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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: 2045-2057. Introduction:. Basolateral amygdala (BLA) to medial prefrontal

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Stan B. Floresco and Maric T. Tse

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  1. 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: 2045-2057.

  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 www.umich.edu 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 • 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

  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 (200-950μA, median 650 μA) Figure 1.A

  13. BLA→mPFC(-) Manipulations Dopamine transmission administered via: • VTA Stimulation • Iontophoretic application • Systemic DA receptor agonists • SKF 81297 (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 ~ 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

  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 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

  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 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 feedforwardmPFC 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

  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 81297 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: • 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 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

  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: 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

  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 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

  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 = 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

  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 < 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

  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 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

  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) • 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

  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 = 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

  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

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