Miniature excitatory synaptic currents in cultured ...

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BAY K 8644; Nimodipine; Tetanus toxin. We performed patch clamp recordings in the whole cell mode from cultured embryonic mouse hippocampal neurons.
Brain Research, 518 (1990) 257-268 Elsevier

257

BRES 15539

Miniature excitatory synaptic currents in cultured hippocampal neurons David M. Finch t'4, Robin S. Fisher 2"5 and Meyer B. Jackson 2-4 ~Department of Neurology, Reed Neurological Research Center, 2Mental Retardation Research Center, ~Department of Biology, 4Brain Research Institute, and 5Departments of Anatomy and Cell Biology, Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA 90024 (U.S.A.) (Accepted 21 November 1989) Key words: Miniature synaptic current; Glutamate; Quisqualate; N-methyl-D-aspartate; Synaptic transmission; Ca2+; Dihydropyridine; BAY K 8644; Nimodipine; Tetanus toxin

We performed patch clamp recordings in the whole cell mode from cultured embryonic mouse hippocampal neurons. In bathing solutions containing tetrodotoxin (TTX), the cells showed spontaneous inward currents (SICs) ranging in size from 1 to 100 pA. Several observations indicated that the SICs were miniature excitatory synaptic currents mediated primarily by non-NMDA (N-methyl-o-aspartate) excitatory amino acid receptors: the rising phase of SICs was fast (1 ms to half amplitude at room temperature) and smooth, suggesting unitary events. The SICs were blocked by the broad-spectrum glutamate receptor antagonist y-D-glutamylglycine (DGG), but not by the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (5-APV). SICs were also blocked by desensitizing concentrations of quisqualate. Incubating cells in tetanus toxin, which blocks exocytotic transmitter release, eliminated SICs. The presence of SICs was consistent with the morphological arrangement of glutamatergic innervation in the cell cultures demonstrated immunohistochemically. Spontaneous outward currents (SOCs) were blocked by bicucuUine and presumed to be mediated by GABA A receptors. This is consistent with immunohistochemical demonstration of GABAergic synapses. SIC frequency was increased in a calcium dependent manner by bathing the cells in a solution high in K ÷, and application of the dihydropyridine L-type calcium channel agonist BAY K 8644 increased the frequency of SICs. Increases in SIC frequency produced by high K + solutions were reversed by Cd2÷ and ~o-conotoxin GVIA, but not by the selective L-type channel antagonist nimodipine. This suggested that presynaptic L-type channels were in a gating mode that was not blocked by nimodipine, and/or that another class of calcium channel makes a dominant contribution to excitatory transmitter release.

INTRODUCTION Synaptic transmission occurs through the release of n e u r o t r a n s m i t t e r from vesicles located within synaptic terminals. Transmitter release at the neuromuscular junction has been extensively studied, and is revealed in electrophysiological recordings of stimulus-evoked endplate potentials and in recordings of spontaneous miniature e n d p l a t e potentials (mEPPs). m E P P s are the elem e n t a r y quanta of synaptic transmission, and reflect the presynaptic release and subsequent postsynaptic binding of acetylcholine contained in single vesicles 9'19. (Nonquantal release has also been observed, but produces smaller m e m b r a n e potential changes34.) The frequency of occurrence of m E P P s can be increased by depolarizing terminals with electrical stimulation or bathing in solutions with high [K÷] 39. E v o k e d transmitter release at the n e u r o m u s c u l a r junction and squid giant synapse is dep e n d e n t u p o n the presence of Ca 2÷ and can be initiated by directly injecting Ca 2+ into the presynaptic terminal ~s' 33,38,46. Miniature synaptic currents are a valuable indi-

cator for determining the m o l e c u l a r mechanisms of synaptic transmission. Unlike studies of e v o k e d transmitter release, analysis of these events provides an independent criterion for separating the presynaptic and postsynaptic elements. Thus, the sites of action of modulating agents can be identified and the m o l e c u l a r nature of the targets inferred. A n a l o g o u s vesicular release of n e u r o t r a n s m i t t e r from central neurons 6'32 has not been as well characterized largely due to technical p r o b l e m s associated with recordi n g these small signals. In particular, knowledge about the mechanisms of transmitter release from m a m m a l i a n central neurons and their susceptibility to physiological regulation remains incomplete. Recently, Trussell et al. 7° and B e k k e r s and Stevens 4 r e p o r t e d the presence of miniature spontaneous inward currents (SICs) in whole cell patch clamp recordings from cultured mouse spinal and h i p p o c a m p a l neurons. Evidence for the glutamatergic nature of these currents was p r e s e n t e d , and a differential distribution of active sites at zones of putative synaptic contact was d e m o n s t r a t e d . Miniature spontane-

Correspondence: D. M. Finch, 73-364 CHS, Brain Research Institute, University of California, Los Angeles, CA 90024, U.S.A. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

258 o u s c u r r e n t s c a n also b e r e c o r d e d f r o m b r a i n slices :~< 4~.6o.6~ a n d d i s s o c i a t e d n e u r o n s ~7. In t h e p r e s e n t w o r k , we h a v e u s e d t h e p a t c h c l a m p t e c h n i q u e 26 to e x a m i n e p u t a t i v e m i n i a t u r e s y n a p t i c inw a r d c u r r e n t s o c c u r r i n g s p o n t a n e o u s l y in m o u s e h i p p o c a m p a l cell c u l t u r e s . S i n c e t h e r e is c o n s i d e r a b l e e v i d e n c e t h a t g l u t a m a t e is a n i m p o r t a n t e x c i t a t o r y t r a n s m i t t e r in t h e h i p p o c a m p u s m'~ 1,24, we f u r t h e r t e s t e d t h e h y p o t h e s i s t h a t t h e SICs r e p r e s e n t e d v e s i c u l a r r e l e a s e o f g l u t a m a t e or a glutamate-like transmitter substance. We studied the role o f g l u t a m a t e r e c e p t o r s u b t y p e s , a n d e x a m i n e d t h e d e p e n d e n c e o f t h e S1Cs o n Ca 2+. Part o f this w o r k was p r e s e n t e d in a b s t r a c t f o r m e°.

MATERIALS AND METHODS

Primary cultures of fetal mouse hippocampus were prepared using the method of Swaiman et al. 66. Briefly, the hippocampus was dissected from the fetal telencephalon (18 day gestational age), treated for 30 rain at 37 °C with 0.25% trypsin in isosmolar calcium-free saline and then triturated. Cells were plated at a density of 106 cells for each 35 mm Falcon 'Primaria' dish in minimum essential medium (MEM) with 10% each of horse and fetal calf serum. The medium was changed after 5 days to MEM containing 10% horse serum, 10 ktM fluorodeoxyuridine and 50 ~M uridine. The medium was shifted 2 days later to MEM with 10% horse serum which was then changed twice weekly. Cultures were used 1-5 weeks after plating. During electrophysiological experiments, cells were bathed at room temperature (22-24 °C) in physiological saline buffered at pH 7.2 ('normal bath', composition listed in Table l), and visualized with an inverted phase contrast microscope. Patch pipets of borosilicate glass with resistances of 10-20 MI2 and filled with a potassium gluconate solution buffered at pH 7.2 ('normal pipet,' Table I) were used for recording. A membrane seal (usually 5 GI2) was achieved by gentle suction. The membrane under the pipet tip was then ruptured by further brief suction to obtain whole-cell recordings. The membrane potential was clamped at -60 mV with an EPC-5 patch clamp amplifier (List Electronics). To block sodium-dependent action potentials and currents, tetrodotoxin (TTX) was included in the bathing solutions at a concentration of 0.16 or 3.13 IzM. Spontaneous or evoked action currents or action potentials were never observed with either concentration of TTX. Drugs were administered either by 'focal' or 'bath' application. For focal application, drugs were ejected from a patch pipet placed 5-25/~m from the soma using pressure from a Picospritzer (General Valve Corp., 1-5 psi or 0.07-0,35 kg/cm2). For bath application,

drugs were applied by exchanging the m~rmal bath with drugcontaining bath using a continuous perfusion system. The stated drug concentrations for focill applications indicate the concentra lions in the pipets, which were diluted to an unknown extent m ihc bath. Stock solutions of drugs were prepared at a concentration ol 10 raM, stored frozen in the dark and diluted into bathing solutions just prior to the initiation of each experimcul BAY K 8,644 and nimodipine were dissolved in DMSO. Duc lo the photosensitive nature of these compounds, cxpcriments with them were performed in indirect light. The DMSO concentration after dilution ranged from 0.01 to 1% (usually {}.[)[ 0. I g{), and DMSO was applied in the absence of drug as a control. Stock solutions of other compounds were dissolved in normal bath. BAY K 8;644 and nimodipine were donated by Dr. A. Scriabine of Miles Pharmaceuticals. Excitatory amino acid antagonists were obtained from Cambridge Research Biochemicals, tetanus toxin from Calbiochem, ~o-conotoxin GVIA from Peninsula Labs, and other compounds from Sigma Chemicals. Data were recorded on a VCR-based tapc system (Unitrade), a chart recorder, or both instruments. The resistance of the pipet plus the cell was monitored at intervals throughout the experiments with 10 mV, 200 ins hyperpolarizing command pulses to verify that changes in membrane currents rcflected experimental manipulations rather than changes in seal properties. Resting potential was measured at the beginning of each recording and at intervals throughout the experiments to allow assessment of cell viability. For data analysis requiring measurement of the frequency of spontaneous currents, only events greater than twice the baseline noise were counted from the chart recordings. Figures were prepared from chart records or, when faithful reproduction of faster signal components was required, from plots obtained from the VCR system (3 kHz filtering) using a digital oscilloscope and digital plotter. The neurophysiological experiments were complemented by morphological analyses of hippocampal neurons after 21 days in culture. Marker molecules of glutamate (glutaminase, GLU; a synthesizing enzyme for glutamate, see ref. 16) and y-aminobutyric acid (GABA) (glutamic acid decarboxylase, GAD; the synthesizing enzyme for G A B A ) were detected by immunohistochemical methods. These markers are present in cell bodies of neurons, and in punctae visible under light microscopy that likely represent excitatory (GLU) and inhibitory (GABA) synaptic contacts. The cultures were fixed by immersion in 4% paraR)rmaldehyde. They were stained by unlabeled antibody peroxidase-antiperoxidase immunohistochemical methods as detailed in previous reports 2t47. The antibodies directed against GLU (rabbit antirat kidney GLU) were obtained from N. Curthoys ~4. The antibodies directed against GAD (sheep anti-rat brain GAD-sheep anti-GAD complex) were obtained from I.J. Kopin 5~. After labeling with 3,3'-diaminobenzidine, the coverslips bearing the cultured cells were mounted on large rectangular coverslips for light microscopical examination. The results for each marker were replicated 5 times. "Specific' labeling was considered to be the differential staining between these cultures and comparable control cultures treated with non-immune serum from the appropriate species.

TABLE I Solution names and compositions

Concentrations in mM. Bathing solutions contained TI'X.

Normal pipet Normal bath 0 Ca High K High K, 0 Ca

Potassium gluconate

NaCl

KCI

140 -

1 138 138 126 126

4 4 16 16

MgCl 2

CaCl 2

EGTA

HEPES

1

1 3 l 3

0.5

5

10

2 0 2 0

5

10 10 10 l(I

5

259 RESULTS

In control cultures, 92% of the cells showed SICs and 20% of the cells showed SOCs. Accordingly, most results

Voltage clamp recordings were obtained from 129 neurons, with a mean age of 20 days from plating. The mean resting potential of 72 cells recorded with normal bath and pipet solutions (Table I, TTX in the bath), and no prior experimental manipulations was -54 + 5.0 (S.D.) mV. In the absence of TTX, recordings were characterized by action currents and small, apparently random, fast, inward currents occurring in the absence of any stimulation or drug application (Fig. 1A 0. These small currents are referred to here as spontaneous inward currents (SICs). The rising phase of SICs was fast and smooth suggesting unitary events (see Fig. 1 of succeeding paper2°"). Spontaneous outward currents (SOCs) were also observed but at a much lower frequency. Action currents disappeared in TTX (Fig. 1A2). With the exception of Fig. 1, all figures show data collected in the presence of TTX.

presented here concern only SICs. The mean frequency of SICs was 0.81/s, and the mean frequency of SOCs was 0.01/s. SIC frequency was temperature-dependent. In 4 experiments in which cells were held at a temperature of 34-37 °C (instead of the usual 22-24 °C), the mean SIC frequency was 15.8/s. Accurate reversal potentials were difficult to determine due to the variable size and intermittent nature of the currents, but SICs appeared to reverse near 0 mV and SOCs appeared to reverse between -60 and -80 mV. SIC frequency was independent of the holding potential. Amplitudes and half-amplitude rise and fall times of SICs were measured in 7 representative cells. Rise times were measured from onset to 1/2 of the peak amplitude. Fall times were measured from peak amplitude to 1/2 of the peak amplitude on the decaying phase. The modal SIC amplitude was about 10 pA (Fig. 3), but SICs varied from just greater than the noise level of 1-3 pA to more than 100 pA (Figs. 2, 3). The mean SIC amplitude was 17.5 pA + 5.2 (SD). The SIC rise time averaged about 1 ms, and the fall time was about 10 ms. Thus, even small events were highly asymmetric, and not rectangular, as would be expected if they were caused by the opening of a single channel. There were no consistent correlations between SIC rise time and amplitude nor between SIC fall time and amplitude (Fig. 4). In some of the cells, the largest SICs tended to show short rise and fall times, but there were many smaller events with similar short time courses (Fig. 4). SOCs showed a slower time course than SICs (Fig. 2A4).

Characteristics of miniature spontaneous currents The SICs and SOCs were one or two orders of magnitude smaller than action currents (Fig. 1). Fig. 1 shows that action currents appeared to be triggered by SICs or by events larger than the SICs (A1, curved arrow). Similarly, spontaneous action potentials recorded under current clamp (0 nA) appeared to be often triggered by a class of depolarizations larger than those seen in TTX (Fig. 1B1, curved arrow). These larger events were not studied further but presumably represented the effects of synchronous transmitter release triggered by presynaptic action potentials.

A

B

Voltage Clamp

Current Clamp Cell # 30622 3 2

100 pA

No l-IX 200 msec

J

40 mV

200 msec

,IF &

2 TTX

• ~

• ~

Fig. 1. Comparison of recordings in the absence and in the presence of TFX (0.16/~M). The records in column A were obtained under voltage clamp (holding potential of -60 mV): note the large inward action currents (shown as a downward deflection) in the absence of TTX (Al); and the much smaller SICs seen both without and with TTX (A~ and A2, filled triangles). Records in column B were obtained from the same cell at rest under current clamp (0 current injected), and show action potentials in the absence of TTX (B0, and much smaller spontaneous depolarizations without and with TTX (B t and B2, open triangles). The insets (arrows) show the rising phases at ×20 horizontal resolution. In all subsequent figures, TTX was present in the bathing media, and figures are presented at higher gain to show SICs more clearly.

260

A

B

S p o n t a n e o u s Synapt~c Currents

S p o n t a n e o u s Synaptic Potentials

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rl

2 - , - r - T - T r, -lr[,-

3

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2 mV

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C

Fig. 2. SICs and SOCs (column A, downward and upward deflections, respectively) recorded under voltage clamp; and spontaneous depolarizations and hyperpolarizations (column B, upward and downward deflections, respectively) recorded under current clamp, all from the same cell. Trace A 4 shows that SICs (double arrow) were in general faster than SOCs (single arrow).

Immunohistochemistry Our pharmacological experiments (described below) indicate that the transmitter for SICs is glutamate and the transmitter for SOCs is G A B A . In order to obtain anatomical correlates to our physiological investigations, we carried out immunohistochemical studies to localize GLU and G A D in our cell cultures (see Materials and Methods). GLU and G A D were specifically labeled in all of the tested cultures (Fig. 5). Both markers were localized in cell bodies with neuronal morphology, and in punctae (neuritic terminals). GLU-immunoreactive cell bodies and punctae were always more frequent than GAD-immunoreactive cell bodies and punctae.

Four different arrangements of GLU- and GADimmunoreactive cell bodies and punctae were obtained in these experiments. (1) GLU-positive punctae were 1o-

70 r = -0.02. p > 0.7 60 50 40

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Fig. 3. Amplitude histogram of SICs in a representative cell, held at -60 mV in normal bath. 2 pA binwidth. The modal amplitude was 8-10 pA. The smallest events ( < 2 pA) could often not be clearly distinguished from baseline noise.

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261

Fig. 5. Photomicrographs of hippocampal neurons after 21 days in dissociated culture (brightfield transmission illumination, 100 x oil objective). A: a glutaminase immunoreactive cell (*) is contacted by glutaminase immunoreactive punctae (arrowheads) along somatic (s) and neuritic (n) regions of its profile. B: a glutaminase non-immunoreactive cell (*) is contacted by glutaminase immunoreactive punctae (arrowheads) along somatic (s) and neuritic (n) regions of its profile. C: a glutamic acid decarboxylase immunoreactive cell (*) is contacted by glutamic acid decarboxylase immunoreactive punctae (arrowheads) along somatic (s) and neuritic (n) regions of its profile.

cated next to GLU-positive cell bodies (Fig. 5A). (2) GLU-positive punctae were located next to GLU-negative cell bodies (Fig. 5B). (3) GAD-positive punctae were located next to GAD-negative cell bodies (not shown). (4) GAD-positive punctae were located next to GAD-positive cell bodies (Fig. 5C). Thus both candidate glutamatergic and G A B A e r g i c neurons appeared to receive both glutamatergic and G A B A e r g i c synaptic contact. The distal glutamatergic synapses shown here on neurites might have produced smaller, slower, synaptic events than somatic synapses due to electrotonic decay and imperfect space clamp. This might have contributed to the variable amplitudes and rise and fall times of SICs. However, the absence of a correlation of SIC rise or fall times with amplitude suggested that the inherent variability in SIC amplitude was large relative to the variability produced by differential electrotonic decay (see also ref. 29).

Effect of amino acid antagonists and agonists In order to identify the receptors mediating SICs and SOCs, we tested the actions of amino acid receptor-

specific ligands. Bath application of the G A B A A receptor antagonist bicuculline methiodide (100 /~M) blocked SOCs almost completely without affecting SICs. This result indicates that SOCs are mediated by G A B A A receptors. 7-D-Glutamylglycine ( D G G ) is a broad spectrum excitatory amino acid blocker and 2-amino-5 phosphono-

A

B

C

Normal Bath

1 mM DGG

Wash

i

.

_,

..

SICs

2

,,,, I

"T

; .......

--r'--'--

Glutamate

Response

c~l # 30762-1-t

Fig. 6. Block of SICs and glutamate response by the broad spectrum glutamate receptor antagonist y-D-glutamylglycine (DGG). Baseline SICs and responses to 100 ms puffs of 10/~M glutamate (arrows) were obtained in normal bath (A1.2). Then, 1 mM of DGG was perfused into the dish (B), followed by a wash with normal bath (C). Note that DGG almost totally blocked SICs, and substantially reduced the glutamate response, with partial recovery during the wash. All data from the same cell.

262

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no effect on SICs was seen, quisqualate (QUIS) responses were also not visibly affected, but N M D A

2

l Quisqualate

l NMDA

V H = -30 mV

6o pA 20 se~ Cell # 30760 1 2

Fig. 7. Effect of the NMDA antagonist 5-APV on SICs and on responses to NMDA and QUIS. Baseline SICs (At), responses to puffs of QUIS (A2, 2/aM, 100 ms puffs at arrow), and responses to puffs of NMDA (A3, 100#M, 500 ms duration puffs at arrow) were obtained in normal bath. Then, 100 ~M of 5-APV was perfused into the dish (B), followed by wash with normal bath (C). The response to NMDA was reduced to only 22% of the baseline value (B3), with partial recovery (C3), while SICs and the QUIS response were not clearly affected by 5-APV (rows 1 and 2). Records in D illustrate the voltage dependence of the NMDA response, due to Mg~-+ block at negative holding potentials 43'5°. All data from the same cell.

valeric acid (5-APV) is a selective antagonist for the N M D A receptor ~2't5. We found that 1 mM D G G blocked SICs very effectively (Fig. 6) without reducing SOCs. Lower concentrations of D G G were generally less effective. Concomitant with blockade of SICs was a strong reduction in responses to 10 # M pressure applied gluta-

A

reduced the glutamate response by 62% with a recovery to 82% of that observed prior to antagonist application. The experiments with D G G indicate that SICs arc mediated by an excitatory amino acid receptor. In order to estimate the extent to which N M D A and n o n - N M D A receptors contribute to SICs, we tested the effect of 5-APV in 7 cells with concentrations ranging from 0.01 to 1.0 mM. Generally, 5-APV had no effect on SICs except at 1 mM, where a slight effect was seen. In two experiments in which 0.1 mM 5-APV was bath-applied,

1

I

~

rr

3

Wash

1

1 NMDA Response

mate (Fig. 6). In 3 cells tested, perfusion of 1 mM D ( ; ( ;

C

100 ~.tM 5APV

responses were blocked by 75-100% (Fig. 7). Since N M D A and n o n - N M D A receptors have a strikingly different voltage dependence, the effect of voltage was examined. Depolarization should enhance N M D A receptors by relief of voltage d e p e n d e n t block by Mg2+ 43,50. Depolarization from - 6 0 to - 3 0 mV increased N M D A responses by an average of 220% (three cells, Fig. 7D), while SIC amplitudes were reduced by 27% and Q U I S responses were reduced 23%. The percent reductions in SICs and Q U I S responses were indistinguishable. The mean SIC decay time was also not significantly affected by depolarization. Thus, under the conditions of this study, S1Cs are primarily n o n - N M D A receptorgenerated currents (but see Discussion). Additional insight into the nature of the receptor mediating SICs was obtained by using desensitizing concentrations of agonists. In 6 out of 8 cases, local application of Q U I S ( l - 1 0 # M ) eliminated SICs (Fig. 8A), presumably by desensitizing Q U I S receptors ~5`°~-Ttt.

Date and Preineubation:

Quisqualate Pipet Down

UP

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i,

4/17/87 None

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