Stan B. Floresco and Maric T. Tse

(2007) The Journal of Neuroscience 27: 2045-2057.

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

Kudos to Russ Carpenter’s Presentation

www.umich.edu

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

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

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)

Figure 1.C

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

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

1. Duration of inhibitiona. Longest period of spontaneous firing cessation within

the first 200 ms after BLA stimulation

2. Onseta. Timing of suppression after BLA stimulation in ms

3. Percentage of inhibition of spontaneous firing rate

a. Ratio of average spontaneous firing rate post BLA stimulation to average pre-stimulation rate(200 ms each)

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

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.V

TA Stimulation

2.I

ontophoretic application

3.S

ystemic DA receptor agonistsa) SKF 81297 (D1)b) Quinpirole (D2-like)c) Bromocriptine (D2)d) PD168,077 (D4)

BLA stimulation intensities:

Evoked complete cessation of firing Onset ~30 ms Duration ~ 150-200ms Evoked inhibition: 2-3 sweeps of 100-200 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

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.BRepeated 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

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

Repeated measures ANOVA- Baseline vs. Post-DA manipulation = within measures

Designed to determine if receptor specificity involvement

SKF 81297 – 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 (-): 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 BLA-evoked inhibition or the BLA→mPFC(-) spontaneous firing rates over time

D1 agonist SKF 81297 (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 = 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 BLA-evoked 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

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

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

BLA stimulation frequency dependency trials:

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 suppressionAt 200 ms, still significantly reduced evoked firing probability

38±13%; p = 0.041

Figure 4.B

*p < 0.05**p < 0.01

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