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Dopamine D3 receptor modulation of prefrontal cells and circuits
- 1. Dopamine D3 receptor modulation of prefrontal cells and circuits N E U R O S C I E N C E T H E S I S S E M I N A R F R I D A Y , S E P T E M B E R 2 2 , 2 0 1 7 R E B E C C A C L A R K S O N B E N D E R L A B
- 2. Prefrontal cortex supports high level cognitive processes Adapted from Arias-Carrión et al., 2010 Prefrontal cortex • Working memory • Attention • Decision-making
- 3. Adapted from Arias-Carrión et al., 2010 Prefrontal cortex Prefrontal cortex is a central hub in a distributed system • Prefrontal circuits integrate information about internal and external conditions to guide decision-making.
- 4. Adapted from Arias-Carrión et al., 2010 Nucleus accumbens Thalamus Amygdala Prefrontal cortex Spinal cord VTA Prefrontal cortex is a central hub in a distributed system • Prefrontal circuits integrate information about internal and external conditions to guide decision-making. • Prefrontal cortex is extensively interconnected with other brain regions.
- 5. Prefrontal dopamine is critical for normal cognitive function • Dopaminergic input is essential for both fear and reward-related learning. Adapted from Arias-Carrión et al., 2010 VTA Prefrontal cortex source of prefrontal dopamine input fibers that release dopamine
- 6. Dysfunctional prefrontal dopamine signaling à • Schizophrenia • Depression • Parkinson’s • Drug addiction Prefrontal dopamine is critical for normal cognitive function Adapted from Arias-Carrión et al., 2010 VTA Prefrontal cortex source of prefrontal dopamine input fibers that release dopamine
- 7. PFC pyramidal neurons receive dense dopaminergic input VTA Adapted from Arias-Carrión et al., 2010 source of prefrontal dopamine input fibers that release dopamine Neurons: highly specialized cells within the brain that communicate with each other Prefrontal cortex
- 8. VTA Prefrontal cortex PFC pyramidal neurons receive dense dopaminergic input
- 9. PFC pyramidal neurons receive dense dopaminergic input
- 10. PFC pyramidal neurons receive dense dopaminergic input
- 11. PFC pyramidal neurons receive dense dopaminergic input
- 12. Pyramidal neurons have many kinds of receptors
- 13. Dopamine acts via D1-family and D2-family receptors Dopamine Receptors D1-family D2-family
- 14. Dopamine acts via D1-family and D2-family receptors Dopamine Receptors D1-family D2-family Gas/olf Gai/o
- 15. Dopamine acts via D1-family and D2-family receptors Dopamine Receptors D1-family D2-family Gas/olf Gai/o Adenylyl cyclase
- 16. Dopamine Receptors D1-family and D2-family both have multiple subtypes D1-family D2-familyPharmacology
- 17. Dopamine Receptors D1-family and D2-family both have multiple subtypes D1-family D2-familyPharmacology D1 D5 D2 D3 D4
- 18. Dopamine Receptors D1-family D2-family D1 D5 D2 D3 D4 D1-family and D2-family both have multiple subtypes Genetically-modified “transgenic” mice
- 19. Dopamine Receptors D1-family D2-family D1 D5 D2 D3 D4 Dopamine receptor subtypes in PFC Genetically-modified “transgenic” mice
- 20. Dopamine Receptors D1-family D2-family D1 D5 D2 D3 D4 Dopamine receptor subtypes in PFC Genetically-modified “transgenic” mice
- 21. D3R role in prefrontal function Dysfunction in prefrontal D3R signaling may play a major role in neuropsychiatric disorders D3Rs Schwartz et al., 2000; Newman et al., 2012; Joyce and Millan, 2005; Gross et al., 2013; Glickstein et al., 2005; Loiseau and Millan, 2009; Watson et al., 2012
- 22. D3Rs D3R role in prefrontal function • Many antipsychotics have equal affinity for D3R and D2R Dysfunction in prefrontal D3R signaling may play a major role in neuropsychiatric disorders Schwartz et al., 2000; Newman et al., 2012; Joyce and Millan, 2005; Gross et al., 2013; Glickstein et al., 2005; Loiseau and Millan, 2009; Watson et al., 2012
- 23. D3Rs D3R role in prefrontal function • Many antipsychotics have equal affinity for D3R and D2R • Pro-cognitive effect of decreased prefrontal D3R activation Dysfunction in prefrontal D3R signaling may play a major role in neuropsychiatric disorders Schwartz et al., 2000; Newman et al., 2012; Joyce and Millan, 2005; Gross et al., 2013; Glickstein et al., 2005; Loiseau and Millan, 2009; Watson et al., 2012
- 24. D3Rs D3R role in prefrontal function • Many antipsychotics have equal affinity for D3R and D2R • Pro-cognitive effect of decreased prefrontal D3R activation • Very high D3R affinity for dopamine (>20x higher than D2R) Dysfunction in prefrontal D3R signaling may play a major role in neuropsychiatric disorders Schwartz et al., 2000; Newman et al., 2012; Joyce and Millan, 2005; Gross et al., 2013; Glickstein et al., 2005; Loiseau and Millan, 2009; Watson et al., 2012
- 25. D3Rs D3R role in prefrontal function • Many antipsychotics have equal affinity for D3R and D2R • Pro-cognitive effect of decreased prefrontal D3R activation • Very high D3R affinity for dopamine (>20x higher than D2R) Dysfunction in prefrontal D3R signaling may play a major role in neuropsychiatric disorders The cellular mechanisms by which D3Rs modulate prefrontal function remain unclear Schwartz et al., 2000; Newman et al., 2012; Joyce and Millan, 2005; Gross et al., 2013; Glickstein et al., 2005; Loiseau and Millan, 2009; Watson et al., 2012
- 26. D3Rs D3R role in prefrontal function Dysfunction in prefrontal D3R signaling may play a major role in neuropsychiatric disorders Thalamus Amygdala Spinal cord VTA Nucleus accumbens • Many antipsychotics have equal affinity for D3R and D2R • Pro-cognitive effect of decreased prefrontal D3R activation • Very high D3R affinity for dopamine (>20x higher than D2R) The cellular mechanisms by which D3Rs modulate prefrontal OUTPUT remain unclear Schwartz et al., 2000; Newman et al., 2012; Joyce and Millan, 2005; Gross et al., 2013; Glickstein et al., 2005; Loiseau and Millan, 2009; Watson et al., 2012
- 27. D3Rs Key questions remain concerning mPFC D3R function 1) Where is the D3R in the prefrontal cortex?
- 28. D3Rs Key questions remain concerning mPFC D3R function 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R- expressing neurons?
- 29. D3Rs Key questions remain concerning mPFC D3R function 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R- expressing neurons? Electrophysiology = electrical properties of biological cells and tissues Action potentials (APs)
- 30. D3Rs Key questions remain concerning mPFC D3R function 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R- expressing neurons? 3) What are the downstream targets of these D3+ neurons?
- 31. D3Rs Key questions remain concerning mPFC D3R function 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R- expressing neurons? 3) What are the downstream targets of these D3+ neurons? 4) How do D3Rs modulate prefrontal neurons?
- 32. D3Rs Key questions remain concerning mPFC D3R function 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R- expressing neurons? 3) What are the downstream targets of these D3+ neurons? 4) How do D3Rs modulate prefrontal neurons? 5) What are the functional consequences of D3R signaling on neural activity?
- 33. D3Rs Key questions remain concerning mPFC D3R function 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R- expressing neurons? 3) What are the downstream targets of these D3+ neurons? 4) How do D3Rs modulate prefrontal neurons? 5) What are the functional consequences of D3R signaling on neural activity? What do we already know about D1R and D2R expression?
- 34. Orientation to mouse prefrontal cellular anatomy
- 35. Orientation to mouse prefrontal cellular anatomy
- 36. Orientation to mouse prefrontal cellular anatomy Coronal section
- 37. Orientation to mouse prefrontal cellular anatomy Coronal section Coronal brain slice
- 38. Orientation to mouse prefrontal cellular anatomy medial prefrontal cortex (mPFC) Coronal brain slice
- 39. Orientation to mouse prefrontal cellular anatomy medial prefrontal cortex (mPFC) Coronal brain slice
- 40. Orientation to mouse prefrontal cellular anatomy
- 41. Prefrontal cortical circuits contain diverse cell types
- 42. Prefrontal cortical circuits contain diverse cell types Pyramidal neurons send excitatory projections across the brain Nucleus accumbens Prefrontal cortex Thalamus Amygdala Spinal cord VTA
- 43. Two major classes of prefrontal projection neuron Pyramidal neurons send excitatory projections throughout the brain Spinal cordAmygdala StriatumCortex
- 44. Two major classes of prefrontal projection neuron Two major classes: Pyramidal neurons send excitatory projections throughout the brain
- 45. Two major classes of prefrontal projection neuron 1) Pyramidal tract (PT) = subcortical Two major classes: Pyramidal neurons send excitatory projections throughout the brain
- 46. Two major classes of prefrontal projection neuron 1) Pyramidal tract (PT) 2) Intratelencephalic (IT) = cortical Two major classes: Pyramidal neurons send excitatory projections throughout the brain 1) Pyramidal tract (PT) = subcortical
- 47. Two major classes of prefrontal projection neuron 1) Pyramidal tract (PT) 2) Intratelencephalic (IT) Two major classes: Pyramidal neurons send excitatory projections throughout the brain
- 48. What do we know about D1R and D2R expression in L5 PFC?
- 49. D1 D1 Contralateral cortex D1R is expressed on callosally-projecting (IT) pyramidal neurons Gee, Ellwood et al., 2012 Projection targets of D1R- and D2R-expressing L5 neurons
- 50. Thalamus D2 D2 D2R is expressed on subcortically-projecting (PT) pyramidal neurons Gee, Ellwood et al., 2012 Projection targets of D1R- and D2R-expressing L5 neurons
- 51. D1R- and D2R-dependent modulation in PFC D1R- and D2R-expressing neuron populations are electrophysiologically distinct D2 D2 D1 D1 D1+ D2+ Gee, Ellwood et al., 2012
- 52. D1R- and D2R-dependent modulation in PFC D1R- and D2R-expressing neuron populations are differentially affected by dopamine D2 D2 D1 D1
- 53. D1R- and D2R-dependent modulation in PFC D1-receptor dependent: • Increase in AP firing • Depolarization • Increase in input resistance Seong and Carter 2012 D1 D1
- 54. D1R- and D2R-dependent modulation in PFC D2-receptor dependent: • Increase in AP firing • Requires synaptic stimulation Gee, Ellwood et al., 2012; Robinson and Sohal 2017 D2 D2
- 55. D1R/D2R D1R and D2R role in prefrontal cortex 1) Where are they in the prefrontal cortex? 2) What are their electrophysiological properties? 3) What are their downstream targets? 4) How do these receptors modulate prefrontal neurons? 5) What are the functional consequences?
- 56. D1R/D2R D1R and D2R role in prefrontal cortex 1) Where are they in the prefrontal cortex? 2) What are their electrophysiological properties? 3) What are their downstream targets? 4) How do these receptors modulate prefrontal neurons? 5) What are the functional consequences? Layer 5 Distinct properties IT vs PT Increase AP firing via different mechanisms
- 57. 1 How are D3Rs distributed anatomically? Results Outline D3R DISTRIBUTION
- 58. 1 How are D3Rs distributed anatomically? 2 ELECTROPHYSIOLOGICAL PROPERTIES OF D3R-EXPRESSING NEURONS Results Outline What are the electrophysiological characteristics of these neurons? Do they differ from neighboring neurons? D3R DISTRIBUTION
- 59. 3 Are D3Rs expressed on a specific class of projection neuron? PROJECTION PATTERNS
- 60. 3 4 Are D3Rs expressed on a specific class of projection neuron? PROJECTION PATTERNS D3R-DEPENDENT MODULATION Do D3R-expressing pyramidal neurons undergo subtype- specific modulation?
- 61. 3 4 5 Are D3Rs expressed on a specific class of projection neuron? PROJECTION PATTERNS Do D3R-expressing pyramidal neurons undergo subtype- specific modulation? What are the functional consequences of D3R modulation? D3R IMPACT ON NEURONAL OUTPUT D3R-DEPENDENT MODULATION
- 62. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION Dopamine receptor distribution in mPFC
- 63. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION mPFC Dopamine receptor distribution in mPFC
- 64. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing cells in mPFC D3-Cre transgenic mouse Experimental method: mPFC
- 65. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing cells in mPFC D3-Cre transgenic mouse + Cre-dependent virus with fluorescent marker Experimental method: DIO-EYFP
- 66. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing cells in mPFC D3-Cre transgenic mouse + Cre-dependent virus with fluorescent marker Experimental method: DIO-EYFP
- 67. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing cells in mPFC D3-Cre transgenic mouse + Cre-dependent virus with fluorescent marker Experimental method: DIO-EYFP
- 69. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION Dopamine receptor distributions (D1R/D2R/D3R) D1R-expressing (whole brain) D2R-expressing (mPFC) D3R-expressing (mPFC)
- 70. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION L5 distribution differs between D1R+/D2R+/D3R+ cells
- 71. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION L5 distribution differs between D1R+/D2R+/D3R+ cells
- 72. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION L5 distribution differs between D1R+/D2R+/D3R+ cells
- 73. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION L5 distribution differs between D1R+/D2R+/D3R+ cells
- 74. 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION L5 distribution differs between D1R+/D2R+/D3R+ cells Distinct L5 laminar profiles suggests separate neuronal populations
- 75. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Berger et al., 2001, 2003 [HCN = hyperpolarization-activated cation] Receptor expression correlated with intrinsic properties Hyperpolarizing (negative) current injection can activate HCN channels, revealing “h-current” h-current affects neuronal functional properties: • Synaptic integration • AP backpropagation
- 76. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current”
- 77. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current” Rebound Sag
- 78. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current”
- 79. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current”
- 80. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current” Type 2
- 81. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current” Type 2
- 82. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current” Type 2
- 83. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES D2+ cells can be distinguished by “h-current” Type 2 Type 1 or Type 3
- 84. Sorting D1+ and D3+ cells is a little trickier… 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES
- 85. Sorting D1+ and D3+ cells is a little trickier… 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES
- 86. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Cannot be distinguished by latency to peak rebound D1+ and D3+ neurons have overlapping sag/rebound properties
- 87. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES 1) Are these separate populations? Cannot be distinguished by latency to peak rebound D1+ and D3+ neurons have overlapping sag/rebound properties
- 88. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES 1) Are these separate populations? 2) Can we use electrophysiology to predict dopamine receptor expression in unlabelled neurons? Cannot be distinguished by latency to peak rebound D1+ and D3+ neurons have overlapping sag/rebound properties
- 89. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Dt D1+ and D3+ neurons have overlapping sag/rebound properties
- 90. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES DmV (exp. fit) D1+ and D3+ neurons have overlapping sag/rebound properties
- 91. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Sag and rebound properties significantly differ, but have a lot of overlap D1+ and D3+ neurons have overlapping sag/rebound properties
- 92. Using AP firing properties to distinguish D1+ and D3+ neurons 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons?
- 93. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES • AP bursting • Spike train shape • AP waveform shape Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons? Using AP firing properties to distinguish D1+ and D3+ neurons
- 94. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES • AP bursting • Spike train shape • AP waveform shape Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons? Using AP firing properties to distinguish D1+ and D3+ neurons
- 95. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Using AP firing properties to distinguish D1+ and D3+ neurons ISI 1 ISI 2 ISI = inter-spike interval
- 96. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES • AP bursting • Spike train shape • AP waveform shape Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons? Using AP firing properties to distinguish D1+ and D3+ neurons
- 97. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Action potential threshold • AP bursting • Spike train shape • AP waveform shape Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons? Using AP firing properties to distinguish D1+ and D3+ neurons
- 98. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Action potential threshold • AP bursting • Spike train shape • AP waveform shape Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons? Using AP firing properties to distinguish D1+ and D3+ neurons
- 99. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Action potential threshold Using AP firing properties to distinguish D1+ and D3+ neurons
- 100. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES • AP bursting • Spike train shape • AP waveform shape Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons? Using AP firing properties to distinguish D1+ and D3+ neurons
- 101. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES • AP bursting • Spike train shape • AP waveform shape Can AP firing properties help distinguish D1R- and D3R-expressing pyramidal neurons? Using AP firing properties to distinguish D1+ and D3+ neurons
- 102. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Using AP firing properties to distinguish D1+ and D3+ neurons
- 103. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Rate of rise Using AP firing properties to distinguish D1+ and D3+ neurons
- 104. No single variable completely separates the populations 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES
- 105. No single variable completely separates the populations 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES 1) Are these separate populations? 2) Can we use electrophysiology to predict dopamine receptor expression in unlabelled neurons?
- 106. No single variable completely separates the populations 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Combining information from all 5 variables might be more effective at discriminating D1+ and D3+ neuronal phenotypes…
- 107. Supervised machine learning for classification Multiple Electrophysiological Features D1 or D3 receptor expression ? Use Linear Discriminant Analysis to see if classification can be done through a linear combination of features. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Feature 1 Feature2
- 108. Supervised machine learning for classification Multiple Electrophysiological Features D1 or D3 receptor expression ? Use Linear Discriminant Analysis to see if classification can be done through a linear combination of features. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Feature 1 Feature2
- 109. Supervised machine learning for classification Multiple Electrophysiological Features D1 or D3 receptor expression ? Use Linear Discriminant Analysis to see if classification can be done through a linear combination of features. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Feature 1 Feature2
- 110. Supervised machine learning for classification Multiple Electrophysiological Features D1 or D3 receptor expression ? Use Linear Discriminant Analysis to see if classification can be done through a linear combination of features. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Feature 1 Feature2
- 111. Supervised machine learning for classification Multiple Electrophysiological Features D1 or D3 receptor expression ? Use Linear Discriminant Analysis to see if classification can be done through a linear combination of features. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Feature 1 Feature2
- 112. Supervised machine learning for classification Multiple Electrophysiological Features D1 or D3 receptor expression ? Use Linear Discriminant Analysis to see if classification can be done through a linear combination of features. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Feature 1 Feature2
- 113. Supervised machine learning for classification Multiple Electrophysiological Features D1 or D3 receptor expression ? Use Linear Discriminant Analysis to see if classification can be done through a linear combination of features. 1 2 3 4 5 ELECTROPHYSIOLOGICAL PROPERTIES Feature 1 Feature2
- 114. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 Results of linear discriminant analysis D1 D3
- 115. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 Results of linear discriminant analysis Feature 1 Feature2 With only two features, we can easily visualize the separation between two classes… But what about five features? D1 D3
- 116. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 Results of linear discriminant analysis Feature 1 Feature2 With only two features, we can easily visualize the separation between two classes… But what about five features? D1 D3
- 117. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 Results of linear discriminant analysis D1 D3 Feature 1 Feature2
- 118. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 Results of linear discriminant analysis Type 1 Type 3 D1 D3 Feature 1 Feature2
- 119. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 Results of linear discriminant analysis D1 D3 Type 1 Type 3 Feature 1 Feature2
- 120. D1+/D2+/D3+ neurons classified as T1/T2/T3 D1+ (n = 92) D2+ (n = 35) D3+ (n = 157) ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5
- 121. D1+/D2+/D3+ neurons classified as T1/T2/T3 D1+ (n = 92) D2+ (n = 35) D3+ (n = 157) ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 Given some electrophysiological overlap, can we confirm D1+/D3+ separation another way?
- 122. Breeding strategy: D3-Cre mice x D1-tdtomato mice ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5x D3-Cre D1-tdTomato D3-Cre x D1-tdTomato
- 123. Anatomical segregation consistent with electrophysiology ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 D1R-expressing D3R-expressing
- 124. Anatomical segregation consistent with electrophysiology ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 D1R-expressing D3R-expressing
- 125. Anatomical segregation consistent with electrophysiology ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 D1R-expressing D3R-expressing
- 126. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing pyramidal neurons comprise a distinct cell class from D1R- and D2R-expressing neurons: Recap: mPFC D3R distribution and electrophysiology
- 127. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing pyramidal neurons comprise a distinct cell class from D1R- and D2R-expressing neurons: • Different L5 laminar distribution Recap: mPFC D3R distribution and electrophysiology
- 128. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing pyramidal neurons comprise a distinct cell class from D1R- and D2R-expressing neurons: • Different L5 laminar distribution • Distinct electrophysiological phenotype (Type 3 vs. Type 1/Type 2) Type 1 Type 3 Recap: mPFC D3R distribution and electrophysiology
- 129. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing pyramidal neurons comprise a distinct cell class from D1R- and D2R-expressing neurons: • Different L5 laminar distribution • Distinct electrophysiological phenotype (Type 3 vs. Type 1/Type 2) • Anatomically separate from D1R+ neurons Recap: mPFC D3R distribution and electrophysiology
- 130. ELECTROPHYSIOLOGICAL PROPERTIES 1 2 3 4 5 DOPAMINE RECEPTOR DISTRIBUTION D3R-expressing pyramidal neurons comprise a distinct cell class from D1R- and D2R-expressing neurons: • Different L5 laminar distribution • Distinct electrophysiological phenotype (Type 3 vs. Type 1/Type 2) • Anatomically separate from D1R+ neurons What are the projection patterns of D3R-expressing pyramidal neurons? Recap: mPFC D3R distribution and electrophysiology
- 131. Two major classes of prefrontal projection neuron 1) Pyramidal tract (PT) 2) Intratelencephalic (IT) D1 D1 D2 D2 • Subcortical projections: • Thalamus • Brainstem • Spinal cord • Cortical projections • Contralateral cortex • Amygdala • Striatum 1 2 3 4 5 PROJECTION PATTERNS
- 132. Two major classes of prefrontal projection neuron 1) Pyramidal tract (PT) 2) Intratelencephalic (IT) D1 D1 D2 D2 PT IT • Subcortical projections: • Thalamus • Brainstem • Spinal cord • Cortical projections • Contralateral cortex • Amygdala • Striatum 1 2 3 4 5 PROJECTION PATTERNS
- 133. Two major classes of prefrontal projection neuron 1) Pyramidal tract (PT) 2) Intratelencephalic (IT) D1 D1 D2 D2 • Subcortical projections: • Thalamus • Brainstem • Spinal cord • Cortical projections • Contralateral cortex • Amygdala • Striatum 1 2 3 4 5 PROJECTION PATTERNS D3 ? PT IT
- 134. Experimental technique: Orthograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 AAV-DIO-ChR2-EYFP Inject into prefrontal cortex D3-Cre::Ai14 transgenic mouse + Cre-dependent virus with fluorescent marker
- 135. Experimental technique: Orthograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 D3-Cre::Ai14 transgenic mouse + Cre-dependent virus with fluorescent marker AAV-DIO-ChR2-EYFP Inject into prefrontal cortex
- 136. Experimental technique: Orthograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 D3-Cre::Ai14 transgenic mouse + Cre-dependent virus with fluorescent marker AAV-DIO-ChR2-EYFP Inject into prefrontal cortex à D3R-expressing cells are infected
- 137. Experimental technique: Orthograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 D3-Cre::Ai14 transgenic mouse + Cre-dependent virus with fluorescent marker AAV-DIO-ChR2-EYFP Inject into prefrontal cortex à D3R-expressing cells are infected Virus is trafficked down the axonal fibers
- 138. Prefrontal D3+ cells are transfected with DIO-ChR2-EYFP PROJECTION PATTERNS 1 2 3 4 5
- 139. Prefrontal D3+ cells are transfected with DIO-ChR2-EYFP PROJECTION PATTERNS 1 2 3 4 5
- 140. Prefrontal D3+ neurons project to contralateral cortex PROJECTION PATTERNS 1 2 3 4 5
- 141. Prefrontal D3+ neurons have cortical and subcortical targets PROJECTION PATTERNS 1 2 3 4 5
- 142. Prefrontal D3+ cells are transfected with DIO-ChR2-EYFP PROJECTION PATTERNS 1 2 3 4 5 Cells transfected across cortical layers
- 143. Experimental technique: Retrograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 D3-Cre::Ai14 transgenic mouse Ctb-488 Inject into cortical or subcortical region Amygdala + retrograde tracer in downstream brain regions
- 144. Experimental technique: Retrograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 Ctb-488 Inject into cortical or subcortical region Amygdala D3-Cre::Ai14 transgenic mouse + retrograde tracer in downstream brain regions
- 145. Experimental technique: Retrograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 Ctb-488 Inject into cortical or subcortical region Amygdala D3-Cre::Ai14 transgenic mouse + retrograde tracer in downstream brain regions
- 146. Experimental technique: Retrograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 Ctb-488 Inject into cortical or subcortical region Virus is trafficked from axon terminal to cell body Amygdala D3-Cre::Ai14 transgenic mouse + retrograde tracer in downstream brain regions
- 147. Experimental technique: Retrograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 mPFC coronal brain slice D3-Cre::Ai14 transgenic mouse + retrograde tracer in downstream brain regions
- 148. Experimental technique: Retrograde viral tracing PROJECTION PATTERNS 1 2 3 4 5 mPFC coronal brain slice D3-Cre::Ai14 transgenic mouse + retrograde tracer in downstream brain regions
- 149. PROJECTION PATTERNS 1 2 3 4 5 Verify targeting in 4 potential downstream targets Contralateral mPFC Nucleus accumbens core Basolateral amygdala Mediodorsal thalamus
- 150. PROJECTION PATTERNS 1 2 3 4 5 Projection neurons in mPFC: Overlap with D3R+ neurons Contralateral mPFC Nucleus accumbens core Basolateral amygdala Mediodorsal thalamus Injection location mPFC coronal section L1 L2/3 L5a L5b L6 D3-Cre::Ai14 Ctb-488 (retrogradely-labeled)
- 151. PROJECTION PATTERNS 1 2 3 4 5 Contralateral mPFC Nucleus accumbens core Basolateral amygdala Mediodorsal thalamus Injection location mPFC coronal section L1 L2/3 L5a L5b L6 Projection neurons in mPFC: Overlap with D3R+ neurons D3-Cre::Ai14 Ctb-488 (retrogradely-labeled)
- 152. PROJECTION PATTERNS 1 2 3 4 5 Contralateral mPFC Nucleus accumbens core Basolateral amygdala Mediodorsal thalamus Injection location mPFC coronal section Projection neurons in mPFC: Overlap with D3R+ neurons L1 L2/3 L5a L6 L5b
- 153. PROJECTION PATTERNS 1 2 3 4 5 Contralateral mPFC Nucleus accumbens core Basolateral amygdala Mediodorsal thalamus Injection location mPFC coronal section Projection neurons in mPFC: Overlap with D3R+ neurons L1 L2/3 L5a L6 L5b
- 154. PROJECTION PATTERNS 1 2 3 4 5 Contralateral mPFC Nucleus accumbens core Basolateral amygdala Mediodorsal thalamus Injection location mPFC coronal section Projection neurons in mPFC: Overlap with D3R+ neurons L1 L2/3 L5a L6 L5b
- 155. PROJECTION PATTERNS 1 2 3 4 5 L5 D3R-expressing neurons are an IT population D1 D1 D2 D2 IT PT IT D3 • Contralateral cortex • Nucleus accumbens
- 156. D1 D1 D2 D2 PT IT IT D3R-dependent modulation in prefrontal cortex D2 D2 D1 D1 1 2 3 4 5 D3R MODULATION Do D3R-expressing pyramidal neurons undergo subtype-specific modulation? D3
- 157. D3 1 2 3 4 5 D3R MODULATION D3R-dependent modulation in D3R-expressing neurons
- 158. D3 1 2 3 4 5 D3R MODULATION D3R-dependent modulation in D3R-expressing neurons Axon initial segment (AIS): Site of action potential initiation
- 159. 1 2 3 4 5 D3R MODULATION D3R-dependent modulation in D3R-expressing neurons Experimental Method
- 160. 1 2 3 4 5 D3R MODULATION D3R-dependent modulation in D3R-expressing neurons Experimental Method
- 161. 1 2 3 4 5 D3R MODULATION D3R-dependent modulation in D3R-expressing neurons Experimental Method
- 162. 1 2 3 4 5 D3R MODULATION D3R-dependent modulation in D3R-expressing neurons Experimental Method
- 163. 1 2 3 4 5 D3R MODULATION D3R-dependent modulation in D3R-expressing neurons Experimental Method AP-evoked AIS calcium
- 164. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 D3R-dependent modulation in D3R-expressing neurons
- 165. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 Wash on quinpirole (D2- family receptor agonist) D3R-dependent modulation in D3R-expressing neurons
- 166. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 Wash on quinpirole (D2- family receptor agonist) Reduction in AIS Ca D3R-dependent modulation in D3R-expressing neurons
- 167. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 This modulation also occurs in Type 3 neurons D3R-dependent modulation in D3R-expressing neurons
- 168. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 Mice lacking the D3R do not show this effect D3R-dependent modulation in D3R-expressing neurons
- 169. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 D3R-dependent modulation in D3R-expressing neurons
- 170. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 D3R modulation does not occur in neighboring cells
- 171. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 D3R modulation does not occur in neighboring cells
- 172. 1 2 3 4 5 D3R MODULATION D2 D2 D1 D1 D3R modulation does not occur in neighboring cells
- 173. D3R IMPACT ON NEURONAL OUTPUT Functional impact of D3R modulation 1 2 3 4 5 Does D3R modulation at the AIS affect neuronal output?
- 174. D3R IMPACT ON NEURONAL OUTPUT Functional impact of D3R modulation 1 2 3 4 5 Does D3R modulation at the AIS affect neuronal output? Experimental methods: Evoke APs in D3R-expressing neurons à 1) Bath application of quinpirole (D2-family agonist) 2) Local application of Ni to AIS (approximates modulatory affect) OR
- 175. D3R IMPACT ON NEURONAL OUTPUT Functional impact of D3R modulation 1 2 3 4 5 Does D3R modulation at the AIS affect neuronal output? Experimental methods: Evoke APs in D3R-expressing neurons à 1) Bath application of quinpirole (D2-family agonist) 2) Local application of Ni to AIS (approximates modulatory affect) OR
- 176. D3R IMPACT ON NEURONAL OUTPUT Functional impact of D3R modulation 1 2 3 4 5 Simulated synaptic input
- 177. D3R IMPACT ON NEURONAL OUTPUT Functional impact of D3R modulation 1 2 3 4 5 Simulated synaptic input
- 178. D3R IMPACT ON NEURONAL OUTPUT Functional impact of D3R modulation 1 2 3 4 5 Simulated synaptic input
- 179. D3R IMPACT ON NEURONAL OUTPUT Functional impact of D3R modulation 1 2 3 4 5
- 180. D3R IMPACT ON NEURONAL OUTPUT Selective suppression of high frequency AP bursts 1 2 3 4 5
- 181. D3R IMPACT ON NEURONAL OUTPUT Selective suppression of high frequency AP bursts 1 2 3 4 5
- 182. D3R IMPACT ON NEURONAL OUTPUT Selective suppression of high frequency AP bursts 1 2 3 4 5
- 183. D3R IMPACT ON NEURONAL OUTPUT Selective suppression of high frequency AP bursts 1 2 3 4 5
- 184. Conclusions 1 2 3 4 5 D3R IMPACT ON NEURONAL OUTPUT PROJECTION PATTERNS D3R MODULATION ELECTROPHYSIOLOGICAL PROPERTIES DOPAMINE RECEPTOR DISTRIBUTION 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R-expressing neurons? 3) What are the downstream targets of these D3+ neurons? 4) How do D3Rs modulate prefrontal neurons? 5) What are the functional consequences of D3R signaling on neural activity?
- 185. Conclusions 1 2 3 4 5 D3R IMPACT ON NEURONAL OUTPUT PROJECTION PATTERNS D3R MODULATION ELECTROPHYSIOLOGICAL PROPERTIES DOPAMINE RECEPTOR DISTRIBUTION 1) Where is the D3R in the prefrontal cortex? Superficial cortical layers, in particular L2/3 and L5a
- 186. Conclusions 1 2 3 4 5 D3R IMPACT ON NEURONAL OUTPUT PROJECTION PATTERNS D3R MODULATION ELECTROPHYSIOLOGICAL PROPERTIES DOPAMINE RECEPTOR DISTRIBUTION 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R-expressing neurons? • Low sag and rebound • No initial doublet • Broad, slow APs • Steadily depolarizing AP threshold during train Electrophysiologically-distinct phenotype from D1R- and D2R-expressing neurons
- 187. D1 D1 D2 D2 PT IT IT Conclusions 1 2 3 4 5 D3R IMPACT ON NEURONAL OUTPUT PROJECTION PATTERNS D3R MODULATION ELECTROPHYSIOLOGICAL PROPERTIES DOPAMINE RECEPTOR DISTRIBUTION 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R-expressing neurons? 3) What are the downstream targets of these D3+ neurons? D3 IT cell type • Projections to contralateral cortex and nucleus accumbens
- 188. Conclusions 1 2 3 4 5 D3R IMPACT ON NEURONAL OUTPUT PROJECTION PATTERNS D3R MODULATION ELECTROPHYSIOLOGICAL PROPERTIES DOPAMINE RECEPTOR DISTRIBUTION 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R-expressing neurons? 3) What are the downstream targets of these D3+ neurons? 4) How do D3Rs modulate prefrontal neurons? Decrease in AP-evoked calcium influx at the AIS
- 189. Conclusions 1 2 3 4 5 D3R IMPACT ON NEURONAL OUTPUT PROJECTION PATTERNS D3R MODULATION ELECTROPHYSIOLOGICAL PROPERTIES DOPAMINE RECEPTOR DISTRIBUTION 1) Where is the D3R in the prefrontal cortex? 2) What are the electrophysiological properties of D3R-expressing neurons? 3) What are the downstream targets of these D3+ neurons? 4) How do D3Rs modulate prefrontal neurons? 5) What are the functional consequences of D3R signaling on neural activity? Selective reduction in high frequency AP bursting
- 190. Conclusions 1 2 3 4 5 D3R IMPACT ON NEURONAL OUTPUT PROJECTION PATTERNS D3R MODULATION ELECTROPHYSIOLOGICAL PROPERTIES DOPAMINE RECEPTOR DISTRIBUTION This provides a framework for understanding cellular and subcellular substrates of prefrontal dopaminergic modulation How do antipsychotic drugs impact prefrontal function (and output) via both D3R and D2R? How does regulation of D3+ bursting impact intracortical processing?
- 191. Thank you! Bender lab Kevin Bender Current Members Jiggy Athilingam Roy Ben-Shalom Ken Burke Caroline Keeshen Anna Lipkin Perry Spratt Former Members Alayna Liptak Gina Rinetti Vargas Sungchil Yang Funding sources: NSF, NIDA, NIMH Sohal Lab Steven Gee Thesis Committee Stephan Lammel Roger Nicoll Vikaas Sohal