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The Journal of Neuroscience, October 1, 1998, 18(19):8003-8015
Electrophysiological Characterization of GABAergic Neurons in the
Ventral Tegmental Area
Scott C.
Steffensen1,
Adena L.
Svingos2,
Virginia
M.
Pickel2, and
Steven J.
Henriksen1
1 The Scripps Research Institute, La Jolla, California
92037, and 2 Cornell University Medical College, New
York, New York 10021
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ABSTRACT |
GABAergic neurons in the ventral tegmental area (VTA) play a
primary role in local inhibition of mesocorticolimbic dopamine (DA)
neurons but are not physiologically or anatomically well characterized.
We used in vivo extracellular and intracellular recordings in the rat VTA to identify a homogeneous population of
neurons that were distinguished from DA neurons by their rapid-firing, nonbursting activity (19.1 ± 1.4 Hz), short-duration action
potentials (310 ± 10 µsec), EPSP-dependent spontaneous spikes,
and lack of spike accommodation to depolarizing current pulses. These
non-DA neurons were activated both antidromically and orthodromically by stimulation of the internal capsule (IC; conduction velocity, 2.4 ± 0.2 m/sec; refractory period, 0.6 ± 0.1 msec) and
were inhibited by stimulation of the nucleus accumbens septi (NAcc).
Their firing rate was moderately reduced, and their IC-driven activity
was suppressed by microelectrophoretic application or systemic
administration of NMDA receptor antagonists. VTA non-DA neurons were
recorded intracellularly and showed relatively depolarized resting
membrane potentials ( 61.9 ± 1.8 mV) and small action potentials
(68.3 ± 2.1 mV). They were injected with neurobiotin and shown by
light microscopic immunocytochemistry to be multipolar cells and by electron microscopy to contain GABA but not the
catecholamine-synthesizing enzyme tyrosine hydroxylase (TH).
Neurobiotin-filled dendrites containing GABA received asymmetric
excitatory-type synapses from unlabeled terminals and symmetric
synapses from terminals that also contained GABA. These findings
indicate that VTA non-DA neurons are GABAergic, project to the cortex,
and are controlled, in part, by a physiologically relevant NMDA
receptor-mediated input from cortical structures and by GABAergic
inhibition.
Key words:
ventral tegmental area; nondopamine; intracellular; immunocytochemistry; GABA; neurobiotin; refractory period; superexcitability
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INTRODUCTION |
The mesolimbic dopamine (DA) pathway
originating in the ventral tegmental area (VTA) and projecting to the
nucleus accumbens septi (NAcc) is considered to be a fundamental
component in the neural system underlying natural rewarding behaviors
and the reinforcing properties of drugs of abuse, including the
psychostimulants, opioids, and alcohol (Wise, 1996 ; Koob and Le Moal,
1997). Neurochemical lesions of the VTA produce deficits in exploratory
behavior, locomotor activity, and measures of cognitive function (Le
Moal et al., 1969, 1975; Simon et al., 1979, 1980). It has been
suggested that these deficits may result from abnormal regulation of
the electrical activity of midbrain DA neurons (Kalivas and Duffy,
1993).
The anatomical projections of DA neurons in the substantia nigra pars
compacta (SNc; nucleus A9) and adjacent VTA (nucleus A10) have been
carefully examined (Fallon and Moore, 1978; Beckstead et al., 1979;
Swanson, 1982). Dopamine neurons in the midbrain have been identified
by tyrosine hydroxylase (TH) immunoreactivity and by certain
electrophysiological and pharmacological characteristics, including (1)
long-duration (2-5 msec), biphasic or triphasic action potentials, (2)
a low rate (2-9 impulses/sec) of spontaneous activity marked by
bursting episodes characterized by spike-amplitude decrement, (3)
relatively slow axonal conduction velocity (~0.5 m/sec), (4)
inhibition of spontaneous activity by DA receptor agonists and
subsequent reversal by DA receptor antagonists (Wang, 1981a,b; Grace
and Bunney, 1983; Chiodo, 1988), and (5) a marked inward rectification
in response to an applied hyperpolarizing current (Grace and Onn, 1989;
Lacey et al., 1989).
In the VTA, neurons without detectable TH immunoreactivity lie in close
proximity to TH-labeled cells and are presumed to be GABAergic neurons
(Nagai et al., 1983; Otterson and Storm-Mathisen, 1984; Mugnaini and
Oertel, 1985). Neurons having nondopaminergic physiological properties
have also been identified in this region. These properties include (1)
relatively high rates of spontaneous activity (>10 impulses/sec), (2)
action potential durations of <1 msec, and (3) lack of the inward
rectification after-hyperpolarizing current (Grace and Onn, 1989; Lacey
et al., 1989). These neurons appear to represent a heterogeneous
population whose neurochemical identity, projections, innervation, and
physiological significance are less clear than that of DA neurons in
the VTA.
Current evidence supports the contention that non-DA neurons play a
significant role in the regulation of DA neurons in the VTA. Thus, the
neurochemical, electrophysiological, and hodological characterization
of VTA non-DA neurons is essential to an understanding of the
reinforcing and rewarding properties of drugs of abuse as well as the
natural rewarding behaviors ascribed to DA neurons (Wise et al., 1992;
Wise, 1996). Thus, we sought to identify the physiological properties,
transmitter content, and synaptic connectivity of a homogeneous
population of non-DA neurons in the VTA. Physiologically characterized
neurons were filled with neurobiotin and processed for electron
microscopic immunocytochemical labeling of GABA or TH to test
specifically the hypothesis that the identified neurons were
GABAergic.
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MATERIALS AND METHODS |
Subjects and surgical preparation
Male Sprague Dawley rats (250-350 gm) were anesthetized with
halothane (3.0-4.0%) and placed into a stereotaxic apparatus. Body
temperature was monitored and maintained at 37.0 ± 0.1°C by a
feedback-regulated heating pad. The skull was exposed, and holes were
drilled to accommodate placement of stimulating and recording
electrodes. The dura was opened over recording sites to prevent
breakage of micropipettes. Halothane anesthesia was maintained at
0.75% after surgery.
Extracellular recordings
Extracellular potentials were recorded by a single 3.0 M NaCl-filled micropipette [5-10 M ; inner diameter
(i.d.), 1-2 µm] cemented 20-40 µm distal to a seven-barrel
micropipette (30-80 M barrels) and amplified with an Axon
Instruments Axoprobe-1A microelectrode amplifier/headstage.
Microelectrode assemblies were oriented, via stereotaxic coordinates,
into the VTA (from bregma: 5.6-6.0 mm posterior; 0.5-1.0 mm lateral;
and 7.0-8.5 mm ventral) with a Burleigh piezoelectric microdrive.
Single-unit activity was filtered at 1-3 kHz (3 dB) for
"filtered" recordings and at 0.1-10 kHz for "unfiltered"
recordings. The potentials were displayed on analog and digital
oscilloscopes. Square-wave constant-current pulses (50-1000 µA; 0.15 msec duration; average frequency, 0.1 Hz) were generated by a Grass
PSIU6 isolation unit controlled by a MASTER-8 Pulse Generator or
by computer. The internal capsule (IC) (from bregma: 2.5 mm
posterior; 2.5-3.0 mm lateral; and 6.0-7.0 mm ventral) and NAcc (from
bregma: +1.5 mm anterior; 1.4 mm lateral; and 5.5-7 mm ventral) were
stimulated with insulated, bipolar stainless steel electrodes.
Single-unit activity
We evaluated spikes that had a >5:1 signal-to-noise ratio.
Extracellularly recorded action potentials were discriminated with Mentor N-750 or Fintronics spike analyzers and converted to
TTL-level pulses. Single-unit potentials, discriminated spikes,
and stimulation events were captured by National Instruments NB-MIO-16
A/D digital input/output, and counter/timer data acquisition boards in
Macintosh-type computers. Potentials were digitized at 20 kHz and
12-bit voltage resolution.
Analysis of responses
Waveforms, discriminated spikes, and stimulation events were
processed with National Instruments LabVIEW software.
Interspike-interval or peristimulus spike histograms were constructed
for determinations of firing rate and single-unit modal (e.g., bursting
vs nonbursting) activity. The histograms were normalized to the number
of spikes before and after drug or experimental treatment (1 sec epoch; 2000 spikes; 2 msec bin width). A cell was considered to be bursting if
its pattern of discharge was characterized by multiple action potentials over a short time period (10-15 msec) with spike-amplitude decrement and spike-interval increment. For determinations of the
probability of the occurrence of a driven spike across stimulus levels,
peristimulus spike histograms were generated at 0.5 Hz stimulation and
averaged over 40 trials (±100 msec window; 2 msec bin width). The
number of driven spikes at each stimulus level was determined by
rectangular integration using IGOR Pro software.
Spike duration was determined by measuring the time between half-peak
amplitude for the falling and rising edges of the unfiltered extracellular spike. The criteria for antidromicity were (1) the lack
of an EPSP preceding the spike (seen best in intracellular recordings),
(2) near-constant latency at threshold stimulus levels, (3) faithful
following of five pulses at 200 Hz, and, most importantly, (4)
collision between spontaneously occurring and IC-evoked spikes. Collision tests were performed by triggering the stimulator with an
unfiltered spontaneous spike and by adjusting the spontaneous-spike to
driven-spike interval such that stimulation of the axon of the cell
failed to produce a spike that reached the somatic recording electrode.
The absolute refractory period r of VTA non-DA neurons was
calculated by the following formula: r = I 2L, where I is the
interval from the ogive of the spontaneous spike (the fastest falling
edge; usually approximately one-half of the peak amplitude of the
spike) to the ogive of the driven spike and L is the latency from the stimulus artifact to the ogive of the driven spike. We accomplished this by setting I at 2L,
where 100% extinction of the driven spike was evident at twice the
threshold stimulus level, and by increasing I in 0.2 msec
increments until a driven spike occurred.
Histology
After recording, cell locations were labeled by electrophoretic
application of pontamine sky blue 2% (alternating ±10 µA current) in 3 M NaCl. After the experimental procedures, the animals
were killed with a lethal dose of halothane anesthesia (5%), and the brains were removed and preserved in 10% formalin. Histological inspection of the dye injection was accomplished by microscopic inspection of 50 µm cryostat-sectioned slices. The location of the IC
and NAcc stimulation sites was determined by passing ±3.0 mA current
through the bipolar stimulating electrode for 5 sec each and by
examination of the lesion site by microscopic inspection of 50 µm
cryostat-sectioned tissue slices with the aid of potassium ferri/ferrocyanide staining. The results for control and drug treatment
groups were derived from calculations performed on the driven and
spontaneous activity and were expressed as mean ± SEM. The
results were compared before and after drug treatment by use of the
two-tailed t test, at each point.
Drug preparation and delivery
For in situ microelectrophoretic application in the
VTA, we dissolved D,L-2-aminophosphonovalerate (APV;
20 mM; Research Biochemicals, Natick, MA) in 0.9% saline,
loaded the solution into seven-barrel glass micropipettes (barrel i.d.,
0.5-1.0 µm), and microelectrophoretically administered it by current
injection (25-50 nA) through the micropipettes.
Intracellular recordings and dye labeling
For intracellular recordings, VTA non-DA neurons were impaled
with sharp micropipettes (1.0 mm outer diameter, WPI borosilicate capillary tubing with filament) that were pulled on a vertical pipette
puller and filled with 4% neurobiotin and 1.0 M filtered (0.22 µm Millipore filter; Bedford, MA) potassium acetate (impedance, 50-70 M). The animals were prepared in a manner similar to that used for extracellular recording; however, halothane anesthesia was
increased to 1% from 0.75% to mitigate movement artifacts. The
electrodes were lowered to 6500 µm below the brain surface and
subsequently advanced at 3-5 µm steps while monitoring bridge balance and direct current potential with an Axon Instruments Axoprobe-1A microelectrode amplifier. The bridge was balanced and
monitored by capacitance neutralization with 0.5 Hz, 20 msec duration,
0.5 nA current pulses. After electrode impedance changes of ~20
M, as indicated by a negative deflection during the current pulse,
the electrode was "buzzed" with high-frequency current and
concomitantly advanced 3-5 µm until impalement. Because of the high
spontaneous firing of VTA non-DA neurons, extracellularly recorded
spikes were usually evident. Impaled neurons often evinced a quiescent
period after impalement with recovery to full spike amplitude (usually
60-80 mV) and resting potential (usually 55 to 60 mV) within 2-5
min. To aid in the recovery, we temporarily used 0.5 to 1.0 nA
constant hyperpolarizing current. Spike accommodation was studied at
depolarizing levels up to +4.0 nA. After electrophysiological characterization, impaled neurons were labeled with neurobiotin by
passing depolarizing current (+1.0-1.5 nA; 33 Hz; 20 msec duration) for 10-20 min.
Immunocytochemical methods
Antisera. For the detection of GABA immunoreactivity,
a rat antiserum was used that was raised against GABA conjugates and that has been well characterized and published extensively (Bayer and
Pickel, 1991). To confirm the selective labeling of GABAergic neurons,
we used a highly specific monoclonal TH antibody produced in mice
(Incstar, Stillwater, MN) for the immunolabeling of DA neurons.
Tissue fixation and immunocytochemistry. Rats were killed
with an overdose of halothane (5%) and rapidly perfused transcardially in the following order: (1) 3 ml of heparinized saline, (2) 60 ml of
3.75% acrolein in 2% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, and (3) 120-240 ml of 2% paraformaldehyde in PB.
The brains were removed from the cranium and post-fixed in 2%
paraformaldehyde in PB. Perfused brains were cut through the VTA,
according to the corresponding plates from the Paxinos and Watson rat
brain atlas (1986), into 30-40 µm coronal sections on a
Lancer Vibratome. Tissue sections were incubated in 1% sodium borohydride in 0.1 M PB for 30 min to bind to active
aldehydes. To enhance antibody penetration, we incubated tissue
sections in a cryoprotectant solution (25% sucrose and 3.5% glycerol
in 0.05 M PB) and then freeze-thawed the sections
successively in (1) Freon, (2) liquid nitrogen, and (3) room
temperature PB. To reduce background labeling, we incubated sections in
0.5% bovine serum albumin (BSA) in 0.1 M Tris-buffered
saline (TBS) for 30 min. After tissue sections were freeze-thawed, they
were rinsed several times in TBS and incubated overnight at room
temperature in an ABC/TBS solution (Hsu et al., 1981).
After several rinses in TBS, tissue sections were incubated in a
solution of 3,3'-diaminobenzidine (DAB; 22 mg per 100 ml of TBS) and
0.01% H2O2 for 6 min to visualize the
neurobiotin. Tissue sections were analyzed by light microscopy for the
presence of a filled cell and its processes. Filled cells usually were contained within four to six tissue sections. Tissue sections containing filled cells were separated into those to be processed for
light microscopy and those to be processed for dual labeling with
either GABA or TH. Tissues processed for light microscopy were rinsed
in 0.05 M PB, mounted on gelatin-coated slides, dehydrated via a series of graded ethanols, and coverslipped. Tissue sections were
examined using a Nikon light microscope equipped with differential interference contrast optics.
We incubated tissue sections processed for dual-labeling electron
microscopy in either rat anti-GABA (1:1000) or mouse anti-TH (1:1000)
in a solution of 0.1% BSA in TBS overnight at 4°C. We then rinsed
them in TBS and incubated them for 3 hr in either anti-rat or
anti-mouse colloidal gold-labeled 1 nm secondary IgG (1:50) for
detection of GABA or TH, respectively. The sections were then incubated
in 2% glutaraldehyde and reacted with a silver-intense solution
(Amersham, Arlington Heights, IL) for an empirically determined amount
of time. Sections were then incubated for 60 min in 2% osmium
tetroxide in 0.2 M PB, dehydrated in a series of ethanols
and propylene oxide, and flat-embedded in Epon 812 between two pieces
of Aclar plastic. We then sectioned the flat-embedded tissue sections
(40-50 nm) on an LKB ultramicrotome from the outer surface of the
plastic/tissue interface using a diamond knife (Diatome, Inc.). Tissue
sections were collected on to copper mesh grids, counterstained with
lead citrate (Reynolds, 1963) and uranyl acetate, and examined with a
Phillips CM-10 electron microscope.
Data analysis. We analyzed sections cut from the region of
interest based on the maximal labeling of antigens and the
morphological quality of the tissue. A Dage video camera and associated
software were used to obtain images of the regions to be sectioned for electron microscopy. We used at least 10 grids, containing 2-10 thin
sections that had been collected from all vibratome sections. All
electron micrographs were taken from the tissue-Epon surface. Fields
containing labeling for the antigens were categorized according to the
following criteria (Peters et al., 1991): (1) profile type (i.e., somata, dendrites, axons, or glia), (2) subcellular associations (i.e., plasma membranes, smooth endoplasmic reticulum, etc.), (3) types
of synaptic contacts (asymmetric, excitatory type and symmetric,
inhibitory type) (Carlin et al., 1980), and (4) association between
differentially labeled profiles (i.e., colocalization, apposition,
etc.). To test the specificity of the immunocytochemical methods, we
replaced the primary antiserum with preimmune or normal serum. These
controls do not definitively preclude cross-reactivity with other
proteins; therefore we refer to "like-reactivity" because of this
possibility.
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RESULTS |
Extracellular electrophysiological characterization of VTA
nondopamine neurons
VTA non-DA neurons recorded extracellularly in
halothane-anesthetized rats were distinguished from VTA DA neurons by
location, spontaneous activity, axonal conduction velocity, refractory
period, and orthodromic-driven activity. The most distinguishing
feature of VTA non-DA neurons was their fast spontaneous activity
(19.1 ± 1.4 Hz; n = 122) relative to DA neurons
and their uninterrupted phasic activity characterized by alternating
0.5-2 sec ON and 0.5-2.0 sec OFF periods (mean period, 0.43 ± 0.07 Hz; Fig. 1). No bursting activity,
defined as spike-amplitude decrement and spike-interval increment,
was observed in any of the VTA non-DA neurons studied. Unfiltered
recordings of VTA non-DA neuron spikes revealed biphasic action
potentials, characterized by a prominent, initial negative-going
component followed by a small positive-going component (Fig. 1,
top). The mean duration of the negative-going spike measured
at half-maximal amplitude was 310 ± 10 µsec (n = 93). VTA non-DA neurons were found in clusters of neurons whose spontaneous activity appeared to be homogeneous. This is demonstrated by the two neurons recorded simultaneously in Figure 1. In this case,
although both neurons are phasic, they are not simultaneously phasic.
Although simultaneously recorded neurons were often not synchronous,
the baseline voltage noise level recorded within a cluster during the
ON phase was often twice that of the baseline activity recorded
outside a cluster. Up to 10 neurons could often be recorded in one
electrode tract, but, typically, the best two neurons per tract and one
to two tracts per animal were studied. After extracellular
electrophysiological evaluation of the last recorded neuron in each
animal, pontamine sky blue was microelectrophoretically applied at the
recording site, and the brains were examined for their location. The
extracellular recording sites were localized to the VTA and to the
borders of adjacent structures according to the Paxinos and Watson
stereotaxic atlas (Fig. 2).
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Figure 1.
Extracellular electrophysiological
characterization of dopamine and nondopamine neurons in the ventral
tegmental area. Top, Unfiltered recordings of a
VTA DA neuron evoked by stimulation of the NAcc (left)
and of a spontaneous non-DA neuron (right) are shown.
Calibration bar applies to both. VTA DA neurons were slow-firing (<1
Hz), bursting neurons that were driven by NAcc stimulation with spike
durations of >500 µsec (arrow indicates NAcc stimulus
artifact). VTA non-DA neurons were relatively fast-firing, nonbursting
cells that evinced negative-going spikes and were characterized by
spike durations of <500 µsec. VTA non-DA neurons were not driven by
NAcc stimulation. Bottom, Under halothane anesthesia,
VTA non-DA neurons evinced pronounced and persistent phasic activity as
demonstrated by the two simultaneously recorded VTA non-DA neurons in
the filtered trace.
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Figure 2.
Recording sites of VTA nondopamine
neurons. The neuropil surrounding extracellularly recorded VTA non-DA
neurons was stained with pontamine sky blue after electrophysiological
evaluation. VTA non-DA neurons (filled circles) were
localized along the rostrocaudal and dorsoventral extent of the VTA
[shown here at 5.3, 5.6, 5.8, 6.0, and 6.3 mm from bregma (Paxinos and
Watson, 1986)]; however, they tended to be encountered in
clusters from 200 to 1000 µm dorsal to DA neurons. ip,
Interpeduncular nucleus; ml, medial lemniscus;
pn, paranigral nucleus; rmc, red nucleus,
magnocellularis; snc, substantia nigra pars compacta;
snr, substantia nigra pars reticulata;
vta, ventral tegmental area.
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Afferents and efferents of VTA nondopamine neurons
Stimulation of the IC evoked both antidromic and orthodromic VTA
non-DA spikes (Fig. 3). Antidromicity was
determined by collision studies and by response to high-frequency
stimulation. This proved at times to be problematical because of the
short latency of activation of VTA non-DA spikes from the IC (2.1 ± 0.2 msec; n = 23) and their high spontaneous firing
rate. In the example depicted in Figure 3A, IC stimulation
evoked a VTA non-DA spike (unfiltered recording) with short latency.
The IC-driven spike was extinguished by a preceding spontaneous spike
that occurred at a spontaneous-spike to driven-spike interval that was
less than double the latency of the evoked spike (Fig. 3B).
If we assume a linear path from the stimulating to the recording
electrode, the conduction velocity for VTA non-DA spikes driven
antidromically from the IC was 2.4 ± 0.2 m/sec (n = 23). The refractory period was 0.6 ± 0.1 msec (n = 15) as determined by collision test (see Materials
and Methods). Stimulation of the IC elicited orthodromic spikes in most
VTA non-DA neurons with a mean latency of 3.2 ± 0.3 msec
(n = 72) in which no collision occurred. Paired-pulse
analysis of spikes driven from the IC revealed an inhibitory period for
orthodromic spikes (filtered recordings) extending from 3 to 20 msec
(Fig. 3C; n = 8) and a superexcitable period
for antidromic spikes (filtered recordings) also extending from 3 to 20 msec (Fig. 3D; n = 8).
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Figure 3.
VTA nondopamine neuron reciprocal input to cortex:
extracellular recordings. A, Stimulation of the
IC elicits a VTA non-DA spike (filled
circle). B, A spontaneous spike that precedes IC
stimulation extinguishes the driven spike. Calibration bar in
A applies to B. The
asterisk signifies where the driven spike would have
occurred had there been no collision. C, Stimulation of
the IC also elicits orthodromic VTA non-DA spikes and produces a period
of inhibition of an orthodromic spike that extends to 20 msec
(C) when tested with equipotent paired stimuli at
a 2× threshold stimulus level. D, IC stimulation
produces a period of superexcitability for an antidromic spike when
tested with equipotent paired stimuli at a 0.5× threshold stimulus
level. Arrows indicate IC stimulus artifacts. The time
between arrows or the interstimulus interval
(ISI) in C and
D is indicated in each graph.
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NMDA receptor-mediated input to VTA non-DA neurons
Figure 4A shows a
strip-chart recording of a representative VTA neuron whose firing rate
decreased after in situ microelectrophoretic application of
the competitive NMDA receptor antagonist APV. The firing rate of all
VTA non-DA neurons was significantly reduced by APV 35.7 ± 7%
(Fig. 4A; p < 0.001;
n = 13) or by systemic administration of the
noncompetitive NMDA antagonist MK-801 36.5 ± 6% (0.5 mg/kg; p < 0.001; n = 8; data not
shown). The effects of APV were also tested on the activity of VTA
neurons driven orthodromically by IC stimulation. APV blocked the
occurrence of IC-driven spikes (Fig. 4B).
High-frequency stimulation of the IC evoked multiple spiking of
orthodromic VTA non-DA neurons whose number of discharges exceeded the
number of stimulation pulses in the train (Fig.
5A). The latency of some
driven spikes elicited by high-frequency stimulation often exceeded the
typical latency of a single-stimulus spike by two orders of magnitude
(i.e., 2-200 msec). Multiple spiking was strongly dependent on
frequency because 200 Hz stimulation evoked discharges whereas 50 and
400 Hz did not. The number of pulses in the train and the number of
trains were held constant at 10 for all frequencies. Systemic
administration of the noncompetitive NMDA antagonist MK-801 (0.5 mg/kg)
significantly reduced the multiple discharging associated with
tetanic stimulation of the IC (Fig. 5B; p < 0.001; n = 4). Systemic administration of MK-801 (0.1 mg/kg) reduced multiple discharging at 200 Hz by 44%
(p < 0.05; n = 3) compared with
78% with 0.5 mg/kg MK-801.
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Figure 4.
Local application of NMDA receptor antagonists
attenuate spontaneous and cortical input to VTA nondopamine neurons.
A, The rate meter above shows the firing
rate of a VTA non-DA neuron before and after in situ
microelectrophoretic application of the NMDA antagonist APV
(horizontal gray bar). APV significantly decreased the
VTA non-DA firing rate. B, Microelectrophoretic
application of APV markedly decreased the occurrence of orthodromic VTA
non-DA spikes evoked by stimulation of the IC. Asterisks
indicate significance level (p < 0.001).
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Figure 5.
Systemic administration of NMDA receptor
antagonists attenuate frequency-dependent multiple spike discharges
evoked by stimulation of cortical input to VTA nondopamine neurons.
A, These peristimulus spike histograms demonstrate the
effects of high-frequency stimulation of the IC on VTA non-DA spike
discharges. Each histogram represents 10 cumulated epochs of IC
stimulation (0.1 Hz; bin width, 2 msec). Stimulation of the IC with 10 pulses at 400 Hz did not evoke spike discharges but produced a 100 msec
period of inhibition of spontaneous firing (top),
whereas the same number of pulses at 200 Hz elicited multiple spike
discharges that occurred with latencies nearly an order of magnitude
greater than the single spike latency of 2-3 msec
(middle). The inset shows a
representative filtered recording of a VTA non-DA neuron after
high-frequency stimulation (200 Hz; 10 pulses) of the IC. Systemic
administration of the NMDA receptor antagonist MK-801 markedly
suppressed the multiple discharging of this VTA non-DA neuron
(bottom). The horizontal bar in each
histogram represents the stimulus train. B, Summary of
the VTA non-DA spike discharges produced by high-frequency IC
stimulation shows that the number of spike discharges is a function of
the frequency of stimuli. For all frequencies, the number of pulses and
the number of epochs were held constant at 10 while varying the
interval between pulses. Although 50 and 400 Hz evoked little or no
spike discharges, 200 Hz markedly and 100 Hz moderately increased
discharging. Systemic administration of MK-801 significantly reduced
VTA non-DA spike discharging produced by IC stimulation across
frequencies. Asterisks indicate significance level
(p < 0.001).
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Inhibition of VTA non-DA neurons by nucleus accumbens input
Stimulation of the NAcc produced a consistent inhibition of VTA
non-DA spontaneous activity that extended for 75 ± 11 msec (n = 5). Occasionally, VTA non-DA spikes could be
driven by stimulation of the NAcc; however, the threshold for
activation was often so high (>2.0 mA) and the fidelity of activation
was so low that this excitatory input was not considered to be an
important one for this study.
Intracellular recording and labeling
After successful impalement of VTA non-DA neurons, their passive
membrane properties and response to IC stimulation were characterized in vivo. VTA non-DA neurons had a mean resting membrane
potential of 61.9 ± 1.8 mV (n = 18), and their
mean spike amplitude was 68.3 ± 2.1 mV (n = 18).
The ON period of spontaneous VTA non-DA activity was accompanied by a
9.4 ± 0.9 mV depolarization (Fig. 6A; n = 8). Spontaneous and orthodromic IC-evoked VTA non-DA intracellular spikes were preceded by an EPSP (Fig. 6B) whose mean
amplitude was 7.6 ± 0.3 mV (Fig. 6C). There appeared
to be little or no spontaneous EPSP activity during the OFF phase (Fig.
6A).
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Figure 6.
VTA nondopamine neuron spontaneous activity is
dependent on synaptic input. A, A representative 2.5 sec
trace of a VTA non-DA neuron recorded intracellularly in
a halothane-anesthetized rat is shown. The resting membrane potential
of this VTA non-DA neuron was 61 mV, and the spike amplitude was 70.5 mV. This and all other VTA non-DA neurons were characterized by
pronounced phasic ON and OFF activity under halothane anesthesia (see
Fig. 1). The ON phase of activity was accompanied by a 10.6 mV
depolarization. B, The time base during the period
indicated by the horizontal black line in
A is expanded to show the individual spikes. An EPSP
appeared to precede every spontaneous spike. The voltage axis is the
same as that in A. C, The amplitudes of
each EPSP during the period indicated by the horizontal gray
line in A were measured and plotted in the
histogram and were characterized by a normal distribution of
spontaneous EPSP amplitudes. The mean EPSP amplitude was 7.6 ± 0.3 mV (n = 68).
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As demonstrated in the extracellular recordings in Figure 3,
stimulation of the IC evoked both antidromic and orthodromic VTA non-DA
spikes. We characterized the reciprocal nature of the corticotegmental
input and the tegmentocortical output of intracellularly recorded VTA
non-DA neurons in vivo. Figure
7 shows both orthodromic (Fig.
7A) and antidromic (Fig.
7B,C) VTA non-DA spikes driven by
stimulation of the IC in two separate neurons recorded intracellularly. The resting membrane potential of the orthodromic-driven spike in
Figure 7A was 62 mV, whereas that of the antidromic-driven spike in Figure 7, B and C, was 65 mV.
Antidromicity was determined by the collision test in Figure
7C. Orthodromic and spontaneous, but not antidromic, VTA
non-DA spikes were preceded by an EPSP.
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Figure 7.
VTA nondopamine neuron reciprocal input to cortex:
intracellular recordings. A, Similar to that in the
extracellular recordings in Figure 3, stimulation of the IC
(arrowheads indicate stimulus artifact) consistently
evoked intracellularly recorded VTA non-DA spikes at short latency. In
this example, the driven spike (filled circle)
appeared to be orthodromic because a spontaneous spike occurred within
a spontaneous-spike to driven-spike interval that was less than twice
the latency of the driven spike plus the refractory period. In
addition, a small EPSP preceded the driven spike. The spike at the
far right was a spontaneous spike and was not
time-locked to successive stimuli. B, This VTA non-DA
spike doublet was driven antidromically from the IC. Spike doublets
occurred more with intracellular than with extracellular recordings.
Note that there is little or no EPSP preceding the doublet of short
latency spikes even though the spontaneous spikes are accompanied by
EPSPs. C, The doublet of IC-driven spikes is
extinguished by a spontaneous spike that occurs within an interval that
was less than twice the latency of the first driven spike plus the
refractory period. The asterisk indicates where the
spikes would have occurred in the absence of collision. The calibration
bars in A apply to B and
C.
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VTA non-DA neurons responded to depolarizing current steps with
multiple spike discharges characterized by a lack of accommodation (Fig. 8A). The number
of spike discharges was monotonically related to current intensity
(Fig. 8B). Table 1
summarizes the cellular properties of extracellularly and
intracellularly recorded VTA non-DA neurons.
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Figure 8.
Lack of accommodation in VTA nondopamine neurons.
This neuron had a resting membrane potential of 64 mV.
A, Increasing levels of depolarizing current produced
multiple VTA non-DA spiking. Current steps are shown
above the traces. The calibration bar
(top) is the same for all traces.
B, Summary of the input/output response for spiking
produced by depolarizing current demonstrates the lack of accommodation
of VTA non-DA neurons.
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Morphological, ultrastructural, and immunocytochemical
characterization of VTA non-DA neurons
All neurons were first characterized via extracellular
electrophysiological criteria. After successful impalement, neurobiotin was iontophoretically injected in the neurons for time periods from 5 to 30 min. Of the 22 neurons recorded intracellularly and labeled with
neurobiotin in vivo, we were able to find 10 of them with
light microscopy. It appeared that a minimum of 10 min of iontophoretic
application combined with intracellular spike activity, both
spontaneous and current driven, was required to identify neurobiotin-labeled neurons in the tissue. Neurobiotin-labeled neurons
were characteristically multipolar, with two or more dendrites seen in
a single plane of section (Fig. 9).
Ultrastructural analysis showed that neurobiotin-filled somata and
dendrites contained sparse immunogold labeling for GABA (Fig.
10). Neurobiotin- and GABA-labeled
dendrites received asymmetric excitatory-type synapses from unlabeled
terminals. In addition, these dendrites received synaptic input from
other terminals that formed symmetric inhibitory-type synapses. These
latter terminals also sometimes contained GABA immunoreactivity. In
tissue processed for TH, neurobiotin-filled cell bodies and dendrites
did not contain immunogold-silver labeling for TH.
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Figure 9.
Neurobiotin labeling of VTA nondopamine neurons
in vivo. Light micrograph of a neurobiotin-labeled non-DA
neuron in the VTA. This is the same neuron studied
electrophysiologically in Figure 8. This neuron was characteristic of
all neurons identified electrophysiologically as VTA non-DA neurons and
was multipolar in shape with few dendritic processes
(D) branching from its soma
(S).
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Figure 10.
VTA nondopamine neurons contain GABA
immunoreactivity. Electron micrograph of the neuron in Figure 9
shows two dendrites (GABA-D and
GABA/NB-D) that contain immunogold-silver particles for
GABA (arrows). GABA/NB-D also contains
peroxidase reaction product for neurobiotin, indicative of the
physiologically characterized non-DA neuron. The filled and nonfilled
GABA dendrites are linked by two common axon unlabeled terminals
(UTs), which appear to form asymmetric synapses. The
gold-silver particles for GABA are sparse but are indicative of
specific labeling because there is virtually a total absence of
spurious particles in the tissue. Scale bar, 0.4 µm.
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DISCUSSION |
VTA nondopamine neurons are GABAergic projection neurons
Using electrophysiological and anatomical techniques, we have
identified, with stringent criteria, a homogeneous population of non-DA
neurons in the VTA. They are rapid-firing, nonbursting neurons with
reciprocal innervation from the cortex and inhibitory input from the
NAcc indicating that these neurons influence and are influenced by
cortical and limbic structures. They also contain GABA immunoreactivity
and receive excitatory-type synapses from unlabeled terminals and
symmetric inhibitory-type synapses from terminals that sometimes
contain GABA immunoreactivity. These findings indicate that VTA non-DA
neurons are GABAergic and are also subject to GABA inhibition. The
prevailing view is that VTA non-DA neurons are local circuit
interneurons (Beart and McDonald, 1980; O'Brien and White, 1987;
Churchill et al., 1992); however, VTA non-DA neurons may also project
to the cortex and ventral striatum (Thierry et al., 1980). Furthermore,
the neurotransmitter used by both local and projection neurons in the
VTA, similar to that used by non-DA neurons in the substantia nigra, is
thought to be GABA (Nagai et al., 1983; Otterson and Storm-Mathisen,
1984; Mugnaini and Oertel, 1985). This is supported by studies showing that GABAergic terminals provide synaptic input to DA neurons in the
VTA (O'Brien and White, 1987; Carlsson and Carlsson, 1990; Bayer and
Pickel, 1991).
VTA non-DA neurons were driven antidromically by IC stimulation,
indicating that they were not mere local circuit interneurons but that
they project to cortical sites as well. Regardless of whether or not
the input to VTA non-DA neurons was orthodromic or antidromic, brief,
high-frequency stimulation of the IC elicited multiple spike
discharging, which was optimal at 200 Hz. One possible explanation for
this marked frequency-dependent hyperexcitability is that the interval
corresponding to 200 Hz (i.e., 5 msec) is within the superexcitable
period for the antidromic input, whereas 2.5 and 20 msec intervals,
corresponding to 400 and 50 Hz, are near or beyond the time domain for
the superexcitable period for the antidromic input. Superexcitability
is a ubiquitous feature of excitable membranes in which the threshold
for spike elicitation by a conditioned stimulus is lowered by either a
depolarization-induced local increase in external potassium
concentration or by a hyperpolarization-induced reduction in sodium
inactivation (Raymond and Lettvin, 1978). The 5 msec interval
corresponding to the 200 Hz stimulation is also within the inhibitory
period for the IC-driven orthodromic input in which multiple
discharging of VTA non-DA neurons would not be expected; therefore, a
role for disinhibition can probably be discounted. It is reasonable
that the multiple discharging of VTA non-DA neurons results from
feedforward excitation from neighbor VTA non-DA neurons that are
activated antidromically by IC stimulation.
VTA nondopamine neuron spontaneous firing is dependent on
synaptic input
The two most distinguishing features of VTA non-DA neurons under
halothane anesthesia were their relatively fast firing rates and their
phasic activity. We have recently demonstrated in preliminary studies
that this persistent phasic activity seems to result from the
anesthesia because the activity of VTA non-DA neurons in freely behaving rats is regular and contingent on cortical arousal (Steffensen et al., 1996). Moreover, in the anesthetized preparation, neither IC
stimulation nor application of NMDA antagonists modified the phasic
activity. VTA non-DA neurons were characteristically fast-firing neurons, perhaps because of their somewhat depolarized resting membrane
potential. Nearly every VTA non-DA spike was preceded by an appreciable
EPSP, suggesting that the spontaneous activity of these neurons is
strongly regulated by excitatory input.
Many studies have emphasized the role of intrinsic membrane properties
to explain the rate and pattern of firing of midbrain DA neurons (Grace
and Bunney, 1984a,b; Llinas et al., 1984; Kita et al., 1986; Nedergaard
and Greenfield, 1992; Kang and Kitai, 1993). There are, however,
significant differences between the spontaneous activity of SN and VTA
DA neurons in vitro and in vivo (Bunney et al.,
1973; Wilson et al., 1977; Kita et al., 1986), suggesting that afferent
input plays a role in modulating the activity, particularly the firing
pattern, of these neurons. In support of this hypothesis, most VTA
non-DA neurons in the in vitro slice preparation are
quiescent (Wang and French, 1995). The VTA non-DA neurons described in
the in vitro study share similar characteristics to those
described here, including an action potential duration of <1.0 msec, a
mean resting membrane potential of 60.8 ± 2.6 mV, and a lack of
rectification to hyperpolarizing current steps. When considered
together, these findings strongly suggest that the firing rate of VTA
non-DA neurons is a function of afferent input.
Corticotegmental NMDA receptor-mediated input to VTA
nondopamine neurons
During the OFF component of phasic activity, EPSPs were not
evident in VTA non-DA neurons, suggesting either that the intrinsic activity of these neurons is coincident with excitatory input or that
spiking results from an EPSP that is predominantly NMDA receptor-mediated and under a voltage-dependent Mg2+
block typical of neurons at 60-65 mV, the characteristic average resting membrane potential of these neurons. This situation would be
somewhat unique because EPSPs are usually associated with an initial
non-NMDA receptor-mediated current that is followed by NMDA
receptor-mediated currents. Local or systemic application of NMDA
receptor antagonists moderately reduced the firing rate of VTA non-DA
neurons and markedly reduced VTA non-DA spikes elicited by single and
tetanic stimulation of the IC, supporting a role for NMDA receptors in
mediating glutamatergic corticotegmental neurotransmission under both
physiological and supraphysiological conditions. There is substantial
evidence demonstrating that both DA and non-DA neurons receive
monosynaptic innervation from prefrontal cortical neurons (Christie et
al., 1985; Sesack and Pickel, 1992) and that stimulation of the
prefrontal cortex evokes excitatory responses on VTA DA and non-DA
neurons (Thierry et al., 1979, 1980). NMDA and non-NMDA receptors,
including AMPA and kainate receptors, have also been
located on VTA DA and non-DA neurons and are activated by glutamate
receptor agonists (Kalivas et al., 1989; Seutin et al., 1990; Wang and
French, 1993, 1995).
It has been proposed that VTA DA neurons may be regulated by both a
direct excitatory corticofugal input to DA neurons and, indirectly, an inhibitory input comprising corticofugal excitatory inputs onto VTA non-DA neurons (Wang and French, 1995). Therefore, the
excitability of VTA DA neurons would result from the net effect of
direct excitation and indirect inhibition from non-DA neurons by
cortical afferents. The latter may explain why the NMDA receptor blockers MK-801 and phencyclidine excite DA neurons in
vivo (Zhang et al., 1993), increase DA release in the NAcc (Mathe
et al., 1998), and produce hyperlocomotion (Loscher and Honack, 1992). Corticofugal glutamatergic projections to VTA DA neurons (Wolf, 1998)
as well as glutamate receptors (Karler et al., 1989) have also been
implicated in the development of behavioral sensitization to
psychostimulants, an animal model for the intensification of drug
craving believed to underlie human drug addiction. Sensitization results, in part, from a long-term change in mesocorticolimbic DA
transmission and may involve a disinhibition of dopamine neurons (Steketee and Kalivas, 1991). The disinhibition of DA neurons may
result from decreased excitatory corticofugal drive to VTA non-DA
neurons or from increased GABAergic drive from the NAcc onto VTA non-DA
neurons. Because of their wideband firing activity, dependency on NMDA
receptor-mediated cortical input, and inhibitory modulation by the
NAcc, VTA non-DA neurons may contribute to plasticity in the complex
neuronal circuits underlying behavioral sensitization. We hypothesize
that these neurons receive a physiologically relevant NMDA
receptor-mediated input that paces GABA inhibition to DA neurons in a
manner similar to the role played by thalamic inputs to substantia
nigra pars reticulata GABAergic neurons in mediating inhibition of SNc
DA neurons (Tepper et al., 1995).
Potential role for VTA nondopamine neurons in mesocorticolimbic
neurotransmission and reward
We have recently reported preliminary work indicating that VTA
non-DA firing rates increase markedly during the onset of movement and
decrease markedly with select anesthetics (Steffensen et al., 1996). In
addition, the firing rate of VTA non-DA neurons decreased 42% during
slow-wave sleep and increased 114% during REM sleep, relative to
wakefulness (Lee et al., 1997). These findings suggest that VTA non-DA
neurons may regulate cortical arousal and psychomotor systems.
Furthermore, recent preliminary work has indicated that VTA non-DA
firing rates also decrease during the acquisition of heroin
self-administration behavior but soon after desensitize, providing
further evidence that VTA non-DA neurons are involved in
psychomotor-related events (Lee et al., 1996). If an increase in the
mesolimbic dopaminergic tone is important in brain-reward mechanisms
(Wise and Rompre, 1989), it is reasonable that GABA inhibition of DA
neurons by VTA non-DA neurons may be an important mechanism of
regulation. In support of this hypothesis, it has recently been
demonstrated that self-administration of GABA antagonists into the VTA
is blocked by D2 receptor antagonists (David et al., 1997).
It is unlikely that ascending DA fibers serve as the transducers of
intracranial self-stimulation (ICSS) reward. These conclusions are
based mostly on medial forebrain bundle (MFB) refractory period measurements that determined that the primary effector of reward was
not mesencephalic DA neurons (Gallistel et al., 1981). It was
determined that the refractory period of the neurons directly activated
by ICSS lies in the range of 0.4-1.3 msec (Yeomans, 1979), the
conduction velocity along the path was 1.0-4.5 m/sec, the direction
was descending in the MFB, and there was an axonal linkage between the
lateral hypothalamus and the VTA (Shizgal et al., 1982). Collectively,
these conclusions have been known as the "descending path
hypothesis" (Wise, 1980a; Shizgal et al., 1982; Gallistel, 1983).
This makes it likely that the axons were myelinated fibers, unlike
catecholaminergic fibers that are unmyelinated and have refractory
periods in the range of 1.8-2.0 msec (Guyenet and Aghajanian, 1978;
Maeda and Mogenson, 1981). Thus, it has been hypothesized that the
neuronal transducers of reward send fibers that descend and synapse on
DA neurons of the VTA (Wise, 1980b; Murray and Shizgal, 1994), where
their anatomical dispersion corresponds to the dispersion of VTA DA
neurons (Wise, 1982). GABAergic VTA non-DA neurons had conduction
velocities of 2.4 ± 0.2 m/sec and absolute (measured at 2×
threshold) refractory periods averaging 0.6 ± 0.1 msec. The
relatively fast conduction velocity and the short refractory period of
these neurons are within the range for a neuronal transducer of
reward.
Dopamine-independent mechanisms may be involved in the reinforcing
properties of drugs of abuse because findings from several laboratories
have questioned the notion that DA-dependent mechanisms are the final
common pathway in the processes mediating reinforcement. These studies
have shown that chemical destruction of DA terminals in the NAcc with
6-OHDA had no effect on morphine or heroin self-administration (Ettenberg et al., 1982; Pettit et al., 1984; Dworkin et al., 1988).
Furthermore, a lack of DA involvement in drug reinforcement has also
been demonstrated for oral ethanol self-administration (Rassnick et
al., 1993) and conditioned place preference (Cunningham and Noble,
1992; Risinger et al., 1992) as well as for cocaine self-administration
(Goeders and Smith, 1983) and conditioned place preference (Spyraki et
al., 1982; Mackey and Van der Kooy, 1985). More recently, the role of
DA in cocaine self-administration has been called into question by
studies demonstrating that DA-transporter knock-out mice continue to
self-administer cocaine (Rocha et al., 1998). Taken together, these
studies and the findings presented here provide evidence of the
existence of non-DA pathways that also play a role in mediating the
reinforcing or rewarding properties of drugs.
| |
FOOTNOTES |
Received April 27, 1998; revised July 10, 1998; accepted July 16, 1998.
This work was supported by Public Health Service Grants AA10075 to
S.C.S., DA08301 to S.J.H., and DA04600 and MH40342 to V.M.P. and by an
Aaron Diamond postdoctoral fellowship to A.L.S. We thank Dr. Antonieta
Lavin for invaluable training with intracellular recordings in
vivo and Eric Colago for histological assistance.
Correspondence should be addressed to Dr. Scott C. Steffensen,
Department of Neuropharmacology (CVN-13), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
| |
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Top
Abstract
Introduction
