From Single Synapses to Clinical Studies: Therapeutic Developments from Optogenetics

Antonello Bonci, MD NIDA/NIH [email protected] Dopamine

Apathy motivation

Aggressive behaviors Sexual behaviors Appetitive behaviors

Reward Deficiency Syndrome

Parkinson’s disease ADHD Schizophrenia Sensory neglect Learning and memory LTP and dopamine

AMPAR dopamine CaMKII glutamate Ca++ NMDAR

Bonci and Malenka, 1999; Overton and Clark, 1999 The time course of VTA ‐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 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

100m

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