From Single Synapses to Clinical Studies: Therapeutic Developments from Optogenetics
Antonello Bonci, MD NIDA/NIH [email protected] Dopamine Addiction
Apathy motivation
Aggressive behaviors Sexual behaviors Appetitive behaviors
Reward Deficiency Syndrome
Parkinson’s disease ADHD Schizophrenia Sensory neglect Learning and memory LTP and dopamine neurons
AMPAR dopamine neuron CaMKII glutamate Ca++ NMDAR
Bonci and Malenka, 1999; Overton and Clark, 1999 The time course of VTA cocaine‐dependent synaptic plasticity
Ungless et al., Nature (2001) LTP
3 hours 1 day 3 days 5 days 1 week 3 weeks 3 months time The time course of VTA cocaine‐dependent synaptic plasticity
Ungless et al., Nature (2001) Chen et al., Neuron (2008) LTP Cocaine self‐administration Single cocaine
3 hours 1 day 3 days 5 days 1 week 3 weeks 3 months time The time course of VTA cocaine‐dependent synaptic plasticity
Chen et al., Neuron (2008) LTP Cocaine self‐administration
Stuber et al., Science (2010) How Reward related learning
3 hours 1 day 3 days 5 days 1 week 3 weeks 3 months time The other players
Alterations in microglia have been described in a variety of psychiatric diseases
From Aguzzi, Barres and Bennet, Science (2013) Basic properties of microglial cells in the midbrain and accumbens
Lindsay de Biase
Mirror project: Wendy Xin (astrocytes) TH Iba1 = Microglia NG2 = Oligodendrocyte Precursors Not shown – Oligodendrocytes, Astrocytes deBiase et al., under submission Regions of interest for study of basal ganglia (BG) microglia
(knock‐in) • P58‐62 CX3CR1 EGFP • ♂ and ♀ Jackson Labs • perfusion‐fixed TH
SNc
NAc SNr VTA m
Medial Lateral
deBiase et al., under submission Density of microglia varies dramatically across BG nuclei
NAc VTA SNc SNr TH * P < 0.02, all comparisons; (sequential Bonferroni correction) *
m * EGFP * *
m
deBiase et al., under submission Morphology of microglia varies dramatically across BG nuclei
* P < 0.006 all comparisons ex. NAc vs. SNr and VTA vs. SNc
deBiase et al., under submission Quantitative analysis of microglial cell morphology Electrophysiology to probe microglial membrane properties in acute brain slices
(knock‐in) CX3CR1 EGFP Jackson Labs
• P35‐42 • ♂ and ♀ • Horizontal midbrain section
MT
Anterior
Medial
deBiase et al., under submission SNr microglia express more voltage-activated conductances
10% 0% 60% (2/21 cells) (0/7 cells) (12/20 cells)
deBiase et al., under submission Analysis of gene expression in microglia FACS isolation of microglia from specific basal ganglia nuclei
NAc VTA SN Ctx
Ion Torrent RNA sequencing Whole Transcriptome Analysis (collaboration with NIAAA) Relative regional differences in microglial cell density are established early in development and persist throughout life
P12P58‐62 18 mo
deBiase et al., under submission Microglial responses to aging are not uniform across the BG 57%
18 mo P58‐62 77% 100% 78%
Clustering Abnormal morphology (prevalent in midbrain) Under analysis A biomarker for aging? Microglial cell density is not correlated with neuronal density
NAc VTA SNc SNr EGFP
100m
NeuN
deBiase et al., under submission SUMMARY: Distinct microglial phenotypes across the basal ganglia
DENSITY EGFP
m SUMMARY: Distinct microglial phenotypes across the basal ganglia
MORPHOLOGY SUMMARY: Distinct microglial phenotypes across the basal ganglia
MEMBRANE PROPERTIES Summary
• Microglia within the BG exhibit regionally specific phenotypes and may possess differential capacity to respond to CNS insults
• Foundation for defining how microglia impact synaptic transmission and synaptic plasticity within the BG / reward circuitry in both physiological and pathological contexts (substance abuse, stress-dependent behaviors, psychiatric diseases, neurodegeneration) Part III: from optogenetic studies to a treatment against cocaine craving
Courtesy of Ed Boyden, MIT Optogenetics can be also used to silence neurons
Courtesy of Ed Boyden, MIT Part II: from optogenetic studies to a treatment against cocaine craving
Kusumoto et al., PNAS (2015) Seif et al., Nature N. (2013) Chen et al., Nature (2013) Insula PFC
Stuber et al., Nature (2011)
BLA Accumbens McDevitt et al., Cell R. (2014) V. Subiculum Hipp Dorsal Britt et al., Neuron (2012) Raphe
VTA
Stuber et al., J Neurosci (2010) LH Kempadoo et al, J Neurosci (2013) An optogenetic study inspired by human studies based on the broad concept of “hypofrontality” on cocaine abusers
PFC Billy T. Chen
Decision making processes Evaluation of expected outcome Inhibitory control Self-administration paradigm
1. Rats are trained to self-administer cocaine on a seek-take chain schedule (about 2 months) with progressively longer Random Intervals.
2. At the very end of training, rats receive 4 sessions of non-contingent foot shock in 30% of trials
Take Seek
RI 5 Cocaine RI 30 (30 inf. or 6hrs) RI 60 1. Rats are divided into non-compulsive and compulsive groups
shock sensitive
Shock‐resistant
Chen et al., Nature (2013) The prelimbic cortex is involved in many physiological functions, including decision making processes
Adapted from Vertes, Neuroscience 2006; 142 1‐20) In vivo Patch‐Clamp recordings in the prelimbic cortex
CourtesyCourtesy ofof EdEd Boyden,Boyden, MITMIT Hypoactivity in prelimbic PFC neurons after long-access cocaine self-administration
Naive
5,0 Naive 4,0 Cocaine Cocaine
3,0
2,0
1,0 Baseline firing freq. (Hz) 0,0
Thanks to Rob Froemke, NYU Long‐access to cocaine decreases excitability of deep‐layer prelimbic neurons
16 Naive Naive 12 Sensitive Resistant 8 Sensitive 4 Number of APs Number of
Resistant 0 0 200 400 input (pA)
Chen et al., Nature (2013) Hypothesis:
If hypoactivity of the prelimbic cortex is causally linked with cocaine seeking, then
Optogenetic activation of the prelimbic area should reduce cocaine seeking Laser stimulation procedure
‐ 4 days of “shock” training
‐ On day 5, ChR stimulationin the PLCx (@1 Hz) throughout the seek period
Seek Take 30%
Cocaine
Chen et al., Nature (2013) Prelimbic photostimulation decreases compulsive cocaine seeking only after Shock sessions
Chen et al., Nature (2013) Summary
• ~30% of rats will self-administer cocaine despite negative consequences (SR rats).
• Neurons in the prelimbic region of the PFC are significantly hypoactive active after prolonged cocaine self-administration, with shock resistant rats being the least active.
• Activation of prelimbic neurons via optogenetic decreases cocaine seeking and taking behavior in shock resistant rats
• Inhibition of prelimbic neurons via optogenetics in shock sensitve rats triggers cocaine seeking during shock sessions
• Therapeutic applications testable, immediately