Synopsis

Electrical deep brain stimulation and chemogenetics are increasingly used with simultaneous fMRI. This lecture will introduce both techniques, discuss the strengths/weaknesses, and make suggestions to pilot studies.

Speaker Information

Name: Yen-Yu Ian Shih

Lab: http://shihlab.org

email: shihy@unc.edu

Highlights

• Electrical deep brain stimulation (DBS) is a clinically used technique allowing time-locked manipulations of neural circuits at high frequency.
• Chemogenetics allows remote control of selected cellular activity via engineered receptor–ligand pairs.
• Both techniques have their unique strengths and drawbacks when combined with fMRI.

Target audience

Objective

• To introduce DBS, chemogenetics, and their use in preclinical fMRI.
• To discuss the pros and cons and the do’s and don’ts with these techniques.
• To share the speaker’s experience on how to get these experiments started in your lab.

Electrical deep brain stimulation (DBS)

What is electrical deep brain stimulation?

Deep brain stimulation (DBS) is a well-established neurosurgical therapy for multiple neurological and psychiatric disorders. Despite its growing use, the circuit mechanisms underlying the therapeutic effects of DBS are poorly understood, representing a major challenge to refinement for enhanced efficacy and reduced side effects. In human patients, an electrode is stereotactically guided to a target cerebral nucleus and high frequency (~130 Hz) electrical stimulation is typically delivered through a pacemaker-like subcutaneous stimulating device. It is most commonly employed in the treatment of Parkinson’s disease (PD), generally in cases where other medical therapies have become inadequate or dyskinesias have become intolerable. When applied for the symptomatic treatment of PD, the subthalamic nucleus (STN) or internal globus pallidus (GPi) are frequently targeted, often resulting in a marked reduction in several hallmark PD symptoms, including resting tremor and rigidity.

What are the pros?

1. Translational opportunity: DBS is a clinically proven approach. Research addressing how DBS exerts its therapeutic effects will allow optimization of this procedure to enhance therapeutic outcomes, reduce unwanted side-effects, and be adapted to other disorders. fMRI represents one of the few tools that maps brain-wide circuit dynamics and thus is ideal to help understand DBS mechanisms.
2. High throughput: Entry risk is low because no viral injections or development of genetic animal models needed. When used to map circuit network, experiments can be relatively high throughput by using multichannel electrodes.
3. High-fidelity modulation: DBS allows neural modulation at very high frequency, which is often needed to achieve its therapeutic efficacy in many neurological and neuropsychiatric disorders. With regards to brain circuit network studies, high frequency modulation (>100 Hz) remains to be challenging for other techniques such as optogenetics.

What are the cons?

1. Safety: Do not risk human/animal subject safety without careful consideration of SAR. Repeated high power RF pulses could induce severe thermal tissue damage. Many research devices are also not MR compatible and require careful evaluation.
2. Susceptibility artifacts: With only a few exceptions, DBS often uses metal based electrodes and many of those create susceptibility artifacts.
3. Lack of specificity: DBS creates a field of modulation that affects multiple cell types within the same brain volume. It also has no directionality when modulating fiber projections.

Getting started in your lab:

1. Useful background information: http://dx.doi.org/10.1089/brain.2013.0193
2. Make your own electrode: Many electrodes are commercially available for MRI applications (despite susceptibility artifacts may vary). DBS electrodes can also be simply fabricated in house for preclinical MR applications. A protocol worth considering can be found at: http://dx.doi.org/10.1002/mrm.25239
3. Surgical implantation: High precision stereotaxic surgery is crucial for the success of the study. An example of implantation surgery can be found at: http://dx.doi.org/10.3791/51271
4. Suggested proof-of-concept pilot fMRI experiments: Ventral posterior thalamic complex has well-documented projections to the cortex and is suggested for the pilot experiment as DBS target. Stimulation at 25 Hz, 50 µs pulse-width and 1 mA is suggested in lightly anesthetized rats/mice. If successful, robust activation should be observed in the ipsilateral sensory cortex. An example of such study can be found at: http://dx.doi.org/10.1016/j.brs.2013.11.001

Chemogenetics

What is chemogenetics?

Chemogenetics has been defined as a method by which proteins are engineered to interact with previously unrecognized small molecule chemical actuators. Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) is a unique class of chemogenetic receptors that exploit genetic mutations of muscarinic acetylcholine or κ-opioid G-protein coupled receptors (GPCRs) receptors that have high affinity to physiologically inert ligands such as clozapine-N-oxide (CNO) or Salvinorin B (SALB). The most widely used DREADDs are:

• hM3Dq (or Gq-DREADD), which enhances neuronal firing via intracellular calcium release.
• hM4Di (or Gi-DREADD) or KORD (or KOR-DREADD), which inhibits neuronal firing via potassium-channel-induced hyperpolarization and/or presynaptic release of neurotransmitters.

What are the pros?

1. Cell type and projection-specific manipulations: Similar to optogenetics, chemogenetics allows cell type specific modulation by using viral or transgenic approach. Projection specific targeting can be achieved by intracranial microinfusion of DREADD actuators or the use of retrograde virus such as CAV-Cre or rAAV2-retro-Cre to projection terminals with Cre-dependent virus to the cell bodies for more selective DREADD expressions.
2. Multiplexed and bidirectional modulation: The development of KOR-DREADD has made it possible for inhibition of two distinct circuits using separate ligands (CNO or SALB) at different time points in a single subject. It is also possible to activate a circuit using hM3Dq with CNO and suppress another using KORD with SALB. The combination with optogenetics has also advance the toolbox to interrogate selective neural circuit functions.
3. Less invasive, prolonged control of cellular activity, and translational opportunity: A major strength of the DREADD technology is that brain implant or hardware is not needed (with the exception of using intracranial microinfusion for ligand delivery). Recently, transgenic mouse lines have been developed, requiring no surgeries for DREADD expression. Signaling through DREADDs is markedly different and slower compared to most of the other brain modulation tools, which provides prolonged modulation of cellular activity that is not otherwise feasible. This is crucial for clinical translation. GPCRs represent the canonical targets of at least 30% of the top 200 best-selling drugs in the United States. Therefore, the ability to precisely control GPCRs in defined cell types offers a tremendous opportunity to advance treatment.

What are the cons?

1. Potential changes in basal activity: GPCRs have multiple ligand-dependent and independent states ranging from fully inactive, partially active, or fully active. High levels of expression of an engineered protein might have effects in the absence of chemical activation.
2. Desensitization/receptor down regulation: Following repeated dosing with a DREADD chemical actuator, many groups have observed diminished responses due to receptor desensitization and downregulation.
3. You have one shot: Although the pharmacokinetics (or simply the length of “activation state”) may vary according to the types of receptors, actuators, cell types, physiology and species, most of the DREADD experiments perform only one injection of actuator (CNO or SALB) per experimental run. Rapid, repeated control of activity remains challenging for chemogenetics. This is similar to the traditional pharmacological MRI experiments.
4. CNO and SALB side effects: CNO back metabolizes to clozapine which displays agonist affinity for dopaminergic D₁, serotonergic 5-HT_1A, muscarinic M₄, NMDA receptors, and antagonist affinity for dopaminergic D₂, D₄, serotonergic 5-HT_2A, 5-HT_2C, adrenergic α₁, α₂, histaminergic H₁, muscarinic M₁, M₂, M₃, and GABA_A receptors. Additionally, it also displays partial agonist/competitive antagonist affinity at muscarinic M₁-M₅ receptors. These broad-spectrum bindings can result in various side effects on imaging (e.g., responses in off-target areas, significant baseline drifting, suppression of targeted responses) or physiology (e.g., agranulocytosis, seizures, sedation, hypotension, anticholinergic syndrome). Note that new non-CNO actuators have been recently developed such as Compound 21 and perlapine. SALB also retains modest affinity for κ-opioid receptors. Taken together, appropriate control experiments are crucial for chemogenetic studies.

Getting started in your lab:

1. Useful information: DREADD wiki: http://pdspit3.mml.unc.edu/projects/dreadd/wiki/WikiStart; DREADD blog: http://chemogenetic.blogspot.com/; DREADD Primer: http://dx.doi.org/10.1016/j.neuron.2016.01.040
2. Validate DREADD expression: Similar to the optogenetic studies, validation of DREADD expression on target (and off-target) cell type is essential for data interpretation.
3. Control, Control, Control: It is crucial to perform experiments in control subjects without the expression of DREADDs. Generally, CNO dose between 0.1-3 mg/kg has been reported to be pharmacologically and behaviorally inert in mice and rats. SALB dose between 1-10 mg/kg has been reported to alter behavior in KOR-DREADD expressing mice but not control mice. How these doses affect fMRI signals remain unexplored. It is important to study dose-response curve in the proposed model system, and the lowest effective dose of actuators should be used to avoid unwanted side effects.
4. Suggested proof-of-concept pilot fMRI experiments: Expression of virus in sensory-motor cortices is straightforward and thus is suggested for the pilot experiment. Microinjection of 1 µl of purified and concentrated AAV-CaMKIIα-hM3Dq (~10¹² infections units per ml) into the preferred target is a reasonable starting point. Following 4 weeks of expression, inject CNO at 1 mg/kg i.p. or i.v. during fMRI. If successful, robust activation should be observed in the target region. An example of such study can be found at: http://cds.ismrm.org/protected/15MPresentations/abstracts/1908.pdf

Acknowledgements

References

• DBS-fMRI review: http://dx.doi.org/10.1089/brain.2013.0193
• Simple DBS-fMRI electrode fabrication: http://dx.doi.org/10.1002/mrm.25239
• Rodent DBS surgery: http://dx.doi.org/10.3791/51271
• DBS-fMRI pilot study: http://dx.doi.org/10.1016/j.brs.2013.11.001

• DREADD wiki: http://pdspit3.mml.unc.edu/projects/dreadd/wiki/WikiStart
• DREADD blog: http://chemogenetic.blogspot.com/
• DREADD Primer: http://dx.doi.org/10.1016/j.neuron.2016.01.040
• DREADD fMRI pilot study: http://cds.ismrm.org/protected/15MPresentations/abstracts/1908.pdf