1. Stan B. Floresco and Maric T. Tse
Dopaminergic regulation of inhibitory and excitatory transmission in the basolateral amygdala-prefrontal cortical pathway
Stan B. Floresco and Maric T. Tse
(2007) The Journal of Neuroscience 27:

2. Introduction: 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

3. BLA glutamatergic projections to: anterior cingulate, prelimbic, infralimbic cortices and GABAergic interneurons
Kudos to Russ Carpenter’s Presentation

4. Glutamatergic Signaling
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
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

5. Mesocortical Dopamine (DA)
Ventral Tegmental Area (VTA):
Neurons overlap with BLA projections in the mPFC
Modulate BLA activity on mPFC neurons
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

6. How does DA modulate BLA-mPFC inhibitory [BLA→mPFC(-)] and excitatory [BLA→mPFC(+)] activity in vivo?

7. Materials & Methods Male Sprague Dawley rats
SNE-100 Kopf concentric bipolar electrical stimulating
electrodes
mPFC – dorsal border
BLA
VTA
NAc (not all)
Spike 2 software
Master-8 programmable pulse generator
Peristimulus time histograms (PSTHs)

8. Results 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

9. BLA →mPFC(-) Neurons Characterization:
Activation via BLA-evoked polysynaptic parvalbumin-immunoreactive 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

10. BLA-evoked Inhibition Modification Measures
Duration of inhibition
Longest period of spontaneous firing cessation within the first 200 ms after BLA stimulation
Onset
Timing of suppression after BLA stimulation in ms
Percentage of inhibition of spontaneous firing rate
Ratio of average spontaneous firing rate post BLA stimulation to average pre-stimulation rate(200 ms each)

11. BLA→mPFC(-) Neurons In the varying parameters tested:
48 BLA→mPFC(-) neurons tested
Baseline firing rate = 3.3±0.4 Hz
Average duration = 182.7± 11 ms
Average onset = 29.3 ms

12. BLA→mPFC(-) Neurons Single pulse at 0.67 Hz to BLA
Minimum of 50 sweeps
typical 100 – 200 sweeps
Stim. current reduced to
obtain 100 ms inhibition
( μA, median 650 μA)
Figure 1.A

13. BLA→mPFC(-) Manipulations
Dopamine transmission administered via:
VTA Stimulation
Iontophoretic application
Systemic DA receptor agonists
SKF (D1)
Quinpirole (D2-like)
Bromocriptine (D2)
PD168,077 (D4)

14. VTA Modulation of BLA-evoked mPFC(-) Neurons
BLA stimulation intensities:
Evoked complete cessation of firing
Onset ~30 ms
Duration ~ ms
Evoked inhibition: 2-3 sweeps of pulses at 0.67 Hz
Short-term VTA stimulation effects:
Burst pattern: 20 Hz, 4pulse train, 700μA
Delivered 25 ms before single pulse to BLA
Paired stimuli delivered at 10s intervals, 50 sweeps (bursts)
VTA stimulation Results:
Inhibition occurred both prior to and following BLA stimulation, therefore short acting (<200 ms) BLA effects were unable to be determined

15. Results of DA Modulation of BLA→mPFC(-): VTA Stimulation
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

16. PSTH from one neuron before and 2 minutes after VTA repeated burst stimulation
Figure 2.A

17. Results of DA Modulation of BLA→mPFC(-): VTA Stimulation
Figure 2.B
Repeated measures ANOVA
- Baseline vs. Post-DA manipulation = within subject factors

18. Results of DA Modulation of BLA→mPFC(-): VTA Stimulation
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

19. BLA→mPFC(-): Iontophoretic Application of DA
Neurons tested, n = 6 from 4 rats
Effect on BLA-evoked inhibition
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

20. BLA→mPFC(-): Iontophoretic Application of DA
Repeated measures ANOVA
- Baseline vs. Post-DA manipulation = within measures

21. Systemic DA Receptor Agonists
Designed to determine if receptor specificity involvement
SKF – D1 specific
Quinpirole – D2/D4 non-specific
PD-168,077 – D4 specific
Bromocriptine – D2 specific
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 (-): sweeps before and after drug injection
BLA→mPFC (+): sweeps

22. BLA→mPFC(-): Systemic Application of DA
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 BLA-evoked inhibition or the BLA→mPFC(-) spontaneous firing rates over time

23. BLA→mPFC(-): Systemic Application of DA
D1 agonist SKF (0.5mg/kg; n = 5)
No significant effect on any of the three measures
Did not modulate BLA-evoked inhibition
D2-Like: D2, D4 agonist Quinpirole (0.2mg/kg; n = 6)
Significantly weakened BLA-evoked inhibition
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 =
Increased onset of inhibition, p = 0.009
Weakened percentage inhibition of spontaneous firing, p <
D2 agonist Bromocriptine (0.5mg/kg; n = 6)
Weakened percentage inhibition of spontaneous firing, p = 0.003
Did not change onset of inhibition, p = 0.781

24. BLA→mPFC(-): Systemic Application of DA
Figure 3. Administration of D2 or D4 (but not D1) DA receptor agonists attenuates BLA-evoked inhibition of mPFC neurons

25. BLA→mPFC(-): Systemic Application of DA
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 in response to BLA stimulation

26. BLA→mPFC(-): Systemic Application of DA
Two-way between-/within- subjects factorial ANOVA
- between subjects factor: drug treatment
-within subjects factor: baseline and post drug administration

27. BLA→mPFC(+) Neurons Characterization: If response showed:
Fast onset monosynaptic AP response to BLA stimulation
Orthodromic (ortho = true or straight, dromic = running)
Signal to noise ratio of 3:1 minimum
If response showed:
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

28. BLA→mPFC(+) Neurons Submaximal stimulation intensity
μA, 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

29. BLA→mPFC(+) Neurons In the different protocols: 44 BLA→mPFC(+) neurons
Baseline firing rate: 1.9±0.4 Hz
~50% had very low rates of spontaneous firing: 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

30. BLA→mPFC(+): VTA Stimulation
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 ms
Minimum of 25 sweeps
BLA stimulation frequency dependency trials:
BLA stimulation: 20 Hz trains of 5 pulses
Delivered 20 ms after VTA burst stimulation
Combination delivered every 10s

31. BLA→mPFC(+): VTA Stimulation
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 =
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

32. BLA→mPFC(+): VTA Stimulation
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
Modulation Effect:
GABAergic suppression?
DA release suppression?
*p < 0.05
**p < 0.01
Figure 4.B

33. BLA→mPFC(+): Burst Stimulation of VTA
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 <
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

34. BLA→mPFC(+): Burst Stimulation of VTA
Figures 4.D and 4.E

35. BLA→mPFC(+): Iontophoretic Application of DA
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

36. BLA→mPFC(+): Iontophoretic Application of DA

37. BLA→mPFC(+):Systemic Administration of DA Agonists
D1 receptor agonist SKF (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

38. BLA→mPFC(+):Systemic Administration of DA Agonists
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)
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

39. BLA→mPFC(+):Systemic Administration of DA Agonists
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.

40. BLA→mPFC(-): Systemic Application of DA
Two-way between-/within- subjects factorial ANOVA
- between subjects factor: drug treatment
-within subjects factor: baseline and post drug administration

41. Distribution of mPFC(+) and mPFC(-) Neurons
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

42. Antidromic Activated mPFC(+) and mPFC(-) Neurons
n = 22, BLA stimulated
Latency responses compared to mPFC(+) latency responses
Antidromic latency was longer than orthdromic
t(64) = 5.02, p =
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

43. Antidromic Activated mPFC(+) and mPFC(-) Neurons
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

44. Antidromic Activated mPFC(+) and mPFC(-) Neurons
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).
Mean and modal response latencies of BLA-evoked orthodromic excitatory responses (black bars) and BLA-evoked antidromic responses (gray bars).
Distribution of BLA-evoked orthodromic (thick lines) and antidromic (broken lines) response latencies. Bin width, 5 ms.
Figure 7