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Stan B. Floresco and Maric T. Tse
(2007) The Journal of Neuroscience 27: 2045-2057.
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Basolateral amygdala (BLA) to medial prefrontal
cortex (mPFC) circuit involved in:
 Cognitive and emotional processes
 Conditioned fear attainment and extinction
 Differing decision making processes

Disruptions:
 Emotional and cognitive disturbances
 Schizophrenia
 Depression
 Drug addiction
www.umich.edu
Kudos to Russ Carpenter’s Presentation

BLA→mPFC glutamatergic excitatory pathway
 Glu→NMDA→↑Ca++→↑calcineurin→↓DARPP-32
phosphorylation→↑protein phosphatase-1

DARPP-32: potent inhibitor of protein phosphatase-1
 dopamine and cAMP regulated phosphoprotein of MW 32kDA
 http://www.mcmanweb.com/darpp-32.htm

Protein Phosphatase-1 (PP1):
 Cell cycle maintenance, protein synthesis, glycogen storage,
cardiac function , stress recovery, damaged cell apoptosis,
excitation neuron down-regulation of ion pumps and transporters
 Suppression of learning and memory

Ventral Tegmental Area (VTA):
 Neurons overlap with BLA projections in the mPFC
 Modulate BLA activity on mPFC neurons
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DA Receptors:
 D1 expression on mPFC pyramidal cells greater than D2, D4
 Gs →cAMP→PKA→↑DARPP-32 phosphorylation→↓PP1
 D2-like (D2, D4)
 Gi blocks cAMP signaling pathway→↓DARPP-32
phosphorylation→↑PP1
 Increases intracellular Ca++→↑calcineurin activation→↑PP1
 Acts like glutamatergic activation
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Male Sprague Dawley rats
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SNE-100 Kopf concentric bipolar electrical stimulating
electrodes
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mPFC – dorsal border
BLA
VTA
NAc (not all)

Spike 2 software
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Master-8 programmable pulse generator
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Peristimulus time histograms (PSTHs)
4-6 vertical passes through to dorsal mPFC
- BLA stimulation at 0.67Hz, current at 800μA
- 100 pulses delivered when found a responsive neuron
to determine if excitatory or inhibitory
Dorsal/Ventral passes resulted in:
- 4.0±0.6 responsive neurons per electrode track
- n = 167 neurons, 16 rats
- 80% were mPFC(-) in response to BLA stimulation
- 20% were mPFC(+)
BLA input results in an overall net inhibition effect of mPFC
pyramidal neurons
Figure 1.C

Characterization:
 Activation via BLA-evoked polysynaptic parvalbuminimmunoreactive GABAergic interneurons
 Complete cessation of spontaneous firing for 50 ms or more
 Onset of inhibition around 30 ms after stimulation
 Spontaneous firing rate >0.8 Hz
 Similarity to in vitro PFC neuron IPSPs
Duration of inhibition
1.
a.
Longest period of spontaneous firing cessation within the first
200 ms after BLA stimulation
Onset
2.
a.
Timing of suppression after BLA stimulation in ms
Percentage of inhibition of spontaneous firing rate
3.
a.
Ratio of average spontaneous firing rate post BLA stimulation
to average pre-stimulation rate(200 ms each)
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In the varying parameters tested:
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48 BLA→mPFC(-) neurons tested
Baseline firing rate = 3.3±0.4 Hz
Average duration = 182.7± 11 ms
Average onset = 29.3 ms
Single pulse at 0.67 Hz to BLA
Minimum of 50 sweeps
typical 100 – 200 sweeps
Stim. current reduced to
obtain 100 ms inhibition
(200-950μA, median 650 μA)
Figure 1.A
Dopamine transmission administered via:
1.
VTA Stimulation
2.
Iontophoretic application
3.
Systemic DA receptor agonists
a)
b)
c)
d)
SKF 81297 (D1)
Quinpirole (D2-like)
Bromocriptine (D2)
PD168,077 (D4)
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BLA stimulation intensities:
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Evoked complete cessation of firing
Onset ~30 ms
Duration ~ 150-200ms
Evoked inhibition: 2-3 sweeps of 100-200 pulses at 0.67 Hz
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Short-term VTA stimulation effects:

VTA stimulation Results:
 Burst pattern: 20 Hz, 4pulse train, 700μA
 Delivered 25 ms before single pulse to BLA
 Paired stimuli delivered at 10s intervals, 50 sweeps (bursts)
Inhibition occurred both prior to and following BLA
stimulation, therefore short acting (<200 ms) BLA effects
were unable to be determined

Two minutes after VTA burst, stimulated BLA again

n = 11 neurons, 10 rats

Decrease in BLA-evoked inhibition
 Significant reduction in duration of inhibition F(1,10) = 7.96; p = 0.018
 No significant change in onset F(1,10) = 4.31; p = 0.065
 Significant reduction in inhibition of spontaneous firing rate F(1,10) = 5.64;
p = 0.039

Effect returned to baseline after ~10 minutes
 No significant change in baseline firing rate
Figure 2.A
Figure 2.B
Repeated measures ANOVA
- Baseline vs. Post-DA manipulation = within subject factors

Weakening of BLA-evoked inhibition
 No change in baseline firing rate, 2.6 ± 0.5 Hz
 After VTA stimulation, 4.1 ± 1 Hz
 F (1,10) = 1.82, p = 0.207

4 of the 11 neuons tested
 Increase in spontaneous firing, +273 ± 2%
 Little or no change in remaining 7 neurons, -14 ± 15%
 Two-way ANOVA showed no difference in neurons
 F(1,9) = 0.52, p = .489
VTA stimulation induced attenuation of BLA-evoked
inhibition not due to changes in spontaneous firing rates


Neurons tested, n = 6 from 4 rats
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Effect on BLA-evoked inhibition
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
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Substantial reduction in duration F(1,5) = 32.89; p = 0.002
No significant change in onset F(1,5) = 0.43; p = 0.54
No significant % inhibition of spontaneous firing F(1,5) = 2.18; p = 0.20
No significant change in spontaneous firing rate F(1,5)= 2.31; p = 0.189
Iontophoretic application attenuated BLA-evoked
inhibition, but not as succinctly as VTA stimulated modulation
 Spatial restriction contribution?
Figure 2.C
Repeated measures ANOVA
- Baseline vs. Post-DA manipulation = within measures

Designed to determine if receptor specificity involvement
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SKF 81297 – D1 specific
Quinpirole – D2/D4 non-specific
PD-168,077 – D4 specific
Bromocriptine – D2 specific
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Administered via intravenous injection

1 neuron per rat, 1 injection per rat

Stimulation intensities adjusted to baseline BLA-evoked
excitation or inhibition

5 minute period from drug injection to BLA stimulation
 BLA→mPFC (-): 100-200 sweeps before and after drug injection
 BLA→mPFC (+): 40-150 sweeps

Four agonists plus saline control

Treatment by sample interactions resulted in
significant effect for all three measures
 Duration of inhibition F(4,24) = 3.83; p = 0.015
 Onset of inhibition F(4,24) = 3.57; p = 0.020
 Percentage inhibition of firing rate F(4,24) = 4.65; p = 0.006

Saline control had no effect on BLA-evoked
inhibition measures or baseline firing rate
 Repeating single-pulse BLA stimulation did not effect BLAevoked inhibition or the BLA→mPFC(-) spontaneous firing rates
over time

D1 agonist SKF 81297 (0.5mg/kg; n = 5)

D2-Like: D2, D4 agonist Quinpirole (0.2mg/kg; n = 6)
 No significant effect on any of the three measures
 Did not modulate BLA-evoked inhibition
 Significantly weakened BLA-evoked inhibition


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Reduced duration of inhibition, p = 0.007
Increased onset of inhibition, p = 0.002
Weakened percentage inhibition of spontaneous firing, p = 0.012
Therefore can reduce normal BLA induced feedforward mPFC inhibition and enhance BLA driven
excitation pathway
D4 agonist PD-168,077 (1mg/kg; n = 7)
 Weakened BLA-evoked inhibition in all three measures
 Reduced duration of inhibition, p = 0.0003
 Increased onset of inhibition, p = 0.009
 Weakened percentage inhibition of spontaneous firing, p < 0.0001

D2 agonist Bromocriptine (0.5mg/kg; n = 6)
 Reduced duration of inhibition, p = 0.0003
 Weakened percentage inhibition of spontaneous firing, p = 0.003
 Did not change onset of inhibition, p = 0.781
Figure 3. Administration of D2 or D4 (but not D1) DA receptor agonists attenuates BLAevoked inhibition of mPFC neurons

The agonists did not altered the effect of
spontaneous firing rates of mPFC neurons
 D2 and D4 activation weakened BLA-evoked inhibition in a
subpopulation of mPFC neurons
 May then increase effects of excitatory inputs from BLA
 Also found one mPFC(-) neuron that acted as a monosynaptic
mPFC(+) neuron in the presence of D1 agonist SKF 81297 in
response to BLA stimulation
Two-way between-/within- subjects factorial ANOVA
- between subjects factor: drug treatment
-within subjects factor: baseline and post drug administration

Characterization:
 Fast onset monosynaptic AP response to BLA stimulation
 Orthodromic (ortho = true or straight, dromic = running)
 Signal to noise ratio of 3:1 minimum
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If response showed:
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Spike jitter of at least 2 ms minimum
Shift in spike latency with increased amplitude
Followed paired-pulse stimulation (50 Hz) but failed after 400Hz
paired-pulse stimulation (antidromic)
Little to no spontaneous firing rates
Unable to detect BLA-evoked inhibition
Did not analyze feed-forward GABA inhibition
Submaximal stimulation intensity
200-1000μA, median 700 μA
BLA-evoked AP ~ 50-70% at 0.25 Hz
Minimum of 40 sweeps
Evoked firing probabilities:
# of evoked spikes / # pulses delivered
Dopamine transmissions again via:
1. VTA stimulation
2. Iontophoretic application
3. Systemic application of receptor agonists
Figure 1.B

In the different protocols:
 44 BLA→mPFC(+) neurons
 Baseline firing rate: 1.9±0.4 Hz
 ~50% had very low rates of spontaneous firing: 0-0.8 Hz
 Could not determine inhibitory response
 Remaining ~50% displayed evoked EPSP-IPSP-like inhibition
after initial firing
 Only characterized evoked firing effects from DA protocols
 The average latency of evoked excitatory response was 13±0.5 ms

BLA stimulation intensities set to evoke AP ~60-70%
of the time
 Single pulse, 0.25 Hz
 Burst stimulation of VTA 25 ms prior to BLA stimulation
 Some trials adjusted latency to 25-200 ms
 Minimum of 25 sweeps
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BLA stimulation frequency dependency trials:
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BLA stimulation: 20 Hz trains of 5 pulses
Delivered 20 ms after VTA burst stimulation
Combination delivered every 10s
Minimum of 25 sweeps
n = 9, 7 rats
VTA burst stimulation 25s before BLA
single pulse stimulation


 Suppression of BLA-evoked firing -95 ± 4%
 F(1,8) = 76.49, p = 0.0001
 Inhibition did not continue post VTA stimulation

Two minutes post VTA stimulation
 No significant change in evoked firing
probability from baseline F(1,7) = 0.41; p = 0.542

VTA stimulation decreased BLA-evoked
firing, but the duration of the effect
was short lasting
Figure 4.A

Interval adjustment effects on suppression
magnitude evoked firing:
 n = 9, 5 rats
 Two-way repeated measures ANOVA
Significant sample by interval interaction effect F(4,32) = 5.38, p = 0.002
Extending the interval reduced the suppression
At 200 ms, still significantly reduced evoked firing probability
 38±13%; p = 0.041
*p < 0.05
**p < 0.01
Figure 4.B
Modulation Effect:
GABAergic suppression?
DA release suppression?

Effects of VTA burst stimulation on evoked firing
 n = 6, 6 rats
 BLA train stimulation: 5 pulses, 20 Hz
 Two-factor ANOVA
 Significant sample by pulse interaction F(4,20) = 15.49, p < 0.0001
 Increased frequency of BLA stimulation alone
 Significant increase in evoked firing probability, p = 0.006
 Progressive over each pulse in the train
 Burst stimulation of VTA 25 ms prior to BLA train stimulation
 Suppression of firing evoked by the first pulse of BLA train
 Second pulse suppression significantly attenuated compared to the first pulse
 Consequent pulses resulted in no VTA suppression of evoked firing
 End of VTA stimulation to later pulses ~ 100 – 200 ms
 BLA-evoked firing not inhibited
 At 100 – 200 ms, VTA stimulation of single-pulse BLA protocol resulted in
significant suppression of mPFC firing
 Frequency dependent
Figures 4.D and 4.E

n= 3, 3 rats
 100% significant reduction in BLA-evoked firing probability
 Average -36± 4%
 F(1,2) = 67.01, p = 0.014
 No significant change in spontaneous firing rate
 F(1,2) = 11.89, p = .075
DA application weakens BLA-evoked firing in a
subpopulation of mPFC neurons

Suppression via VTA stimulation was greater than via
local application

Extended interval VTA stimulation resembled local
application results


D1 receptor agonist SKF 81297 (0.5 mg/kg)
 n=9
 67% had significant suppression of BLA-evoked firing (6 of 9)
 Magnitude similar to iontophoretic DA application (-36.1± 12%)
 F(1,8) = 7.59, p = 0.024
 No significant change in spontaneous firing rate
 F(1,8) = 0.04, p = 0.847
Figure 5.B

D2-Like receptor agonist Quinpirole (0.2mg/kg)
 n=7
 Did not alter BLA-evoked firing F(1,6) = 0.19, p = 0.678
 Significant increase in baseline firing rate F(1,6) = 6.17, p = 0.048

D4 receptor agonist PD-168,077 (1mg/kg)
 n=7
 Did not alter BLA-evoked firing rate F(1,6) ≤ 1.1, p ≥ 0.335
 Did not alter spontaneous firing rate F(1,6) ≤ 1.1, p ≥ 0.335
Figure 5.A Mean ± SEM firing probability evoked by single-pulse stimulation of the BLA
before drug administration (baseline; white bar) and after systemic administration of
DA agonists selective for D1 (SKF 81297), D2/D4 (quinpirole), or D4 (PD-168,077)
receptors (black bars). *p < 0.05 versus baseline.
Two-way between-/within- subjects factorial ANOVA
- between subjects factor: drug treatment
-within subjects factor: baseline and post drug administration
Antidromic neurons were activated by
stimulating the Nac or the VTA
- some neurons receive either
direct or indirect BLA
projections
- projections then go to
ventral striatum or midbrain
DA cells
Figure 6
n = 22, BLA stimulated
Latency responses compared to mPFC(+) latency
responses


 Antidromic latency was longer than orthdromic
 t(64) = 5.02, p = 0.0001
 Mode (21 ms) higher than orthodromic mode (12ms)
 Orthodromic signals from BLA arrive at mPFC sooner
 Therefore excitation of the BLA probably due to glutamatergic
projections from the BLA to the mPFC and not antidromic activation of
recurrent axon collaterals from the mPFC to the BLA
 BLA-evoked inhibition likely to be due to ascending BLA
glutamatergic pathways
 Electrode placement was caudal BLA
 mPFC projections terminate more in the more rostral BLA
 Latency data suggests excitatory responses were likely ascending
 Inhibition via GABAergic interneurons also ascending glutamatergic pathway
 ~60% of BLA→mPFC(-) had shorter latencies than antidromic
 Points to ascending pathway involvement
A. Five overlaid traces from a
BLA→mPFC(+) neuron that fired
orthodromic spikes after single-pulse
BLA stimulation (left).
Same neuron showing antidromic
spikes after VTA stimulation (right).
B. Mean and modal response latencies of
BLA-evoked orthodromic excitatory
responses (black bars) and BLA-evoked
antidromic responses (gray bars).
C. Distribution of BLA-evoked
orthodromic (thick lines) and
antidromic (broken lines) response
latencies. Bin width, 5 ms.
Figure 7