on a strip chart recorder at resting m e m b r a n e potential (RMP) (-56.0 mV) (A1) and hyperpolarized to - 8 0 m V (A 0. These action potentials were attenuated due to the response time of the recorder. Action potentials, and the underlying excitatory postsynaptic potential, were seen more clearly in pictures taken from an oscilloscope at R M P (A2) and at - 8 0 mV (A3) at the same time. A 2 was recorded at the arrow in A 1, and A 3 at the asterix in A l. The time scale is 10 s for A1, and 200 ms for A 2 and A3; the voltage scale is 40 mV. Constant current pulses were applied when the neuron was hyperpolarized to make sure the bridge was balanced (observed on an oscilloscope trace which is not shown). The current pulses are seen in A 1 at this m e m b r a n e potential transition. These and subsequent recordings were retouched both to remove grid markings and to accentuate the trace. B: action potentials were elicited in response to short depolarizing pulses of 0.5 ms (BI,B2), and to longer pulses of 30 ms (B3). Records from a single neuron in MgTBS at R M P - 5 8 mV. Stimulus 'on' was denoted by the filled arrowhead up, and 'off' by the filled arrowhead down in this and subsequent traces.

TABLE I

Electrophysiological properties of ventral mesencephalic neurons Each entry represents m e a n + standard error of the mean. The n u m b e r of neurons used for each entry is shown in parenthesis. Input resistance was determined by the voltage response to 60 ms hyperpolarizing pulses of current delivered at resting m e m b r a n e potential. T E A and 3-AP refer to t e t r a e t h y l a m m o n i u m and 3-aminopyridine. Asterisks indicate differences from control which were significant at the 0.001 (*), 0.003 (**) and 0.04 (***) levels using the two-tailed Student t-test.

Resting m e m b r a n e potential Overshoot Duration M a x i m u m rate of rise Input resistance

Control (TBS)

TEA/3AP-- TBS

-51.9 +21.5 1.1 173.6 93.9

- 3 7 . 3 + 1.1 (6) mV* +29.0 + 1.6 (5) mV 406.0 + 34.6 (5) ms**

+ 0.1 (81) mV* _+ 0.3 (47) m V _+ 0.01 (45) ms** + 4.8 (26) V/s + 1.3 (46) Mf~***

206.8 _+ 20.8 (6) Mr2***

145 fluorescence, whereas preincubation with a norepinephrine uptake blocker, desmethylimipramine (5 ~ M ) 3,6, had no effect on cellular fluorescence (data not shown). Electrophysiological data were primarily from nondopaminergic neurons as was determined by staining for fluorescent cells after the experiment. The larger neurons were impaled by micropipettes and over half the neurons impaled, had spontaneous generation of action potentials (54% - 44/82 neurons) (Fig. 1A1, A2). This activity was synaptically elicited because membrane hyperpolarization demonstrated excitatory postsynaptic potentials preceding action potential generation (Fig. 1A3). In addition, if neurons were recorded from bathing solution with increased magnesium (Mg TBS) in order to reduce synaptic transmission, less than 20% of the neurons generated action potentials spontaneously (16% - - 6/38 neurons). Depolarization of VM neurons with brief current pulses (0.5 ms) evoked action potentials of short duration (1.1 + 0.01 ms) (mean + S.E.M.) (Fig. 1B1, Table I) with high maximum rate of rise (173.6 + 4.8 V/s) (Fig. 1B2, Table I). Depolarization with a longer pulse (30 ms) demonstrated more clearly the hyperpolarizing afterpotential almost always seen (Fig. 1B3). Action potentials elicited in TBS were dependent on sodium. They could not be evoked following the addition of tetrodotoxin to the bathing medium (0/16 neurons) (Fig. 2A). Indeed, the overshoot of action potentials varied by 60.3 mV per decade change of sodium concentration (Fig. 2E); a value close to that predicted by the Nernst equation. In calcium-free bathing solution, action potentials were of short duration (1.3 + 0.06 ms, n = 7) and maximum rate of rise was unaltered (203.8 + 13.0 V/s, n = 8) (Fig. 2B). With addition of 25 mM tetraethylammonium, 5 mM 3-aminopyridine and 1/~M tetrodotoxin to bathing solution, depolarizing stimulation elicited regenerative responses of long duration (406.0 + 34.6 ms) (Fig. 2C, Table I). Long-duration action potentials could be elicited in almost all neurons if sufficient current was passed to depolarize the neuronal membrane by 30-40 mV for 10-20 ms. Steady membrane hyperpolarization reduced and depolarization increased action potential duration. Action potential duration remained constant if neurons were stimulated at sufficiently long interstimulus intervals (usually 10 s). These action

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Fig. 2. Action potentials could not be elicited from V M neurons in control TBS in the presence of tetrodotoxin (TTX) (1/~M) (A), but could be elicited in calcium free bathing solution (B).

In the presence of T£X (1/~M), tetraethylammonium (TEA) (25 mM) and 3-aminopyridine (3-AP) (5 mM) long-duration action potentials were elicited (C). These action potentials were blocked in the presence of manganese (MgCI2) (5 mM) (D). 'A' and 'D' were 4 and 3 superimposed traces of depolarizing membrane pulses. The stimulus 'off' was not denoted in 'C' because the pulse was of short duration (5 ms). Action potential overshoot varied as a function of extracellular sodium concentration (E). Average action potential overshoot was linearly related to lOgl0 of the sodium concentration. Each point represents the average overshoot amplitude of 5-9 neurons. Standard error of the mean was 2.5, 1.8 and 1.5 mV for 50, 97.5 and 145 mM sodium. The values of overshoot at 50 and 97.5 mM differed from overshoot at 145 mM sodium at the 0.01 and 0.02 levels of confidence using the two-tailed Student t-test, but differed from each other at only the 0.10 level.

potentials were calcium-dependent because a calcium-conductance blocker, manganese (5 mM MnC12), eliminated all regenerative responses (Fig. 2D). Thus, while action potentials of VM neurons were sodium-dependent and tetrodotoxin-sensitive in control bathing medium, long-duration calci-

146 um-dependent action potentials could be evoked if potassium conductance was reduced by tetraethylammonium and 3-aminopyridine, and sodium conductance blocked by tetrodotoxin. This result demonstrates the presence of voltage dependent calcium conductance in these neurons. Many areas of the central nervous system have been prepared in primary dissociated cell culture: forebrain 10, spinal cord 37, hippocampus 32, cerebellum 28, hypothalamus 2, striatum 29 and VM 3.32,34. When studied electrophysiologically, cultured neurons have displayed electrophysiological properties similar to what has been found in in vivo recordings 30. In general, sodium-dependent action potentials have been recorded from neurons in culturelS,36; however, under special circumstances neurons generate calcium-dependent action potentials TM. These calcium-dependent action potentials have been used as models for the presynaptic terminal 11,19 and dendritic branches20, 2~ where calcium conductance plays a greater physiological role. Indeed application of chemicals onto neurons and modification of an electrophysiological response has been shown by Dunlap and Fischbach 1l using chick sensory neurons for norepinephrine, gamma-aminobutyric acid, serotonin, enkephalin and somatostatin; and by Heyer and Macdonald, using mouse spinal cord for penicillin2a, bicuculline20, 21 and the barbiturates ~9 phenobarbital and pentobarbital. Llinas 26 has investigated electrophysiological properties of neurons in pars compacta of the substantia nigra in vitro in guinea pig mesencephalic slices. He demonstrated that these neurons have high input resistance (60-200 Mg2) and that they can generate calcium-dependent action potentials from dendritic sites: one associated with inactivation at resting membrane potentials and the other with more usual calcium conductance characteristics. These calcium conductances fulfil most of the physiological characteristics necessary for the calcium dependent release of dopamine and acetylcholinesterase from dendrites of dopaminergic neurons in substantia nigra. Most probably the calcium-dependent action potentials recorded from VM neurons in cul-

1 Argiolas, A., Melis, M. R., Fadda, F. and Gessa, G. L., Evidence for dopamine autoreceptors controlling dopamine synthesis in the substantia nigra, Brain Research, 234 (1982) 177-181.

ture arise from similar dendritic sites, although we have not investigated that. Prochiantz et al. TM used primary dissociated cell cultures of VM system to study neuronal development of dopaminergic neurons especially the effect of factors released by the target neurons or glia 9.35. The electrophysiological responses of neurons in these cultures has not been investigated until now 22. Our results demonstrate that VM neurons have some properties similar to those found in dorsal root ganglion neurons prepared from primary dissociated cell cultures of mouse spinal cord 18. In these latter cultures, resting potential was -48.8 mV, overshoot was + 28.0 mV, duration was 2.0 ms, maximum rate of rise was 105.2 V/s and input resistance was 49.7 MQ. The longer duration, lower maximum rate of rise and lower input resistance of dorsal root ganglion neurons may reflect more prominent calcium current with subsequent activation of calcium-dependent potassium current. The higher input resistance of VM neurons may also be due to the smaller diameter of these neurons. Nevertheless, the electrophysiological properties of VM neurons are in general similar to those found in other cultured neurons30, and establish that these neurons are appropriate for electrophysiological investigation. In addition, since calcium dependent action potentials can be elicited under special circumstances, VM neurons can be used as a model for presynaptic or postsynaptic dendritic actions. These cultures of VM provide an in vitro model in which it will be possible to determine the electrophysiological interactions between dopamine and other transmitters or neuromodulators, as well as the direct action of dopamine on membrane conductance. We would like to thank Ms. Dianne Davis and Ms. Minerva Feliciano for secretarial assistance, Ms. Diana Cabrera for help with cell culturing, Ms. Marilyn Ilvento for photographic assistance, Dr. Catherine Mytilineou for help with histochemistry, Dr. Kenneth Bergmann for assistance with statistical analyses, and Dr. M. D. Yahr for his support and interest.

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