Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas 77555-1031
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ABSTRACT |
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Neugebauer, Volker, Fatiha Zinebi, Rex Russell, Joel P. Gallagher, and Patricia Shinnick-Gallagher. Cocaine and Kindling Alter the Sensitivity of Group II and III Metabotropic Glutamate Receptors in the Central Amygdala. J. Neurophysiol. 84: 759-770, 2000. G-protein-coupled metabotropic glutamate receptors (mGluRs) are being implicated in various forms of neuroplasticity and CNS disorders. This study examined whether the sensitivities of mGluR agonists are modulated in a distinct fashion in different models of synaptic plasticity, specifically, kindling and chronic cocaine treatment. The influence of kindling and chronic cocaine exposure in vivo was examined in vitro on the modulation of synaptic transmission by group II and III metabotropic glutamate receptors using whole cell voltage-clamp recordings of central amygdala (CeA) neurons. Synaptic transmission was evoked by electrical stimulation of the basolateral amygdala (BLA) and ventral amygdaloid pathway (VAP) afferents in brain slices from control rats and from rats treated with cocaine or exposed to three to five stage-five kindled seizures. This study shows that after chemical stimulation with chronic cocaine exposure or after electrical stimulation with kindling the receptor sensitivities for mGluR agonists are altered in opposite ways. In slices from control rats, group II agonists, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG1) and (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), depressed neurotransmission more potently at the BLA-CeA than at the VAP-CeA synapse while group III agonist, L(+)-2-amino-4-phosphonobutyrate (LAP4), depressed neurotransmission more potently at the VAP-CeA synapse than at the BLA-CeA. These agonist actions were not seen (were absent) in amygdala neurons from chronic cocaine-treated animals. In contrast, after kindling, concentration response relationships for LCCG1 and LAP4 were shifted to the left, suggesting that sensitivity to these agonists is increased. Except at high concentrations, LCCG1, LY354740, and LAP4 neither induced membrane currents nor changed current-voltage relationships. Loss of mGluR inhibition with chronic cocaine treatment may contribute to counter-adaptive changes including anxiety and depression in cocaine withdrawal. Drugs that restore the inhibitory effects of group II and III mGluRs may be novel tools in the treatment of cocaine dependence. The enhanced sensitivity to group II and III mGluR agonists in kindling is similar to that recorded at the lateral to BLA synapse in the amygdala where they reduce epileptiform bursting. These findings suggest that drugs modifying mGluRs may prove useful in the treatment of cocaine withdrawal or epilepsy.
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INTRODUCTION |
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Neurotransmitter
mechanisms may have fundamental similarities but also significant
differences in various models of synaptic plasticity. Understanding the
synaptic and cellular processes in the forms of synaptic plasticity may
provide insight into brain function. Kindling is an established animal
model of human epilepsy in which repeated and focal applications of
initially subconvulsive electrical stimuli to certain brain areas
result in the progressive development of partial and generalized
seizures (Goddard et al. 1969; Racine
1972
). Cessation of chronic cocaine use produces complex
behavioral changes in humans and in animal models (Gawin 1991
). Anxiety and depression are the major symptoms of the
initial phase of cocaine abstinence (Gawin and Ellinwood
1989
), and severe anxiety after recurrent binges and during
withdrawal has been considered to be one of the most important factors
that maintains repetitive cycles of chronic cocaine use.
Previous experiments have suggested that the amygdala is involved in
both temporal lobe epilepsy and in cocaine mechanisms in the brain. The
central nucleus of the amygdala (CeA) in particular is a highly
epileptogenic brain area and, of the amygdaloid nuclei, responds most
rapidly to a kindling stimulus (Le Gal La Salle 1981).
The amygdala is also involved in the learning of stimulus-reward associations (Aggleton 1992
; Gallagher and
Holland 1994
), the acquisition of drug-seeking behavior
associated with cocaine self-administration (McGregor and
Roberts 1993
; Wilson et al. 1994
), and the
behavioral sensitization to cocaine (Kalivas and Alesdatter
1993
; Richter et al. 1995
). Amygdala lesions
(Post et al. 1987
) or MK-801, a glutamate
N-methyl-D-aspartate (NMDA) antagonist, injected
into the amygdala prevented behavioral sensitization to cocaine
(Kalivas and Alesdatter 1993
). The central nucleus of
the amygdala is involved in the reinforcing efficacy of
self-administered cocaine (Caine et al. 1995
;
McGregor and Roberts 1993
) and in reward-related behavior affected by psychostimulants (Robledo et al.
1996
). Amygdala sensitivity to electrical (Neugebauer et
al. 1997a
; Shinnick-Gallagher et al. 1998
) and
cocaine-induced kindling (Post et al. 1987
) and similarities between kindling and behavioral sensitization suggest the
amygdala might be especially vulnerable to repeated cocaine exposure.
G-protein-coupled metabotropic glutamate receptors (mGluRs) play
important roles in neuroplasticity and nervous system disorders (Anwyl 1999; Conn and Pin 1997
;
Knöpfel et al. 1995
; Pin and Duvoisin
1995
), but their role in epilepsy and cocaine dependence has
not been studied in detail. Previous studies in our laboratory in the
basolateral amygdala (Holmes et al. 1996
; Keele
et al. 2000
; Neugebauer et al. 1997a
,b
) and
others in the hippocampal dentate (Klapstein et al.
1999
) have shown that mGluR responses are altered in kindling.
Recent data also suggest, based on changes in mRNA, that mGluRs may
play a role in the biochemical and behavioral effects of cocaine
(Ghasemzadeh et al. 1999
). Eight mGluR subtypes have
been cloned and are classified into groups I-III based on their
sequence homology, agonist pharmacology, and coupling to intracellular
effector systems (Conn and Pin 1997
;
Knöpfel et al. 1995
; Pin and Duvoisin
1995
; Schoepp et al. 1999
). Activation of mGluRs
can produce excitatory or inhibitory effects on neurotransmission depending on the receptor type, synapse, and brain area (Choi and Lovinger 1996
; Conn and Pin 1997
;
Davis and Laroche 1996
; Gereau and Conn
1995a
,b
; Lovinger and McCool 1995
;
Manzoni et al. 1995
; Zheng and Gallagher
1992
; for the basolateral amygdala: Neugebauer et al.
1997a
,b
; Rainnie and Shinnick-Gallagher 1992
).
Since kindling results in an increase in sensitivity of group II and
III mGluR agonists 5-7 days after the last seizure in basolateral
amygdala (BLA) neurons (Neugebauer et al. 1997a) and since cocaine itself can induce kindling (Post et al.
1987
), we hypothesized that, in rats treated chronically in
vivo with cocaine, the sensitivity to mGluR agonists in vitro would be
enhanced particularly in the CeA where cocaine effects had been
reported previously (Caine et al. 1995
; McGregor
and Roberts 1993
; Robledo et al. 1996
). To
ensure that the effects of kindling were similar at different amygdala
synapses, we examined the effect of kindling in CeA neurons. Here we
report that kindling enhances the sensitivity of Group II and III mGluR
agonists in CeA neurons but disprove our hypothesis and instead show
that at the same synapses mGluR agonists are rendered ineffective after
cocaine treatment.
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METHODS |
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Cocaine treatment regimen
Adult male Sprague-Dawley rats (70-90 g) are housed three per
cage with free access to food and water. Rats are either untreated or
receive twice-daily (9:00 and 16:00) injections of saline (0.9%) or
cocaine HCl (15 mg/kg ip; NIDA, Rockville, MD) for 14 consecutive days.
No difference in weight gain of the animals injected with saline or
cocaine is measured over this 14-day period (Gallagher and
Simms, unpublished observations). The animals are monitored behaviorally to ensure that they are responding to cocaine. Previous studies from this group have analyzed behavioral activity and individual neuronal electrophysiological activity during and following 14 days of chronic cocaine treatment (Shoji et al. 1997;
Simms and Gallagher 1996
). Chronic cocaine treatment
results in sensitization of locomotor activity and stereotyped behavior
(e.g., gnawing, rearing, fast repetitive head and/or foreleg movement),
which are rated on a six-point behavioral scale (Gifford and
Johnson 1992
) 15 min after cocaine injection. Twice-daily
injections of cocaine for 2 wk produce behavioral sensitization
(Cunningham 1988
; Shoji et al. 1997
) and
electrophysiological changes in specific brain areas (locus ceruleus,
Harris and Williams 1992
; ventral tegmentum,
Bonci and Williams 1996
; septum, Shoji et al.
1997
, 1998
; nucleus accumbens, Manzoni et al.
1998
, Zhang et al. 1998
) other than the
amygdala. In our studies with the amygdala slice, the
electrophysiological experiments are conducted on the day following the
last injection of cocaine (15 mg/kg ip; twice daily; 14 days) or saline.
Kindling
Rats (70-90 g) are anesthetized with 35 mg/kg pentobarbital
sodium and 145 mg/kg chloral hydrate (Equithesin) and implanted (n = 14 kindled, n = 6 not stimulated)
with tripolar electrodes (Plastics One, Roanoke, VA) into the right BLA
as previously described (Holmes et al. 1996;
Neugebauer et al. 1997a
,b
; Rainnie et al. 1992
). Using the coordinates from Paxinos and Watson
(1986)
, the tips of the two leads are positioned 2.0 mm
posterior and 4.5 mm lateral to Bregma at a depth of 7.3 mm from the
dura surface; the third lead served as a ground for monitoring and/or
recording afterdischarges (ADs). Electrodes are fixed to the skull with dental acrylic (Plastics One). The kindling stimulation of the BLA is
initiated after a postimplantation recovery period of 5 days. The
stimulation consisted of a 2 s train of 60-Hz monophasic square
waves, each 2 ms in duration, administered twice daily at least 8 h apart. Kindling stimulation is applied 50-100 µA above the AD
threshold (200-400 µA). Behavioral seizure severity is rated
according to the ranking scale of Racine (1972)
. The time to the fully kindled state is within a range of 6-14 days [mean:
9.7 ± 0.6 (SE) days, n = 14 animals].
Three to 7 (mean: 4.7 ± 0.4) days after three consecutive
stage-five fully kindled seizures are evoked, animals (180-263 g) are
killed and brain slices prepared for the electrophysiological
experiments. Control slices are obtained from both unoperated
(n = 18) and unstimulated-implanted (n = 6) rats.
Amygdala slice preparation
Brain slices containing the central nucleus of the amygdala
(CeA) are obtained as previously described (Neugebauer et al. 1997a). Rats are decapitated, the brains quickly dissected out and blocked in cold (4°C) artificial cerebrospinal fluid (ACSF) containing (in mM) 117 NaCl, 4.7 KCl, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, and 11 glucose. ACSF is oxygenated and
equilibrated to pH 7.4 with a mixture of 95%
O2-5% CO2. Coronal brain
slices (500 µm) are prepared using a Vibroslice (Campden Instruments, London, UK). After incubation in ACSF at room temperature (21°C) for
1 h, a single brain slice is transferred to the recording chamber and
submerged in ACSF (31 ± 1°C), which superfuses the slice at
~2 ml/min.
Whole cell patch-clamp recording
"Blind" whole cell recordings (Blanton et al.
1989) are obtained from CeA neurons using patch electrodes made
from 1.5 mm borosilicate glass capillaries (1.5 mm OD, 1.12 mm ID;
Drummond, Broomall, PA) pulled on a Flaming-Brown micropipette puller
(P-80/PC, Sutter Instrument, Novato, CA). Recording electrodes are
positioned in the central medial and lateral capsular nuclei under
visual control. The boundaries of the CeA are discerned under light
microscopy; each slice is matched with the corresponding level in
Paxinos and Watson (1986)
. The internal solution of the
recording electrodes (3- to 5-M
tip resistance) contains (in mM) 122 K-gluconate, 5 NaCl, 0.3 CaCl2, 2 MgCl2, 1 EGTA, 10 HEPES, 5 Na2-ATP, and 0.4 Na3-GTP;
pH is adjusted to 7.2-7.3 with KOH and the osmolarity to 280 mmol/kg
with sucrose.
After tight (>2 G) seals are formed and the whole cell
configuration is obtained, neurons are included in the sample if the resting membrane potential is more negative than
55 mV and action potentials overshooting 0 mV are evoked by direct cathodal stimulation. Voltage and current signals are low-pass filtered at 1 kHz with a
4-pole Bessel filter (Warner Instrument, Hamden, CT), digitized at 5 kHz (Digidata 1200 interface, Axon Instruments, Foster City, CA), and
stored on a computer (4DX2-66V, Gateway 2000). Data are also
continuously recorded on a pen chart recorder (Gould 2400, Gould
Instruments, Valley View, OH). Evoked potential and evoked current data
are acquired and analyzed using pCLAMP6 software (Axon Instruments).
Discontinuous single-electrode voltage-clamp (d-SEVC) recordings are
acquired using an Axoclamp-2A amplifier (Axon Instruments) with a
switching frequency of 5-6 kHz (30% duty cycle); gain of 3-8 nA/mV,
time constant 20 ms. Phase shift and anti-alias filter are optimized.
The headstage voltage is monitored continuously on an oscilloscope
(Tektronix, Pittsfield, MA) to ensure precise performance of the amplifier.
Synaptic stimulation
The CeA represents the major output nucleus of the amygdala and
processes information from other amygdala nuclei and from wide-spread
brain areas. We studied two synapses in the CeA: the BLA-CeA and
ventral amygdaloid pathway (VAP)-CeA synapse. Excitatory postsynaptic
currents (EPSCs) are elicited with concentric bipolar stimulating
electrodes (SNE-100, 22 k resistance, Kopf Instruments) placed on
two synaptic pathways to the CeA nucleus. One of the afferent synapses
to the CeA, the VAP-CeA synapse, provides afferent inputs from brain
stem areas (cf. Alheid et al. 1995
; Bernard et
al. 1993
; Harrigan et al. 1994
; T. S. Gray,
personal communication), and the synapse between the BLA-CeA provides
information about intraamygdala communication flow. For stimulation of
the VAP, the electrode is positioned under microscopic control on the
fibers dorsomedial to the CeA and ventral to but outside of the
caudate-putamen (T. S. Gray, personal communication). Electrical
stimuli (150-µs square-wave pulses) are delivered at frequencies
<0.25 Hz. Thresholds for EPSCs and spiking are defined as the
respective intensity, which evoked a response in
5 of 10 trials with
mean amplitude determined from the 10 trial stimulations. Input-output
relations are obtained by increasing the stimulus intensity in 1-V
steps. For evaluation of a drug effect on synaptically evoked
responses, the stimulus intensity is adjusted to 75-80% of the
intensity required for orthodromic spike generation.
Drugs
The following drugs were used:
(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG1),
L(+)-2-amino-4-phosphonobutyrate (LAP4), 7-(hydroxyimino)cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt); 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893); picrotoxin (PTX); (2S)-3-[[(15)-1-(3,4-dichlorophenyl) ethyl]
amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid (CGP 55845); all
purchased from Tocris Cookson, Bristol, UK.
(+)-2-aminobicyclo [3.1.0]hexane-2,6-dicarboxylic acid (LY354740)
was a generous gift from Eli Lilly and Company. Drugs were applied by
gravity-driven superfusion in the ACSF. Solution flow into the
recording chamber (1 ml volume) was controlled with a three-way
stopcock. Applications were 10 min (usually 12-14 min) in duration
to establish equilibrium in the tissue.
Data analysis and statistics
Averaged values are given as the means ± SE. Differences between neurons from fully kindled animals and animals chronically treated with cocaine and control neurons in membrane potential, input resistance, slope conductance, EPSC threshold, and spike threshold are evaluated for statistical significance using a one-way ANOVA followed by a post hoc Dunnett's multiple comparison test. Input-output relationships and concentration-response relationship of the agonists' effects on synaptic transmission are compared between control neurons and neurons either from animals chronically treated with cocaine or from kindled animals using the two-way ANOVAs. Significance of the effects of LCCG1, LY354740, and LAP4 on synaptic transmission in neurons from kindled animals and/or animals chronically treated with cocaine is determined using an F test to analyze the linear regression fitted to the concentration-response data (Prism 3.0, Graph Pad Software, San Diego, CA). Linear analysis is done to show "no effect" and applies only to the cocaine group. Concentration-dependent effects of drugs on the slope conductances are evaluated with repeated measures ANOVA followed by post hoc t-tests where appropriate. Statistical significance is accepted at the level P < 0.05. EC50s and 95% confidence intervals are calculated from sigmoid curves fitted to the cumulative concentration-response data by nonlinear regression using the formula y = A + (B - A)/[1 + (10C/10X)D], where A = bottom plateau, B = top plateau, C = log (EC50), D = slope coefficient. Using the linear curve fit function of pCLAMP6 software (Axon Instruments), slope conductances (in nS) in the absence and presence of agonists are calculated from the linear portion of the current-voltage (I-V) relationships recorded in voltage-clamp mode.
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RESULTS |
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Animals in these studies belonged either to the control group (consisting of naïve untreated animals = 18, neurons = 37; saline injected rats = 8, neurons = 12; or implanted, nonstimulated rats = 6, neurons = 10) or to the fully kindled animal (n = 14; neurons = 23) or chronic cocaine (n = 12; neurons = 22) animal treatment groups. Membrane properties and characteristics of synaptic transmission (Table 1) were compared, in naïve control, saline-injected control and control implanted, nonstimulated animals. No differences were found between the control groups and the data were pooled and termed "controls" (Table 1).
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Chronic cocaine alters membrane properties and synaptic transmission in CeA neurons
Membrane properties and synaptic transmission in neurons from animals chronically treated with cocaine showed several characteristics that distinguished them from control neurons. The resting membrane potential of CeA neurons in brain slices from rats treated chronically with cocaine was, on average, significantly hyperpolarized (Table 1; P < 0.01, Dunnett's test) and the input resistance was significantly lower (P < 0.05, Dunnett's test) compared with control neurons. Accordingly, the average slope conductance calculated from the linear portion of the current-voltage (I-V) relationship was greater in neurons from animals chronically treated with cocaine than in control neurons (P < 0.01, Dunnet's test).
EPSCs were recorded and input-output relationships were obtained by increasing the stimulus intensity in 1-V steps and measuring the peak amplitudes of evoked EPSCs. In Fig. 1A is shown the altered input-output relationships at the VAP-CeA synapses in neurons from animals treated chronically with cocaine. The cocaine treatment led to a significant leftward shift of the curve (Fig. 1A; 2-way ANOVA; VAP-CeA: F = 49.7551,940, P < 0.0001). Chronic cocaine also altered the thresholds for EPSCs and orthodromic spike generation. The EPSC and action potential threshold were significantly lower in neurons from animals treated chronically with cocaine (P < 0.05, VAP-CeA; Table 1). Input-output curves (F = 43.351,893, P < 0.0001) and EPSC threshold (P < 0.01) were also altered at the BLA-CeA synapse (Table 1).
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Kindling alters synaptic transmission but not membrane properties in CeA neurons
Recordings were compared in neurons from control animals and from kindled rats 3-7 days after the last stage-five kindled seizure (Table 1). In contrast to those from animals chronically treated with cocaine, neurons from kindled animals did not show an alteration in resting membrane potential or input resistance or in the linear portion of the input-output relationship (Table 1; Fig. 1B; 2-way ANOVA: F = 0.111,672; P > 0.05). The amplitudes of EPSCs at the VAP-CeA synapse increased from threshold to generation of an action potential current over a stimulus intensity range of 8-18 V in control and from 8 to 15 V in neurons from kindled animals (Fig. 1B). EPSC thresholds (Table 1) did not differ between the groups although thresholds for synaptically evoked action potential current are lower in neurons from kindled animals compared with controls (Table 1). Similar results were obtained at the BLA-CeA synapse (Table 1). These data showed that while kindling and chronic cocaine lower the threshold for action potential firing, chronic cocaine also enhanced evoked synaptic transmission by altering the gain (leftward shift) of CeA synapses.
Group II and group III mGluR agonists depress synaptic transmission in CeA control neurons
GROUP II MGLUR AGONISTS.
In control neurons, a group II agonist, LCCG1, depressed the peak
amplitude of EPSCs evoked at the BLA-CeA (Fig.
2A) and the VAP-CeA (not
shown) synapses. The effect of LCCG1 was slowly reversible, usually
within 20 min of washing with ACSF. A comparison of mGluR agonist
EC50s in Table 2
showed that LCCG1 was more potent at the BLA-CeA synapse than at the
VAP-CeA synapse. Since LCCG1 can have agonist activities at other mGluR
subtypes, including mGluR1, 5, 4a, and 8 (Conn and Pin
1997; Saugstad et al. 1997
; Schoepp et
al. 1997
), we also tested the novel orally active compound LY354740, which is a highly selective group II mGluR agonist
(Schoepp et al. 1997
). LY354740 was more potent than
LCCG1 but had qualitatively similar effects. LY354740 reversibly
depressed EPSCs evoked at the BLA-CeA synapse (Fig. 2B) more
potently than at the VAP-CeA synapse (Table 2). In addition, the
maximum inhibition produced by the group II agonists was not
significantly different from each other as evidenced by the overlapping
95% confidence intervals at the BLA-CeA synapse (LCCG1:
31.82-46.38%; LY354740: 21.15-51.22%). Similar maximum effects of
LCCG1 and LY354740 were measured at the VAP-CeA synapse.
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GROUP II MGLUR AGONIST AND GROUP I
ANTAGONIST.
Recently it has been shown that activation of protein kinase C by group
I agonists blocks the inhibitory effect of group II and group III mGluR
agonists (Macek et al. 1998). It is possible that tonic
endogenous activation of group I mGluRs could influence the inhibitory
effect of group II mGluR agonists. To examine this issue, the effect of
group-II-selective agonist, LY354740, was tested on EPSC amplitude
(Fig. 3A) in the absence
(left) and presence (right) of selective
antagonists for group I, mGluR1, CPCCOEt, (50 µM), and mGluR5,
SIB-1893 (10 µM), in naïve slices. Application of LY354740 (1 µM) for 15 min significantly decreased the EPSC amplitude by 57.8%
(paired t-test, P < 0.001, n = 4), an effect that was reversible in all cells
tested after 15 min of wash with ACSF. In the presence of mGluR1 and -5 antagonists, CPCCOEt and SIB-1893, respectively, LY354740 (1 µM)
showed a similarly significant inhibitory effect on EPSC amplitude
(45.8% decrease; paired t-test, P < 0.001, n = 4). Analysis of the input-output
relationships using a one-way ANOVA with post hoc
Bonferroni comparisons showed no significant difference between the
effects of LY354740 in the presence and absence of the group I mGluR
antagonists. These results suggest that group II mGluR inhibition in
the CeA is not affected by endogenous activation of group I mGluRs.
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GAMMA-AMINOBUTYRIC ACID (GABA) INHIBITION AND GROUP II MGLURS. Our studies were typically performed in the absence of GABA antagonists since inhibition of GABA in the amygdala resulted in epileptiform bursting. To examine whether GABA inhibition affected group II mGluR inhibition, we tested LY354740 (1 µM) on EPSC amplitude before and after treatment with the GABAA antagonist, picrotoxin (PTX, 50 µM) and the GABAB antagonist, CGP 55845 (2 µM, Fig. 3B). Under these conditions, slices become prone to spontaneous bursting. To prevent bursting a low concentration of the non-N-methyl-D-aspartate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX, 3 µM), was added to the superfusing solution. In Fig. 3B is shown the inhibitory effect of LY354740 on the input-output relationship for EPSC amplitude in ACSF (left) and after perfusion with GABAA and -B antagonists for 15 min (right). The data showed that the inhibitory effect of LY354740 on EPSCs elicited with a 12-V stimulus intensity is not different in the absence (72% decrease; paired t-test, P < 0.0001, n = 4) or presence (67.7% decrease; paired t-test, P < 0.0001, n = 4) of GABAA and -B antagonists. One-way ANOVA of the input-output relationships using post hoc Bonferroni comparisons showed that there were no significant differences between the effect of LY354740 in the presence and absence of GABA antagonists, suggesting that GABA inhibition did not influence group II mGluR inhibitory effects.
GROUP III MGLUR AGONIST.
LAP4 depressed the peak amplitude of EPSCs in control neurons
(n = 7), and this effect was readily reversible within
10-12 min of washing with control ACSF (Fig. 2C). Unlike
the group II agonists, however, the concentration response curve
revealed that LAP4 had a lower potency at the BLA-CeA synapse than at
the VAP-CeA synapse (Table 2), but the maximum effect was similar at
both synapses. Furthermore the effects of LAP4 in CeA neurons are more potent than on BLA neurons (cf. Neugebauer et al. 1997a)
(Table 2).
Chronic cocaine treatment renders group II and group III mGluRs functionally ineffective
The inhibitory effects of the group II and the group III agonists
on synaptic transmission of CeA neurons were studied in brain slices
from rats treated chronically with cocaine. Neither the group II
agonists LCCG1 (up to 100 µM; n = 5; see Figs.
4A and
5A) and LY354740 (up to 10 µM; n = 5; see Fig. 5B) nor the group III
agonist LAP4 (100 µM; n = 5; see Figs.
4B and 5C) were able to alter, significantly,
evoked synaptic transmission at the BLA-CeA and the VAP-CeA synapses in
neurons from animals chronically treated with cocaine. Linear
regression analysis of the concentration response curves using a
F test and the runs test (Graphpad Prism 3.0) showed that
the slope was not significantly different from zero and the data were
not significantly nonlinear (P > 0.05 in each case).
These tests indicate that linear regression analysis was appropriate
and there was no significant change of EPSC amplitude associated with
the different agonist concentrations.
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Group II and III agonist responses are enhanced with kindling
The effects of LCCG1 and LAP4 on synaptic transmission were studied in brain slices from kindled animals 3-7 days after the last of three consecutive stage-five seizures.
GROUP II AGONISTS. In kindled neurons, LCCG1 (100 nM) decreased EPSC amplitude to a greater extent than in control neurons (Fig. 6, A and C). Analysis of the concentration-response relationship (Fig. 6C) showed that the EC50 was shifted twofold to the left at the BLA-CeA synapse in kindled (32.8 nM) compared with control (65.8 nM) neurons. In contrast, at the VAP-CeA synapse, the EC50 was shifted 11-fold to the left (Table 2). The maximal effect of LCCG1 on EPSC amplitude was also enhanced in kindled neurons. At the BLA-CeA pathway, LCCG1 depressed EPSC amplitude in control neurons to a maximum value of 38 ± 6% of control; the depression of EPSC was greater after kindling with only 7 ± 4% of the initial EPSC amplitude remaining (Fig. 6C). Likewise significant differences were found between the different concentrations (F = 45.26,70, P < 0.0001) and between control and kindled neurons (2-way ANOVA, F = 25.451,70, P < 0.0001). Similar alterations were recorded at the VAP-CeA synapse.
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GROUP III AGONIST. We compared the depression of synaptic transmission induced by L-AP4 in control and kindled neurons at the two synapses in the central amygdala. LAP4 (1 nM) reduced the amplitude of EPSCs in kindled neurons to approximately the same extent as a higher concentration (100 nM) did in control neurons (Fig. 6B; n = 5). The concentration-response curve for L-AP4 at the BLA-CeA pathway was shifted almost 50-fold to the left in CeA neurons from kindled animals (Fig. 6D; Table 2). Maximal inhibition of EPSC amplitude was not altered with kindling (Fig. 6D). At the VAP-CeA synapse about a fivefold increase in potency was recorded (Table 2). At the BLA-CeA synapse, the differences in the effects of L-AP4 between the different concentrations were significant (2-way ANOVA, F = 20.856,70, P < 0.0001) and between control and kindled neurons (F = 14.861,70, P < 0.0005).
Effects of group II and group III agonists on membrane conductance in neurons from control and from chronic cocaine and kindled animals
GROUP II AGONISTS.
The group II mGluR agonists LCCG1 (10 µM) and LY354740 (
1 µM)
had no significant effect on postsynaptic membrane currents and
membrane conductance in control neurons. Current-voltage
(I-V) relationships for typical control neurons were not
changed during the superfusion of LCCG1 (
10 µM, n = 9) and LY354740 (
1 µM, n = 5), and no significant
changes in slope conductance (repeated-measures ANOVA with post hoc
t-test, P > 0.1) were measured (Fig.
7). Superfusion with higher
concentrations of LCCG1 (100 µM, Fig. 7A) and LY354740 (10 µM, Fig. 7B) increased the slope conductance significantly in control neurons (post hoc t-test following
repeated-measures ANOVA, P < 0.05). At high
concentrations, a small (<20 pA) outward current was observed in four
of seven neurons with LCCG1 (100 µM) and in two of five neurons with
LY354740 (10 µM); in another two neurons, a small inward current
(10-15 pA) was recorded in the presence of LCCG1 (100 µM) and
LY354740 (10 µM). These data showed that LY354740 (<1 µM), and
LCCG1 (<10 µM), while clearly modulating evoked EPSCs, did not
affect postsynaptic membrane conductance.
|
GROUP III AGONIST.
In control neurons, LAP4 neither evoked detectable membrane currents
nor caused changes in the I-V relationship at concentrations up to 100 µM (Fig. 7C). No significant effect of LAP4
(10 µM) on membrane slope conductance was measured
(repeated-measures ANOVA with post hoc t-test,
P > 0.1; see Fig. 7C). At high
concentrations (100 µM), however, LAP4 decreased the slope
conductance significantly (post hoc t-test following
repeated-measures ANOVA, P < 0.05) and evoked a small
(<20 pA) inward current in five of nine control neurons.
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DISCUSSION |
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Whole cell voltage-clamp recordings of CeA neurons were utilized to study in vitro the role of mGluR as modulators of synaptic transmission in brain slices from control animals, fully kindled animals, and animals treated chronically with cocaine. The main findings of this study are: synaptic transmission at two synapses, the BLA-CeA and VAP-CeA, are depressed by group II and III mGluRs agonists; chronic cocaine treatment but not kindling alters membrane properties of CeA neurons; synaptic transmission is enhanced in neurons from both kindled and cocaine-treated animals; sensitivity of group II and group III mGluR agonists is increased in kindling; and the mGluR agonists lose their ability to modulate synaptic transmission in neurons from animals treated chronically with cocaine.
Effects of mGluR activation on synaptic transmission in the CeA nucleus
Differences in agonist potencies between the synapses (Table 2)
may be due to the distribution of mGluR subtypes. Both LY354740 and
LCCG1 are more potent at group II mGluRs but the potency of LCCG1 for
mGluR8 in some systems is equivalent to that of group II mGluRs; its
effects on group I subtypes is 10 times less than at group II
(Schoepp et al. 1999). LY354740 is ~10 times more potent than LCCG1 on group II mGluRs (Schoepp et al.
1999
). Interestingly, the EC50 ratios
LCCG1 to LY354740 for saline-injected control animals at the BLA- and
VAP-CeA synapses is 28:1 and 31:1, respectively, suggesting that both
agonists may act through a similar group II receptor. LAP4 is more
potent on the group III subtypes mGluR4 and mGluR8 than on mGluR7
(Conn and Pin 1997
; Schoepp et al. 1999
), and on cloned mGluR8, LAP4 is 10 times more potent than LCCG1 (Wu et al. 1998
). The agonist potencies for LCCG1 and
LAP4 in the amygdala are greater than measured elsewhere (Anwyl
1999
; Conn and Pin 1997
; Schoepp et
al. 1999
). It has been shown that potency of agonists in cloned
systems is dependent on the amount of receptor expressed
(Hermans et al. 1999
), and it is possible that there are
significant numbers of spare mGluRs in the amygdala. Alternatively,
mGluRs in the amygdala may have a highly efficient coupling mechanism
to their G-protein-mediated second-messenger effectors where high
occupancy of receptors is not needed and/or where amplification of the
response occurs (see Kenakin 1996
). Definitive
determination of mGluR subtypes mediating effects in the CeA awaits the
ready availability of specific agonists and antagonists.
Changes of mGluR subgroups following chronic cocaine exposure
The intriguing finding of this study is that with chronic
cocaine-treatment group II and group III mGluR agonists lose their effectiveness to inhibit synaptic transmission. The functional loss of
sensitivity to group II and group III mGluR agonists may be due to
multiple mechanisms. One possibility is that this loss is due to
changes in the subtype composition. This mechanism is unlikely since
both subgroups are affected equally. An alternative possibility is that
chronic cocaine exposure may interfere with one or more step(s) in the
signal transduction cascade i.e., the G-protein
(Gi/G0)-mediated inhibition
of voltage-dependent calcium currents, negative coupling to adenylyl
cyclase, or, a mechanism downstream from calcium influx through
voltage-gated calcium channels (Conn and Pinn 1997;
Schoppa and Westbrook 1997
; Toms et al.
1996
). Chronic cocaine treatment has been shown to decrease the
levels of the G-protein subunits Gi
and
Go
in brain areas involved in reward
mechanisms (Nestler et al. 1990
, 1993
) and pertussis toxin-sensitive G proteins have been implicated in behavioral sensitization to cocaine (Steketee et al. 1991
). Chronic
morphine increases levels of Gi
and
Go
in the amygdala (Nestler et al.
1989
; Terwilliger et al. 1991
) and cocaine
sensitization is associated with an upregulation of a beta subunit of a
G protein (Wang et al. 1997
). Furthermore, it is
hypothesized that an altered G-protein-dependent mechanism may underlie
the reduced effectiveness of presynaptic metabotropic
GABAB receptors in the septum after chronic
cocaine treatment (Shoji et al. 1997
). Alterations in the adenylyl cyclase/cAMP system, on the other hand, may not account for the chronic cocaine-induced changes of group II and III
effectiveness because extracellular levels of adenosine, due to
elevated cAMP, are increased in the VTA after chronic cocaine treatment
(Bonci and Williams 1996
). Protein kinase C (PKC)
activation has been proposed to inhibit the function of group II and
III mGluRs (Macek et al. 1998
; Swartz et al.
1993
; Tyler and Lovinger 1995
) by uncoupling the
mGluRs from their G proteins (Macek et al. 1998
). Group
I mGluR or adenosine A3 receptor activation can increase a G-protein PKC and subsequently may block group II or III inhibition of synaptic transmission (Macek et al. 1998
). Block of tonic
activation of mGluR1 and -5 with specific antagonists did not affect
group II mGluR inhibition of synaptic transmission in control CeA
neurons, and the presynaptic sensitivity to adenosine is reduced on
cocaine withdrawal (Manzoni et al. 1998
). It is
possible, however, that the higher levels of adenosine (Bonci
and Williams 1996
) may activate A3 receptors, increase
endogenous PKC, and block the inhibitory action of group II and III
mGluR agonists with chronic cocaine treatment. Future experiments are
designed to address this hypothesis.
The loss of mGluR-mediated inhibitory effects may have significant
implications in neuroadaptive changes occurring with chronic cocaine
use. Evidence suggests that drug dependence is associated with
motivational and affective aspects of withdrawal reflected in an
altered brain reward system (Koob et al. 1998). The
amygdala is involved in stimulus-reward associations (Aggleton
1992
; Gallagher and Holland 1994
). Group II
mGluRs may play a role in anxiety since the orally active group II
mGluR agonist, LY354740, has significant anxiolytic properties
(Helton et al. 1998
; Monn et al. 1997
).
Group II mGluR agonists and antagonists also attenuate morphine
withdrawal symptoms (Fundytus and Coderre 1997
;
Fundytus et al. 1997
). If group II mGluR activation is
necessary to modulate anxiety, their loss may contribute to
counteradaptive changes following chronic cocaine, including anxiety,
dysphoria, depression, and irritability (Koob 1996
;
Koob et al. 1998
; Markou and Koob 1991
).
Chronic cocaine-induced changes in electrophysiological properties in CeA neurons
Chronic cocaine treatment enhanced CeA excitatory synaptic
transmission as evidenced by the leftward shift of the input-output relationships recorded at VAP-CeA and BLA-CeA synapses. A lower EPSC
threshold was measured at both synapses, while a lower spike threshold
was recorded only at the VAP-CeA synapse, suggesting that chronic
cocaine differentially altered the gain of CeA synapses. Increase in
glutamate release was measured in the nucleus accumbens after chronic
cocaine treatment (Pierce et al. 1996). In brain slices
from cocaine-treated rats, current-clamped CeA neurons had a more
hyperpolarized membrane potential and their input resistance was
significantly lower compared with control neurons. These findings would
be expected to reduce excitability by shunting the membrane conductance; however, synaptic transmission in chronic cocaine animals
is enhanced, suggesting that the membrane shunting is overcome. Chronic
exposure to cocaine induced similar changes in membrane potential and
input resistance in dorsolateral septum neurons (Shoji et al.
1997
, 1998
; Simms and Gallagher 1996
), and nucleus accumbens neurons (Zhang et al. 1998
). If
changes in membrane properties and synaptic transmission, as measured
in CeA neurons of animals treated chronically with cocaine, also occurs
in cocaine-dependent individuals, these changes may contribute to their
altered emotional status since the CeA is the common output nucleus for
major amygdala functions and modulates various effector systems
involved in the expression of emotional responses through connections
with the forebrain, limbic system, and brain stem (Aggleton
1992
; Alheid et al. 1995
; Gallagher and
Holland 1996
; Maren 1996
).
Synapses and times of withdrawal after chronic cocaine produce
differing electrophysiological changes in membrane properties and/or
synaptic transmission in different preparations. Following a withdrawal
of 7-10 days, changes in synaptic transmission were observed in
neurons from locus ceruleus and ventral tegmental (Bonci and
Williams 1996; Harris and Williams 1992
) but
were not observed in hippocampal (Manzoni et al. 1998
)
or the latter two nuclei (Bonci and Williams 1996
;
Harris and Williams 1992
) 1 day after the last chronic
cocaine injection. After a withdrawal interval of 3 days changes in
voltage-gated sodium current, in membrane potential, and input
resistance were detected in isolated nucleus accumbens neurons from
rats treated chronically with cocaine (Zhang et al.
1998
). Changes in membrane properties and synaptic transmission also occurred after 1 day of withdrawal in the dorsolateral septal nucleus (Shoji et al. 1998
; Simms and Gallagher
1996
) and in the CeA. The septum and amygdala appear to be
sensitive to early withdrawal from chronic cocaine but the persistence
of these effects is not known although limited preliminary studies in
the amygdala suggest that a decrease in the effects of mGluR agonist
are still present after 7 days (Zinebi, Tokarski, and
Shinnick-Gallagher, unpublished observations). Future studies are
directed at determining the development and withdrawal time courses of
these effects.
Kindling-induced changes in electrophysiological properties in CeA neurons
In contrast to chronic cocaine treatment, membrane conductance in
neurons from kindled animals is not altered although the threshold for
action potential generation is lowered in CeA neurons. These data are
similar to findings in BLA neurons (Asprodini et al.
1992; Neugebauer et al. 1997a
; Rainnie et
al. 1992
). One difference in synaptic afferent pathways to the
BLA and the CeA in kindled animals is that input-output relationships,
a measure of information transfer, in the lateral (LA)-BLA synapse
occurs over a <2 V range (Asprodini et al. 1992
;
Neugebauer et al. 1997a
; Rainnie et al. 1992
) while at the VAP-CeA (or BLA-CeA) synapses the
relationships happen over a greater voltage range, suggesting that
within the amygdala there may be a greater amplification of signal
transfer in kindled neurons at the LA-BLA synapse than at the BLA-CeA synapse.
Kindling-induced changes in mGluRs in CeA neurons
In CeA neurons from amygdala kindled animals, the sensitivity to
inhibitory effects of mGluR group II and III agonists were enhanced and
the magnitudes of change were different for the different agonists. The
shift in the EC50s for LCCG1 and LAP4 were 2- and 50-fold, respectively, in CeA neurons at the BLA-CeA synapse, whereas
mGluR group II and III agonists depress synaptic transmission with a
respective 30- and 28-fold increase in potency at the LA-BLA synapse in
BLA neurons from amygdala-kindled animals (Neugebauer et al.
1997a). Interestingly, the inhibitory effects of
L-serine-O-phosphate (LSOP), a group III
agonist, are reduced in hippocampal dentate granule cells with
kindling, suggesting that the effects of kindling on mGluRs may be
different in different parts of the brain (Klapstein et al.
1999
). Comparison between EC50s for LAP4 and LCCG1
showed that LAP4 was 30 times more potent than LCCG1 in kindled neurons at the BLA-CeA synapse and ~4 and 8 times more potent at the VAP-CeA and LA-BLA synapses, respectively. These data suggest that the subtypes
of mGluRs underlying the enhanced inhibitory effects of agonists at the
various amygdala synapses may be different.
Recent data show that the immunoreactivity to mGluR4 is upregulated in
patients with temporal lobe epilepsy (Lie et al. 2000). The inhibitory responses to group II and group III agonists are upregulated in kindling, and, in the BLA, these agents can block epileptiform bursting in kindled animals (Neugebauer et al.
1997a
). In light of these findings, it is reasonable to suggest
that the group II or III agonists may be useful in the treatment of epilepsy.
Chronic cocaine treatment and kindling
The data suggest that CeA synaptic transmission is fine-tuned
through modifications of mGluR subgroups in different models of
synaptic plasticity. Amygdala sensitivity to electrical
(Neugebauer et al. 1997a; Shinnick-Gallagher et
al. 1998
) and cocaine-induced kindling (Post et al.
1987
) and similarities between kindling and behavioral
sensitization to cocaine suggested that alterations in mGluR agonist
action would be analogous in the two models. Chronic cocaine exposure
and electrical kindling alter the sensitivity of mGluR subgroups in
different directions. These findings suggest that the mechanisms
underlying these modifications may be complex. Irrespective of those
mechanisms, drugs that activate group II and group III mGluRs may be
novel therapeutic tools in the treatment of limbic temporal lobe
epilepsy, and those that restore the inhibitory effects may be of value
in chronic cocaine dependence.
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ACKNOWLEDGMENTS |
---|
The authors thank Dr. Darryl Schoepp at Eli Lilly and Company for the generous gift of LY354740 for the cocaine experiments.
This work was supported by National Institutes of Health Grants T32-DA-07287, NS-24643, MH-57875, DA-11991, and NS-38261.
Present address of V. Neugebauer: Dept. of Anatomy and Neurosciences and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, TX 77555-1069.
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FOOTNOTES |
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Address for reprint requests: P. Shinnick-Gallagher (E-mail: psgallag{at}utmb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 December 1999; accepted in final form 27 April 2000.
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REFERENCES |
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