Journal of Physiology (1991), 443, pp.

533-553 With 8 figures Printed in Great Britain

533

MEMBRANE PROPERTIES AND SYNAPTIC RESPONSES OF RAT STRIATAL NEURONES IN VITRO

BY Z.-G. JIANG AND R. A. NORTH From the Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA

(Received 27 November 1990)

SUMMARY

1. A tissue slice containing a section of striatum was cut obliquely from rat brain so as to preserve adjacent cortex and pallidum. Intracellular recordings were.made from 368 neurones, using either conventional or tight-seal configurations. 2. Two types of neurone were distinguished electrophysiologically. Principal cells (96%) had very negative resting potentials (-89 mV) and a low input resistance at the resting membrane potential (39 MQ): membrane conductance (10 nS at -65 mV) increased within tens of milliseconds after the onset of hyperpolarization (99 nS at - 120 mV). Secondary cells (4%) had less negative resting potentials (-60 mV) and a higher input resistance (117 MC2 at the resting potential): hyperpolarization caused an inward current to develop over hundreds of milliseconds that had the properties of H-current. 3. Most principal cells were activated antidromically by electrical stimulation of the globus pallidus or internal capsule. Intracellular labelling with biocytin showed that principal cells had a medium sized soma (10-18 ptm), extensive dendritic trees densely studded with spines and, in some cases, a main axon which extended towards the globus pallidus. 4. Electrical stimulation of the corpus callosum or external capsule evoked a depolarizing postsynaptic potential. This synaptic potential was reversibly blocked by a combination of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 /tM) and DL-2amino-5-phosphonovaleric acid (APV, 30 #M), but was unaffected by bicuculline (30 ftM) and picrotoxin (100 ,zM). The underlying synaptic current had a fast component (time to peak about 4 ms), the amplitude of which was linearly related to membrane potential and which was blocked by CNQX; in CNQX the synaptic current had a slower component (time to peak about 10 ms) which showed voltage dependence typical of N-methyl-D-aspartate (NMDA) receptors. Both currents reversed at -5 mV. 5. Focal electrical stimulation within the striatum (100-300 ,um from the site of intracellular recording) evoked a synaptic potential that was partially blocked (45-95 %) by CNQX and APV: the remaining synaptic potential was blocked by bicuculline (30 ,UM). The bicuculline-sensitive synaptic current reversed at the chloride equilibrium potential. 6. The findings confirm that the majority of neostriatal neurones (principal cells,
MS 8967

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Z.-G. JIANG AND R. A. NORTH

medium spiny neurones) project to the pallidum and receive synaptic inputs from cerebral cortex mediated by an excitatory amino acid acting through NMDA and non-NMDA receptors. These cells also receive synaptic inputs from intrinsic striatal neurones mediated by GABA. A minority of cells (4%) have distinct electrophysiological properties, also receive both types of synaptic input, and are presumed to be interneurones.
INTRODUCTION

Six types of neurones in the striatum have been distinguished according to the size of the soma and the appearance and arrangement of the dendrites (Kemp & Powell, 1971a; Graybiel & Ragsdale, 1983). The best studied is the one which constitutes more than 95% of the cell population in the striatum, being of medium size (9-18 ,m) and densely studded with dendritic spines. The cortical inputs form asymmetrical synaptic contacts with these neurones, mainly on the dendritic spines (Kemp & Powell, 1971 a-c). These medium spiny neurones send their main axons to the fibre bundles beyond the border of the nucleus to terminate in globus pallidus and midbrain destinations (Preston, Bishop & Kitai, 1980; Somogyi, Bolam & Smith, 1981). The axons also extensively arborize throughout a roughly spherical space around the cell bodies, some 250-450 ,tm in radius, and these axonal plexuses provide symmetrical synapses with other striatal neurones, most likely with the same type of cells (Kemp & Powell, 1971 c; Preston et al. 1980; Somogyi et al. 1981). Neurones of this type contain enzymes involved in the synthesis of y-aminobutyric acid (GABA; Ribak, Vaughn & Roberts, 1979; Fisher, Buchwald, Hull & Levine, 1986) and are believed to be inhibitory to the target neurones that they innervate outside the striatum (Yoshida & Precht, 1971; Yoshida, Rabin & Anderson, 1972). The abundance of intrinsic synaptic contacts between this class of cells within the striatum suggests local feedback inhibition among the output cells (Park, Lighthall & Kitai, 1980). Neurones in the striatum receive monosynaptic inputs from the cortex in a topographically organized but extensively overlapping way (Somogyi et al. 1981; McGeorge & Faull, 1989), as well as from the centromedian-parafascicular complex in thalamus (Mehler, 1966; Beckstead, 1984) and substantia nigra in the midbrain (Beckstead, Domesick & Nauta, 1979; Gerfen, Herkenham & Thibault, 1987). In vivo recordings in the cat showed that cortical stimulation induced biphasic synaptic potentials, a depolarization followed by a hyperpolarizing synaptic potential (Hull, Bernardi, Price & Buchwald, 1973; Calabresi, Mercuri, Stefani & Bernardi, 1990b). In vitro recordings in the rat show that intrastriatal stimulation can evoke synaptic potentials having components mediated by excitatory amino acids and by GABA (Cherubini, Herrling, Lanfumey & Stanzione, 1988; Calabresi, Mercuri & Bernardi, 1990a). These results have been interpreted to indicate that the initial depolarizing synaptic potential results directly from the action of an excitatory amino acid released from cortical afferents, and the subsequent inhibitory potential results indirectly from recurrent inhibition (Park et al. 1980). The efferent fibres from the medium spiny neurones project to the globus pallidus (Kemp & Powell 1971d; Walaas & Ouimet, 1989) and substantia nigra (Kemp & Powell, 1971 c; Gerfen, 1985). However, the electrophysiological evidence for
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establishing the medium spiny neurones as the major class of neurone projecting from the striatum is still somewhat weak because in vivo studies found that less than 3 % of the striatal neurones could be excited antidromically (Fuller, Hull & Buchwald, 1975; Kocsis, Sugimori & Kitai, 1977; Preston et al. 1980). The synaptic connections and transmitters used by the five different types of neurones other than the medium spiny neurones are largely unknown, and the basic electrical properties of these different types of neurones have not been extensively studied. The purpose of the present experiments was to determine whether striatal neurones could be further distinguished into subtypes by the ionic currents that they exhibit and the synaptic inputs that they receive. Intracellular recordings in vitro were made from cells in an oblique slice of rat striatum so that adjacent cortex and pallidum could be electrically stimulated. Whole-cell recording was used in some experiments so that the reversal potentials of synaptic currents could be measured more readily than with conventional intracellular microelectrodes. A preliminary report has appeared Jiang & North, 1990).
METHODS

Tissue preparation Male Sprague-Dawley rats were anaesthetized with halothane and killed by a heavy blow to the chest that severed major blood vessels. The brain was quickly removed and slices (thickness 300-400 sum) were cut with a vibratome in cold (1-4 C) physiological saline. An effort was made to preserve longer courses of corticostriatal input fibres (Webster, 1961) and the striatal projecting fibres to globus pallidus and substantia nigra (Voneida, 1960; Preston et al. 1980; Bishop, Chang & Kitai, 1982) by cutting slices obliquely, 30 deg rostral-up to the horizontal plane (Fig. 1). A single slice was mounted between two nylon meshes in a tissue bath, where it was totally immersed in a physiological solution flowing at 2 ml/min and maintained at 36 'C. The solution contained (mM): NaCl, 126; KCl, 2-5; NaH2PO4, 1-2; MgCl2, 1-3; CaCl2, 2-4; glucose, 10; NaH2CO3, 26 and was saturated with 95% 02 and 5% C02. When CoCl2 was added to the solution, NaH2PO4 was omitted. High-resistance microelectrode recording Neurones located in the rostral one-third of striatum were impaled with glass microelectrodes containing potassium chloride (2 M) (resistance 50-150 MQ). In some cases, potassium acetate (2 M), biocytin (2 %) in potassium chloride (1 M), or biocytin (2 %) in potassium acetate (1 M) was used. Membrane potential or current was recorded with an Axoclamp 2A preamplifier. For voltage clamp, the amplifier switched between current passing and voltage sensing at 2-3 kHz (25 % duty cycle). The membrane voltage and current were recorded on a chart-recorder (Gould 2400) with linear frequency response between 0 and 60 Hz. Action potentials and synaptic potentials were also recorded on a digital storage oscilloscope at 1000 points per sweep (Tektronix 2230). A hard copy was then made on an X-Y recorder (Model 200, The Recorder Company; frequency response 0-6 Hz). In practice, the 10-90% rise time of the pen-recorder required four digitized points. Steady-state current-voltage curves were constructed directly on the X-Y recorder using slow depolarizing ramp potentials: in this case the ramp was sufficiently slow (1 mV/s) so as to give the same current as that measured at the end of a 1 or 2 s step.

Tight-seal whole-cell recording Tight-seal whole-cell recordings were made with glass microelectrodes having initial tip resistances of 5-10 MQ, usually rising to 10-20 MQ in the whole-cell configuration. They contained (mM): caesium gluconate, 140; NaCl, 10; CaCl2, 1; MgCl2, 1; HEPES, 10; EGTA, 11; ATP, 1-5 and GTP, 0-3. In some experiments, potassium gluconate was used in place of caesium gluconate. Electrodes were advanced into the tissue without visualization of individual neurones (see Blanton, Lo Turco & Kriegstein, 1989). Seal resistances were typically 2-5 GQ before rupture by suction.
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Z.-G. JIANG AND R. A. NORTH

Whole-cell currents were recorded as described above, except that sampling rates of 15-35 kHz were used. The values of membrane potential were corrected for the liquid junction potential present prior to making a gigaseal.

Electrical stimulation Nerves in the tissue were stimulated electrically by a bipolar concentric electrode (Rhodes Medical Instruments), the tip of which was placed in contact with the slice surface in one of three areas. First, to activate the cortical inputs the electrode was placed on the corpus callosum or
Ventriculus Corpus callosum

N.amudaateenrai

Y! ')

</

lU

\~~~~~~~~~~~N amy gd al o id

t~~~Internal capsule

ce n t r alis/

Thalamus y(: :. - 1 Hippocampus ZAqueduct

Substantia nigra Ventral tegmental area

Fig. 1. Drawing to show the orientation of the brain slice used. The thick line shows the approximate position of the slice, about 30 deg oblique rostral-up to the horizontal plane.
external capsule, whichever was closer to the recording site. Second, for antidromic activation, the electrode was directed to a spot within the globus pallidus or internal capsule. Third, for local intrastriatal stimulation the tip was placed on the surface of the slice within 300 ,tm of the point where the recording electrode entered the tissue. Stimuli were simple rectangular pulses, typically 100 ,us and 8-30 V, from a Grass S88 stimulator. Synaptic potential or current amplitudes before and after adding drugs were measured by averaging responses to four or sixteen stimuli.

Application of drugs and different solutions The solution that passed through the recording chamber could be altered without change in flow rate or temperature to one that contained a drug, or one of different ionic composition. Drugs used were: DL-2-amino-5-phosphonovaleric acid (APV, Sigma), atropine sulphate, baclofen (Ciba), bicuculline methiodide (Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Cambridge Research Biochemicals), hexamethonium (Sigma), ICI 205930 (Glaxo), SCH23390 (Schering), quinpirole (Lilly), SKF38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-lH-3-benzazepine hydrochloride, Smith Kline French), sulpiride (Ravizza), strychnine (Research Biochemicals Incorporated) and tetrodotoxin (TTX, Sigma).

Histology
The methoc of Horikawa & Armstrong (1988) was used. Briefly, biocytin was injected into the cell through a microelectrode filled with a solution containing biocytin (2 %) and potassium acetate (1 M) or biocy in (2 %), potassium chloride (1 M) and Tris buffer (50 mM) (pH 7 0-7 6). In the course of intracellula recording experiments, both depolarizing and hyperpolarizing pulses (50-200 ms, 0 2-1 0 nA) w re passed for 10-50 min. Slices were fixed with Zamboni (0 1 M-phosphate-buffered picric acid an formaldehyde mixture) at room temperature for 30 min and at 4 C overnight. The tissue was pre-treated for 45 min in a 0 5 % solution of Triton X-100 in phosphate-buffered saline and then incubated in either fluorescein-conjugated streptavidin or peroxidase-conjugated

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streptavidin (both in 1:300 dilution, Jackson ImmunoResearch Laboratory) overnight. The fluorescein-stained slices were rinsed and mounted for observation immediately after the incubation. Those treated with peroxidase were reacted with diaminobenzidine tetrahydrochloride (0 5 mg/ml) and H202 (0 01 %) in Tris (01 M, pH 7-6), and mounted for permanent storage and microscopic observation. Labelled cells were either photographed or drawn from individual sections at 250 x or 400 x with a Leitz microscope and a camera lucida.
RESULTS

The results are based on conventional (high-resistance) microelectrode recordings of 268 neurones: these were cells that showed a stable resting potential and an action potential with amplitude larger than 65 mV throughout the period of recording (30 min to 9 h). Whole-cell recordings from eighty-eight neurones were made; when the electrode contained potassium gluconate, the properties of the cells were similar to those observed with the high-resistance electrodes. The results presented pertain to observations made with high-resistance electrodes except where explicitly stated.
A
100
A)
0
CD

E
z

E0

X 10 ms

120 mV

-1 10-100 -90 -80 -70 -60 -50 Resting potential (mV)

100 ms

Fig. 2. Voltage recordings distinguish two types of neurone. A, distribution of resting membrane potentials of 268 striatal neurones. Continuous line is least-squares fit to the sum of two Gaussian functions. B, in a principal cell, the amplitude of hyperpolarizing electrotonic potentials does not increase linearly with the current passed (hyperpolarizing currents were 0-2, 0 5, 0-8, 1-1 and 1-4 nA; depolarizing currents were 0-2, 0 5, 0-8, l-l, 1-4 and 1-7 nA). Resting potential -91 mV. C, in a secondary cell, larger hyperpolarizing electrotonic potentials decline after the first 50 ms (hyperpolarizing currents were 01, 0-2, 03 and 0-4 nA; depolarizing current was 01 nA). Resting potential -60 mV. Highresistance microelectrodes used. In B and C, traces are averages of four, except for the depolarizing electrotonic potential that gave rise to action potentials. The full height of the action potentials is not reproduced; the record was effectively low-pass filtered by playback onto an X-Y recorder (10-90% rise time 0-8 ms in B and 4 ms in A).

Two types of neurone Two types of neurone were noticed during these recordings (Fig. 2); there was no indication that they were differentially located within the tissue slices used. The great majority (principal cells: 257 of 268 cells, 96 %) of neurones were highly polarized (resting potential -89-2+110 mV, n = 200; these and other numerical data are presented as means + S.E.M. for the subset of cells in which quantitative measurements were made). These cells had low input resistances (38-7 + 1-5 MQ, n =
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Z. -G. JIANG AND R. A. NORTH

200) when measured at the resting potential from the amplitude of small ( < 10 mV) hyperpolarizing electrotonic potentials. All twenty-five cells studied with voltage clamp showed prominent inward rectification in the steady-state current-voltage relation (Fig. 3); the slope conductance was 10 0+ 1-4 nS at*- 65 mV and 99-5 + 8-1 nS

AJ

11 nA

1
-130

Potential (mV)

-90

-50

s
(

-2

-3
Fig. 3. Inward rectification of potassium conductance (IK,IR) in principal cells. A, inward rectification is apparent from membrane currents (lower traces, I) evoked by potential steps (upper traces, V). Holding potential was resting potential, -99 mV. Traces reproduced from Gould pen-recorder. B, current-voltage relation for typical principal cell. Points indicate membrane current at termination of 1 s voltage command to the potential indicated (same data as in A). Continuous lines are from an X-Y recorder, and show membrane current in response to a depolarizing voltage ramp (1 mV/s). Increase in the extracellular potassium concentration to 5 and 10 mm strongly depolarizes the cell, increases conductance and shifts positively the potential at which the inflexion occurs in the current-voltage plot. Barium (3 /M) much reduces the inward rectification. Highresistance microelectrodes used.

at - 120 mV (n = 25). When currents were evoked by hyperpolarizing command steps, there was no detectable time-dependent component; thus the rectification develops within the settling time of the single-electrode voltage-clamp amplifier (20-50 ms). Barium (3 ,M) reduced the inward rectification (n = 4) (Fig. 3). Higher potassium concentrations (5 and 10 mm instead of 2-5 mm) shifted to a less negative value the potential at which the conductance increase occurred (Fig. 3). In all these respects, the inward rectification was similar to that seen in olfactory cortex cells by Constanti & Galvan (1983) and described in detail for cells of the rat nucleus
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RAT STRIATAL NEURONES 539 accumbens (Uchimura, Cherubini & North, 1989); they identify it as the inwardly rectifying potassium current (IK, IR). The remaining eleven cells (secondary cells: 4%) had obviously lower resting potentials (-60-0+ 10 mV) and higher input resistances (117+22 MQ). The
A Control I
B

v-~
Barimm

Wash

Caesium

_~ ~ ~
nA _1

40 mV

2s

-110 Potential (mV)

-90

~~~~~~Caesium/

Wash - 1

nA
/ ontrol

120 mV
100 ms
-

-2

Fig. 4. Cation current activated by hyperpolarization (Ih) in secondary cells. A, voltage recordings show marked sag in hyperpolarizing electrotonic potentials; this was little affected by barium (100 ,UM) but completely blocked by caesium (2 mM). B, membrane current (lower traces, I) in response to potential commands (upper traces, V); holding potential was resting potential, -62 mV. Note slow inward current relaxation with hyperpolarizing commands. C, current-voltage relation for the secondary cell. Filled and open circles show currents at beginning and end of command pulse. Continuous lines are current-voltage plots from an X-Y recorder using a depolarizing ramp (1 mV/s), in control solution and in caesium (2 mM). Caesium blocked the slowly developing inward current. High-resistance microelectrodes used.

hyperpolarizing electrotonic potential declined after about the first 100 ms (Figs 2 and 4); therefore, the input resistance values were calculated from the peak amplitude of the electrotonic potential evoked by a 0 5 nA current pulse. This decline in the electrotonic potential was blocked by caesium (2 mm) but unaffected by barium (100 /tM) (Fig. 4). Hyperpolarizing steps from the holding potential (about -60 mV) induced a slow inward current following the instantaneous current jump (Fig. 4). The time course of the development of the inward current was more rapid
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Z.-G. JIANG AND R. A. NORTH with larger hyperpolarizing pulses. Extrapolation of the instantaneous and steadystate current-voltage relation (see Mayer & Westbrook, 1983; Lacey & North, 1988) indicated a reversal potential for the time-dependent component of between -30 and -40 mV (n = 3). Barium (50-100 JaM, n = 3) did not affect the slowly developing inward current, but it was reversibly blocked by caesium (2 mm, n = 3). These findings are sufficient to identify the current as Ih (Mayer & Westbrook, 1983: a similar current was designated IQ by Halliwell & Adams, 1982). The properties of the cells were basically similar with tight-seal, whole-cell recording. When electrodes contained caesium gluconate, the resting potential was initially about -80 mV, the input resistance about 20 MQ and the current-voltage relation showed inward rectification as described above. The inward rectification was fully developed by the end of the capacitative transient, which was typically 10-20 ms in duration (n = 22). The cell depolarized to about -20 mV during the first 30 min after beginning whole-cell recording and the rectification disappeared; the input conductance at this time was 5-10 nS. In six experiments, electrodes contained potassium gluconate; the resting potential was between -90 and -70 mV, the input resistance and the inward rectification were similar to those seen with fine-tipped microelectrodes, and these properties showed no obvious changes during recordings for 30-100 min. One secondary cell studied with whole-cell recording had a stable resting potential of -60 mV and showed typical Ih. A short (2-5 ms) depolarizing current pulse with sufficient intensity (typically 1-5-3-5 nA for principal cells and 200-800 pA for secondary cells) elicited an action potential. The threshold potential for spike initiation was - 57-6 + 2 1 mV (range -51 to -68 mV) for principal neurones (n = 12) and - 55 8 + I 1 mV for secondary neurones (n = 4, range -54 to -58 mV). The amplitude of the action potential was 102 + 1-8 mV (n = 23) and 69-6 + 2-2 mV (n = 7) in principal and secondary cells respectively, and the duration at half-amplitude was 0-83 + 0 04 and 0-64 + 0'04 ms in the same groups of cells. These values are not significantly different (unpaired t test, P > 0 05). After-potentials followed the action potentials in cells of both types. In principal cells (at resting potential, n = 30), the spike after-potential was depolarizing; the amplitude was 19-7 + 0-9 mV and the duration was 27-4 + 29 ms. In secondary cells, the after-potential was hyperpolarizing, with amplitude 8-4 + 0-8 mV and duration 470+ 126 ms (n = 7). Accommodation was not marked in principal cells. Depolarizing current pulses (0 1-1 s) evoked sustained firing at up to 400 Hz (n = 7). However, the maximum frequency of firing observed in secondary cells was only 80 Hz, and in many cases a depolarizing current pulse evoked only a single spike at its onset. Spontaneous repetitive firing was not observed in principal neurones but occurred in four of the eleven secondary cells (at about 0 5 Hz); such spontaneous action potentials lasted throughout the recordings (0 5 to 3-5 h) and were not affected by CNQX and APV (see below).
540

Morphology of principal cells Nine neurones were retrieved for histological observations after they had provided satisfactory intracellular recordings; an example is shown in Fig. 5A. All had membrane properties typical of principal cells, and five of them were excited
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antidromically by stimulation of the globus pallidus or internal capsule (see below). The cells had a polygonal or fusiform soma (maximum diameter 10-20 jtm) with a smooth surface, giving rise to four to six primary aspiny dendrites radiating in various directions. The secondary and higher order branches were often abundantly
A
C

a
b/

'
_ 1
20 mV

J~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10ms
B

1 mm

100 gm

Control Wash

Ca2+ free

40Oms

Fig. 5. Antidromic and synaptic activation of identified projection neurone. A, the cell was antidromically activated by stimulation in globus pallidus (a) and synaptically activated by stimulation in corpus callosum (b). Resting potential -88 mV. B, camera lucida picture of the brain slice showing the location of the tested neurone (arrow) and the two stimulation sites used. a and b show location of stimulating electrodes in globus pallidus and corpus callosum respectively. C, camera lucida drawing of the same cell following intracellular biocytin injection and horseradish peroxidase staining. Soma size was 13 x 8 ,um. Note numerous spines (arrow-heads) on secondary dendrites, and main axon (arrows) extending towards the globus pallidus. The dashed lines show the contours of fibre fasciculi. D, synaptic potential is blocked by calcium-free solution, in another neurone. High-resistance microelectrodes used.

studded with spines and extended to fill a sphere 100-250 Ium in radius. An axon arising from the soma or from a primary dendrite was observed in some cells and could be traced for 45-400 ,tm: axons sometimes arborized extensively within a sphere 200-300 ,um in radius around the soma.
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Z.-G. JIANG AND R. A. NORTH

Antidromic activation from internal capsule or pallidum Stimulation (single pulses of 8-12 V, 100 ,us) evoked an antidromic action potential in fifty-one of seventy-nine principal cells tested (Fig. 5A and B). The stimulating electrode usually had to be repositioned several times before an antidromic response was evoked; however, similar efforts to evoke antidromic action potentials in three secondary cells were unsuccessful. Antidromic spikes appeared in an all-or-none manner as the strength of electrical stimulation was increased: they were unaffected by cobalt (2 mM, n = 3), which blocked synaptic potentials (see below). The antidromic action potential could be fractionated into an initial segment component or completely suppressed with strong membrane hyperpolarization. Indeed, many cells with high resting potentials could only be antidromically activated when a depolarizing current was first applied. The latency of the antidromic spike measured from the onset of the stimulus artifact to the onset of the spike ranged from 0-8 to 2-2 ms: if the axon followed a straight course from the tip of the stimulating electrode to the point of the intracellular electrode, the conduction velocity would be 0-5-1P2 m/s (n = 8). The antidromic action potential was followed by a slow depolarization similar in all respects to the after-depolarization that followed the action potential evoked by directly depolarizing the cell. Even the fractionated antidromic spike (initial segment spike) was followed by an after-depolarization. This slow depolarization was voltage dependent, its amplitude being decreased by cell depolarization and increased by hyperpolarization, as for the depolarizing after-potential following a directly induced action potential. The after-depolarization was not reduced by cobalt (2 mM, n = 4) or cadmium (100 /tM, n = 4), suggesting that it did not include a synaptically mediated component.

Synaptic potentials evoked by stimulating corticostriate fibres Single pulse stimulation of corticostriatal fibres in the corpus callosum or external capsule (Webster, 1961) evoked in most cells a depolarizing potential lasting 20-150 ms (seventy-eight of ninety-one principal, four of six secondary) (Figs 5D and 6A). The latency to the onset was 1-2-5-3 ms; this remained constant when the intensity of the stimulus was altered but became shorter when the stimulating electrode was moved to a point closer to the recording site. From the relation between latency and distance, the conduction velocity was estimated to be 0 49 + 0 05 (range 0-37-0-62) m/s (n 4). The latency was also not changed when the stimulation frequency was varied from 01 to 50 Hz (n = 5). The amplitude of the potential ranged from 2 to 24 mV, and was related to the intensity of stimulation and the distance between the stimulating and the recording sites. Stimulation of the cortex induced a 0-5-2 mV depolarization in three of twenty cells tested and stimulation of the mid-line of the corpus callosum, where crossed corticostriatal fibres would lie, evoked a 1-3 mV synaptic response (n = 5). No slow changes in membrane potential were evoked by single pulse stimuli (n = 200) or even by stimuli comprising trains of pulses (2-32 Hz, 2-5 s, n = 20) of intensity greater than that needed to evoke a maximal fast synaptic potential A stimulus comprising a train of pulses (100 Hz, 1-3 s) induced post-tetanic
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potentiation. The synaptic potential evoked by a single pulse stimulus was increased by 59 + 7-5 % (n = 16) when measured 30 s after the termination of the train. In five of the cells tested, this potentiation was still present after 10 min. The synaptic potential was unaffected by bicuculline (30 ,tM) and picrotoxin (100 ,UM) (n = 22) but reversibly blocked by a combination of CNQX (10 /tM) and
A
a

Bicuculline
b

APV

Bicuculline ~ ~
c

CNQX
~
d

120 mV
2 min

10 mV
20 ms

Bicuculline

APV

CNQX

ba

d~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~Il 120mV
2 min

'orn
40 ms

APV + CNQX b

Bicuculline
HP-50 mV
HP -70 mV
b

HP -50 mV

I500pA
200 s

L500pA
20 ms

Fig. 6. A, synaptic potential evoked by stimulation of corticostriate fibres in the external capsule. The synaptic potential was not affected by bicuculline (30 sM), but was completely blocked by APV (30 #M) and CNQX (10 /M). Rightmost trace shows synaptic potentials recorded at faster sweep speed at times indicated (a-d). Resting potential -95 mV; high-resistance microelectrode. B, both GABA and excitatory amino acids contribute to the synaptic potential evoked by focal stimulation. Upper record, synaptic potentials were elicited by stimulation of the slice surface within the striatum (about 200 ,um away from the recording site). Bicuculline (30 /tM) reduced the amplitude of the synaptic potentials to 67% of control. The remaining synaptic potential was little affected by APV (30 1uM) but completely blocked by CNQX (10 #M). The downward deflections are electrotonic potentials. Resting potential -88 mV; high-resistance microelectrode. Rightmost traces show synaptic potentials at the times indicated (a-d): average of four sweeps is shown in each case. Lower record, a similar experiment to that shown above, but recording with whole-cell tight-seal electrode. Focal intra-striatal stimulation. At -50 mV, the synaptic current was completely blocked by APV (30 /ZM) and CNQX (10 /tM), but at -70 mV the residual GABA component became apparent. Rightmost traces show synaptic currents at times indicated.

APV (30 fzM); these concentrations reduced the amplitude by 93-4 + 0 11 % of control (n = 18) (Fig. 6A). When applied alone, APV (30 /IM) reduced the synaptic potential by 6-2 + 1-8 % (n = 9) when tested at the resting potential (-90 mV) and by 10-8 + 3 0 % at -70 mV (n = 7). CNQX (10 ltM) alone reduced the synaptic potential
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in eight of ten cells tested. This inhibition was mimicked by quinpirole (a D2 receptor agonist; 1-10 JtM, n = 4) though not by SKF38393 (a D1 receptor agonist; 10 n = 3) and was probably presynaptic because depolarizations evoked by application of glutamitic acid were unaffected by dopamine (30 JtM, n = 3) or quinpirole (10 n = 2). Dopamine did not cause any consistent changes in membrane potential of principal cells but it caused a small hyperpolarization (2-4 mV) in three of six secondary neurones tested.

by 8841 + 3-6 % of control (n = 6) at the resting potential. The residual synaptic potential (in the presence of both APV and CNQX) was generally small(<2 mV) and short lasting ( < 10ms). It was not affected by bicuculline (30 JM), picrotoxin (100 /SM), hexamethonium (2 mm), (+ )-tubocurarine (30 /tM), atropine (1 /LM), strychnine (1 /tM), ICS-205930 (1 JtM), sulpiride (1/M) or SCH23390 (1 fLM) (two to four cells tested for each of these compounds). Baclofen (041-3 ftM) reduced the amplitude of the synaptic potential, presumably by the presynaptic action previously reported (Malenka & Kocsis, 1988; Seabrook, Howson & Lacy, 1990): the concentration giving 50% inhibition was 09 + 0-1 /M (n = 8). Dopamine (30-100 /M) also reduced the amplitude of the synaptic potential

gIm,

/LM,

Synaptic potentials evoked by focal stimulation within the striatum Focal stimulation with the tip of the bipolar concentric electrode positioned 200-300,tm from the point at which the recording microelectrode entered the tissue elicited depolarizing synaptic potentials in forty-nine of fifty cells (Fig. 6B). These were 4-38 mV in amplitude and had a duration of 20-300 ms; the amplitude and duration were graded with the intensity of the stimulation, and in almost all cells sufficiently strong stimulation resulted in a synaptic potential that gave rise to an action potential. The synaptic potentials were also reversibly blocked by tetrodotoxin (TTX, 1 ,tM, n = 3) or by a calcium-free solution (n = 4). In sixteen of twenty-five cells tested, the synaptic potential amplitude was reduced to 55-95% of control by bicuculline (30 /LM), picrotoxin (100#M) or a combination of both (Fig. 6B). The inhibition was reversible when bicuculline alone was applied, but the synaptic response did not recover even with washing for 4 h when picrotoxin was used. In bicuculline, APV (30 /tM) reduced the synaptic potential by 7-7 2-0 % (n = 4) and CNQX (10 ftM) reduced the EPSP by 86-1 + 3-5 % (n = 4); the synaptic potential was essentially blocked by a combination of CNQX (10 ,tM) and APV (30 ftM) (amplitude reduced by 92-8 +0-16 % of control, n = 8) (Fig. 6). The synaptic potential of the remaining nine cells was not altered by the GABA antagonists, but was fully blocked by the glutamate antagonists. Conversely, when CNQX (10 ram) and APV (30 /LM) were applied first, they reduced the amplitude of synaptic potential by 56-96% of control (n = 30). The synaptic potential in the presence of CNQX and APV could often (sixteen of twenty cells) be enhanced by repositioning the stimulating electrode and/or by increasing the stimulation intensity (up to 50 V, 041 ms); under such optimal conditions, the synaptic potential amplitude was 11-8 + 1-2 mV and its duration was 227 + 16-5 ms (n = 12). The evoked potential was not further reduced by adding hexamethonium (2 mM), (+)-tubocurarine (30 /LM), atropine (1 /tM), strychnine (1 tM), ICS-205930 (1 #M), dopamine (30 #M), sulpiride (1 /tM) or SCH23390 (1 #M) (two to four cells
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tested with each of these compounds). However, it was almost completely suppressed by bicuculline (30 jtM) and/or picrotoxin (100 /tM) (n = 6). Secondary cells could also be excited synaptically by local intrastriate stimulation (seven of ten cells tested). In two cells, the synaptic response was shown to have components mediated both by an excitatory amino acid and by GABA: in two other cells, there was no effect of bicuculline. It was much more difficult to evoke any synaptic potential in secondary cells than in primary cells: three cells could not be excited synaptically despite several repositionings of the stimulating electrode, and when a response was obtained it was generally of lower amplitude than seen in principal neurones.

Synaptic potentials evoked by stimulation of pallidum or internal capsule Stimulation of the pallidum or internal capsule, in those cases in which an antidromic action potential did not occur, produced a synaptic response similar to that evoked by focal intrastriatal stimulation in six of ten cells. CNQX and APV, as well as bicuculline, were similarly effective, and hexamethonium, atropine, sulpiride, SCH23390 and ICS205930 were without effect. Synaptic potentials were not observed in secondary cells. There is some morphological and electrophysiological evidence for feedback collateral inhibition of striatal output neurones, either directly back onto cell bodies of the output cells or onto excitatory terminals innervating those cells (Ribak et al. 1979; Park et al. 1980; Fisher et al. 1986). It was hypothesized that stimulation in the pallidum might release sufficient GABA to reduce the amplitude of the excitatory synaptic input from the cortex. This was tested in the present study by evoking the excitatory synaptic potential at different intervals after a stimulus to the pallidum. The excitatory synaptic potential was unaffected by pallidal stimulation even though the pallidal stimulus did evoke a fractionated action potential in the cell body (n= 3). Effect of membrane potential on synaptic potentials and currents Glutamate component A synaptic potential mediated by excitatory amino acid was evoked by focal stimulation within the striatum in the presence of bicuculline (30 /tM) and/or picrotoxin (100 JtM), or by stimulation of the corticostriatal inputs in the external capsule (see above). The synaptic potential was reduced in amplitude and markedly slowed in decay by depolarization from the resting potential to -70 mV, presumably as a consequence of the increase in input resistance that occurs in this potential range (Fig. 3). Cell conductance remained constant between -70 and -50 mV, but the synaptic potential amplitude continued to decline; extrapolation from this limited region allowed the reversal potential to be estimated as - 25-5 + 6 1 mV (n = 4). In two similar experiments with electrodes containing potassium acetate the values were -26 and -31 mV. The excitatory synaptic current measured with whole-cell recording reached its peak within 3-8 + 0-16 ms and decayed exponentially with a time constant of 5-0 + 0-4 ms (n = 17; holding potential -60 mV). The peak amplitude was linearly dependent on membrane potential and reversed polarity at - 3-6 + 1-2 mV (n = 32)
18

PHY 443

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(Fig. 7A-C). This value was not significantly different when electrodes contained potassium gluconate. In CNQX (10 tM), the inward synaptic current at -80 or -90 mV was completely blocked, but as less polarized potentials a smaller and slower synaptic current became apparent which also reversed polarity at about

Control

CNQX (10 gm)

1.

. -;,

i1 nA
+40 mV -40
-40 mV__

_-50 mV

-120

40 ms

C
0-5 nA -70

D
1 nA

-50 lo 0~~~~~0 >/ -10 -1 -2

o
-0.5

-1

Fig. 7. Synaptic currents mediated by excitatory amino acid evoked by stimulation of corticostriatal fibres. Tight-seal whole-cell recording with electrode containing caesium. A, the cell was stepped from the holding potential (-40 mV) to different potentials in the range -120 to + 40 mV (lower traces). The resultant membrane currents are shown in the upper traces. At the arrow, a single pulse stimulus was applied to the external capsule. B, the same experiment was repeated in CNQX (10 /eM). C, peak amplitude of synaptic current from A and from B as function of potential. Large currents may be underestimated because of partial escape from clamp (see A). D, current-voltage relation for another neurone before and after adding quisqualate (10 /tM) to the solution.

mV. This current peaked in 10 + 0-4 ms and decayed with a time constant of 111 + 841 ms (n = 10; holding potential + 40 mV). This behaviour is typical of a dual NMDA and non-NMDA component to the synaptic current, as has been described in several other central neurones (Collingridge & Lester, 1989). The currents evoked by superfusion with glutamate (300 or 1000 ,tM), quisqualate (3 or 10 ftM) (Fig. 6D) or NMDA (30 or 100 /LM) reversed polarity respectively at 85 + 1P5 mV (n = 4), -9-0+ 1P8 mV (n = 4) and -6+I0 mV (n = 4).
0
-

GABA component A synaptic potential mediated by GABA was evoked by focal stimulation within the striatum in the presence of CNQX and APV. Even with electrodes that contained potassium acetate, the synaptic potential was depolarizing at the resting potential
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(Fig. 8A). This synaptic potential was reduced in amplitude by depolarization and increased by hyperpolarization. It reversed polarity at -57-5 + 3-5 mV (-47 to -70 mV, n = 6) (Fig. 8). In two cells recorded with potassium chloride in the electrodes, the reversal potentials extrapolated as described above were -32 and
A
-47

-2nA
1n

-57

-65

-74-'0\
-85

+20 mV -90 -70

-40

-10 10 -30 mV

-95

-100

15 mV

20 ms

Fig. 8. Synaptic potential and currents mediated by GABA evoked by focal stimulation within the striatum. CNQX (10 ,UM) and APV (30 /SM) present. A, high-resistance microelectrode recording with an electrode containing potassium acetate (2 M). The synaptic potential is nullified at about -47 mV. B, tight-seal whole-cell recording. The cell was stepped from the holding potential of -40 mV to a different potential in the range -100 to + 20 mV (lower traces). The resultant membrane currents are shown in the upper traces. At the arrow, a single pulse stimulus was applied to the surface of the brain slice within the striatum. C, peak synaptic currents from B as a function of membrane potential. The largest currents are probably underestimated because of escape from clamp.

-30 mV. With whole-cell recording, the synaptic current reversed at -53 + 3.7 mV

GABA was applied by ejecting a few nanolitres from a pressurized pipette into the superfusing solution immediately above the region of the slice from which the recording was made. The pipette contained 100 mM-GABA, and 70 kPa was applied for 50-200 ms. A depolarization was evoked (4-17 mV) that was blocked by bicuculline (30 gM) and picrotoxin (100 ,tM). The depolarization decreased in amplitude as the membrane was depolarized and was nullified at -50 + 5-4 mV (n = 4; electrodes contained potassium acetate). Five similar experiments with whole-cell recording gave a reversal potential of -48+5 mV.
DISCUSSION

(n= 7).

Two types of striatal neurones A main finding of the present study is that two types of neurone can be readily distinguished within the striatum on the basis of membrane properties. In brief, principal cells were highly polarized (about -90 mV), had a higher conductance at the resting potential, and exhibited IK IR (a rapidly developing, barium-sensitive increase in conductance in the potential range -70 to - 120 mV) but not Ih. Secondary cells were less well polarized (about -60 mV), had a lower conductance
18-2
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at the resting potential, and exhibited Ih (a slowly developing, barium-insensitive increase in conductance in the potential range -60 to -100 mV) but not IK JR. Evidence is next discussed in support of the conclusion that the principal cells are medium spiny, GABA-containing projection neurones and that secondary cells are large aspiny interneurones. First, the ratio of the cells observed is about the same as would be expected on the basis of morphological studies that separate striatal neurones according to soma size (Kemp & Powell, 1971 d; Graveland & DiFiglia, 1985). When secondary cells were impaled, the recordings were usually very stable and long lasting, which is also consistent with the cells being large: Wilson, Chang & Kitai (1990) made a similar observation with their in vivo recordings from identified giant aspiny neurones. Second, Wilson et al. (1990) found that the giant aspiny neurones had a higher input resistance than spiny neostriatal neurones; our secondary cells had input resistances (at rest) three times higher than principal cells. The difference in input resistance is in the opposite direction from that which would be expected simply on the basis of cell size, implying even greater differences in the properties of unit area of membrane. Third, there was a marked difference in resting potential of the cells under the present recording conditions. The principal cells have properties similar to those reported for neurones of the ventral striatum (i.e. nucleus accumbens: Uchimura, Higashi & Nishi, 1986; Uchimura et al. 1989), which owe much of their high resting potential to the activation of 'K, IR at rest. In 2-5 mM-potassium, the average resting potential was close to -90 mV but the neurones depolarized markedly as the potassium concentration was raised (Fig. 3; see Uchimura et al. 1989); this makes it difficult to compare the values directly with the membrane potential observed in vivo where the extracellular potassium concentration is generally not known. The conductance IK, IR was previously observed in rat globus pallidum neurones in culture (Stanfield, Nakajima & Yamaguchi, 1985). Secondary cells rested at a potential close to -60 mV, consistent with the absence of IK, IR and the presence of Ih, which would tend to hold the membrane potential at about this level in the face of hyperpolarizing currents (Fig. 3). As mentioned above, it is difficult to compare the present results directly with those of in vivo recordings because of the different conditions: however, it is noteworthy that identified giant aspiny neurones in vivo had potentials 'within a few mV of the spike threshold' whereas spiny neurones had resting potentials 'far from the action potential threshold' (Wilson et al. 1990). The observations are consistent with the interpretation that principal cells, medium aspiny neurones, are highly polarized because they express the inwardly rectifying potassium conductance K, IR whereas the secondary cells, large aspiny neurones, are less polarized because they lack IK, IR but express Ih. A similar distinction between these two cell types has also been observed for neurones in the nucleus accumbens (Uchimura, Cherubini & North, 1990). A fourth difference between the two cell groups was in the after-potentials following the action potential. The principal cells had a depolarizing after-potential. This was considered not to result from release of transmitter because it was not reduced by cobalt (which blocked evoked synaptic potentials, see below). This afterdepolarization was also observed following antidromic activation, even when the antidromic action potential failed to invade fully the cell soma and was recorded as a fractionated spike analogous to the initial segment of a myelinated fibre (Coombs,
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Curtis & Eccles, 1957). Further studies would be necessary to identify the source of the current underlying the after-depolarization but one possibility is capacitative current arising from the discharge of the proximal axon (Blight & Someya, 1985). The action potential of secondary cells, in contrast, was followed by an afterhyperpolarization but this was not studied in detail. The fifth kind of evidence that bears on the distinction between the two cell types relates to the projection of their axons. The majority of principal neurones could be antidromically activated from the pallidum. There are several reasons why antidromic activation might not succeed - the axon may be severed during the slice preparation or the stimulating electrode may not be sufficiently close to the axon within the pallidum - and the present results therefore do not exclude the possibility that all principal cells project to pallidum. This interpretation would be consistent with anatomical studies (Preston et al. 1980; Somogyi et al. 1981) although in previous studies in vivo many striatal neurones could not be excited antidromically. Secondary cells were not antidromically activated from the pallidum, which is consistent with them being aspiny interneurones, but few cells were sampled. The histological studies provide a sixth reason to identify the principal cells as medium spiny neurones. In all respects - soma size, dendritic arborization, heavily spined dendrites - the principal cells conformed to the histological class of medium spiny neurones (see Kawaguchi, Wilson & Emson, 1989; and references therein). No secondary cells were sampled in the present experiments; microelectrodes containing biocytin are less than ideal for studies on membrane properties and were therefore used in only a subset of experiments. However, the remarkable coincidence of the properties of secondary cells described above with those found by Wilson et al. (1990) for a few identified large aspiny neurones supports the view that these cells are in the same group. Previous studies using intracellular recording in vitro have failed to distinguish the two types of neurone seen in the present experiments. In particular, a recent study showed that the properties of neurones located in the striatal matrix did not differ from those of cells in the patches (Kawaguchi et al. 1989). The basic properties of the cells presented in that study were similar to previous accounts (Kita, Kita & Kitai, 1984, 1985; Calabresi, Mercuri, Stanzione, Stefani & Bernardi, 1987a; Calabresi, Misgeld & Dodt, 1987b; Cherubini et al. 1988; Bargas, Galarraga & Aceves, 1988, 1989) and most similar to the properties of our principal cells. It is possible that the relatively low potassium concentration used in the present experiments (2-5 mM) favoured the separation among the two groups on the basis of resting potential and input resistance. Extracellular recordings in vivo provide evidence for two types of neurone in the rat striatum that can be differentiated on the basis of the spike waveform (Nisenbaum, Orr & Berger, 1988). They concluded that the most abundant waveform (Type II: 86% of cells) was generated by a medium spiny neurone; this would therefore correspond to our principal cell.

Synaptic potentials
Our observations confirm and extend those of previous workers (Lighthall & Kitai, 1983; Cherubini et al. 1988; see also Kawaguchi et al. 1989) that rat neostriatal neurones receive synaptic inputs mediated by an excitatory amino acid and by GABA. There are two developments over the earlier observations on the synaptic
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potentials. Antagonists of improved selectivity were used to discriminate between the components mediated by excitatory amino acids and those mediated by GABA, and an oblique slice was used to permit better resolution of the source of the fibres that provided the synaptic input. Thus, stimulation of the external capsule always evoked a synaptic potential that was completely blocked by CNQX and APV, consistent with a strong excitatory input from the cortex (see Introduction). This synaptic potential always comprised two pharmacologically distinct components, mediated by NMDA and non-NMDA receptors. The underlying currents recorded with the whole-cell method had the same reversal potential, consistent with the opening of channels permeable to both potassium and caesium as well as sodium (Nowak, Bregestovski, Ascher, Herbet & Prochiantz, 1984; Mayer & Westbrook, 1987) but the NMDA component showed the typical voltage dependence (Fig. 6C) (Nowak et al. 1984). The slower rise time and longer duration of the NMDA component (Fig. 6A and B) is also characteristic (see Collingridge & Lester, 1989; Westbrook & Jahr; 1989). The synaptic potential mediated by GABA could be isolated by adding CNQX and APV to the perfusing solution; the GABA component was seen in 64 % of cells. The underlying synaptic current reversed at a potential close to the calculated chloride equilibrium potential, as did the response to exogenously applied GABA. This synaptic potential was depolarizing at the resting potential even when potassium acetate was used to fill the recording electrodes, and this may account for an earlier report that only a small proportion of medium spiny neurones showed a hyperpolarizing synaptic input (Lighthall & Kitai, 1983). Any inhibitory action of GABA in these cells (at least, under these conditions) must therefore arise from a shunting of synaptic currents evoked by the excitatory amino acids. It was thought that the after-depolarization following the antidromic action potential (Fig. 5A) might be due to GABA release, in view of the evidence for recurrent inhibition (Park et at. 1980), but the lack of effect of cobalt or cadinium indicates that this is unlikely. Moreover, the glutamate-mediated synaptic potential evoked by stimulating corticostriate fibres was unaffected by a prior antidromic action potential, although GABA release from axon collaterals might have been expected to reduce this synaptic potential by postsynaptic shunting and/or by presynaptic inhibition (through GABAB receptors). On the other hand, in six of ten cells that were not activated antidromically, stimulation of the internal capsule of pallidum could evoke a bicuculline-sensitive synaptic potential. Although the source of the fibres cannot be determined with certainty in the present kind of experiment, this synaptic potential could be due to release of GABA from collaterals of axons of medium spiny neurones projecting from the striatum (Park et al. 1980). The main conclusion of the present work is that a small subpopulation of neostriatal neurones can be distinguished from the abundant medium spiny neurones by the ion conductances expressed. Both classes of cell receive synaptic inputs mediated by excitatory amino acids and by GABA: recordings of synaptic currents indicate that the NMDA, non-NMDA and GABA components of these synaptic inputs operate in a manner similar to that seen in other parts of the mammalian nervous system.

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This work was supported by US Department of Health and Human Services grants DA03160, DA03161 and MH40416.
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