CJH332H1 Lecture Notes - Lecture 15: Stereotactic Surgery, Chlamydomonas Reinhardtii, Dopaminergic
Lecture 15: Molecular Methods II
McGill – elctrodes
• Implanted electrodes to stimulate and excite a group of axons that fire AP, release NT and interact with
receptors to cause behaviors to become apparent
• Stereotactic surgery mistake – missed target and put electrode somewhere different (mice stayed in one area)
• Eletrophsiolog, although poerful, hae liitatios ere’t sure hat e ere stiulatig
Neurotransmitters – technique now
• Often, you cant tell where synapses are coming from (source)
• Striatum receives input from dopaminergic and SN and other glutamatergic
inputs – how do we know what group of neurons are involved/activated?
(electrophysiology very limited)
• Dopamine is neuromodulator (by itself no capacity to open channel/stimulate neuron; work through GPCR)
• VTA → NAc → Prefrontal cortex → CP
- If we stimulate VTA (containing dopamine), why do we see AP/change in membrane potential in NAc (other
channels are being activated)
- Can we prove its only a group of neurons in VTA that excite NAc that does’t iole dopaie?
• Technique uses optogenetics (light activation of channels, not using change in membrane potential)
• Delivery exogenous gene (responsive to light) to an area of the brain interested in
• Channelrhodopsin (channel) activated and turns on neurons that it is expressed in (mCherry is like GFP but red)
• Tyrosine Hydroxylase (TH) is synthetic enzyme/protein for dopamine (we only find TH in dopaminergic neurons)
• Infect the neurons in the VTA
Different types of delivery
• Choice of viruses – what can we package inside them, starting material, how fast the expression shows and how
long it lasts
• Similar to adenovirus, AAV Vector:
- Speed of expression is weeks
- Duration of expression is years
Stereotactic surgery in rats
• Tyrosine hydroxylase (TH) – normal enzyme found in cell, converting tyrosine into dopamine
- VTA is dopaminergic and TH is expressed solely in dopaminergic neurons
• Can we deliver virus to infect only TH-containing groups of neurons? (precision targeting)
• Stain this with an antibody afterwards
• Deliver a virus to infect a small group of neurons using precision targeting in the VTA – contains a gene for a
channel (ChR2)
• ChR2 encodes for a different class of ion channel (not expressed/turned on in the mammalian system)
- These are only stimulated by light activation
• 2 different methods – electrically stimulating cell and examining the synapse OR using the ChR2 to excite the cell
Neurotransmitters – technique now II
• ChR2 (turns on neurons when expression) fused with reporter gene, mCherry (red)
- Not all neurons with TH+ is stained with mCherry, meaning that not all neurons expressions ChR2 (but 80%)
• Measure electrical activity in the NA (since the ChR2+ neurons send their projections to the nucleus accumbens)
• Pulse of light to neurons in VTA cause inward current in NA (Na in, K out)
find more resources at oneclass.com
find more resources at oneclass.com
• Inward current blocked by the AMPA-glutamate receptor blocker (DNQX – AMPA glutamate specific)
• SCH23390 and raclopride block dopamine receptors – no change in post-synaptic cell!
• Suggests that activation of the VTA dopaminergic neurons also release both dopamine AND glutamate on target
neurons but glutamate is what causes activity in post synaptic cell
- Post synaptic cell is responding to glutamate but not to dopamine
How do you solve a problem?
• Electrodes have change in electrical activity spreads in spherical shape – we
stimulate more neurons than we target to stimulate
- Therapeutic effect there but also side effect (other expression)
Recap of the limitations
• Many studies have also, inadvertently, activated other neurons by electrical activation and the same is truer
about lesion studies affecting other neurons
• Level of precision using stimulating electrodes was not sufficient to rule out other biological effects
• Would it be possible to control temporally and specifically which neurons were being activated?
- Respond/cause activation of neurons just as fast as AP would allow us to do
• Are there methods that we can use to control neuronal firing of circuits in a rapid way? – Stanford connection
Basis of optogenetics
• Pond algae contains non-specific cation channel that opens rapidly in response to certain wavelengths of light
- Let’s tur o euros sie the are sittig there i dark usig light!
• Responsive on the order of milliseconds (Chlamydomonas reinhardtii) – same order as normal channels we see
Neuronal activity and light
• Developed by Deisseroth, Boyden, Zhang, Gottschalk and Nagel (more controversial – others Zhuo Hua Pan)
• ChR2 is activated by blue light (many newer modifications via changing the pore region) – generally excitatory
• Halorhodopsins also now generated and used – when expressed and stimulated – generally inhibitory
• Opsins are natively expressed in micro-organisms (algae)
• Using transgenic methods, we can introduce the opsin gene into genome of the experimental anima
- PCR to clone the opsin gene that we are interested in, encoding ChR2
- Via targeted stereotactic viral injection
• Importantly, only certain cells have internal machinery necessary to express opsin protein (researchers control)
- Know which groups of neurons we want to turn off or on
- This is how the selectivity of the method works
Native channelrhodopsins (ChRs)
• Alge are light sensitive in that, when exposed to light, it will swim AWAY from it
• With the retinal portion (light sensitivity) is knocked out, there will be no response
• Retinal will change its conformation when exposed to light to allow flow of ions through the channel
Activation and Inhibition
• With blue light, retinal changes conformation to cause flow of ions through channel and activate the cell
• With yellow or organge light (longer wavelength), halorhodopsins will allow influx of Cl and inactivate the cell
(hyperpolarization/inhibitory)
• Archeorhodopsin (Arch) also a ChR2
- Activation of Arch (yellow/orange light) causes hydrogen OUT
- Resembles a proton pump – not as effective/sensitive as other channels
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
If we stimulate vta (containing dopamine), why do we see ap/change in membrane potential in nac (other channels are being activated) Different types of delivery: choice of viruses what can we package inside them, starting material, how fast the expression shows and how long it lasts, similar to adenovirus, aav vector: Stereotactic surgery in rats: tyrosine hydroxylase (th) normal enzyme found in cell, converting tyrosine into dopamine. These are only stimulated by light activation: 2 different methods electrically stimulating cell and examining the synapse or using the chr2 to excite the cell. Neurotransmitters technique now ii: chr2 (turns on neurons when expression) fused with reporter gene, mcherry (red) Post synaptic cell is responding to glutamate but not to dopamine. How do you solve a problem: electrodes have change in electrical activity spreads in spherical shape we stimulate more neurons than we target to stimulate. Therapeutic effect there but also side effect (other expression)
