How does Deep Brain Stimulation cure Parkinson's Disease? A Cellular Look

Deep Brain Stimulation (DBS) is one of the miracle cures of our lifetime.  Used to treat Parkinson's Disease, and now being applied to depression, it is a drastic but amazingly effective measure.

EDIT 9/23/12: Just because DBS has been shown to be amazingly effective in treating Parkinson's Disease symptoms, that does not mean that it is right for everyone. There are other effective treatments for PD and DBS involves an invasive surgery. In addition, the long term effects of having a stimulating electrode constantly pulsing electricity in your brain are not yet fully known.  In other words, this blog is not giving medical advice, so please don't take it as such.

Deep Brain Stimulation (DBS)

To apply DBS, an electrode is implanted deep in the brain (the SubThalamic Nucleus for Parkinson's Disease).  An implanted wire under the skin connects the stimulation electrode in the brain to a stimulator implanted on the chest. The stimulation can be turned on and off here. 

Here is a short video showing the effect of DBS on and DBS off.  (The actual demonstration starts at 1 minute in, the rest is some arguing in French)


Once the stimulation is turned off, the man has twitches an tremors that prevent him from functioning normally.  As soon as the stimulation is turned back on, he can walk and talk smoothly. 

This is pretty astounding, right? I mean honestly, if I just saw this video and didn't know anything about it, I might not even believe that it was real.  How can electrical stimulation in the brain completely alter this person's ability to move?

Well, scientists around the world are wondering the exact same thing. One recent study made use of optogenetics to dissect the pathways involved in deep brain stimulation.

They first elicited Parkinson's Disease in mice by destroying the dopaminergic cells (in the substantia nigra which feeds into the striatum) on only one side of the brain. Then they give the mice a stimulant drug which makes them hyper. While normal mice would run all over the place on this stimulant, these hemi-parkinson's mice run in a circle because of the imbalance between the two sides of the brain.



or hemi-parkinson's disease (source)

To test whether the treatment you are giving the mouse 'cures' their Parkinson's Disease, you literally count how many times the mouse runs in a circle.  If it runs in a lot of circles, your 'cure' didn't work.

Implanting an electrode into the STN of the mice and applying the stimulation did reduce their circle running, just like the DBS reduces Parkinson's symptoms in humans.  But here's the thing, the STN has a lot of neurons in it, and a lot of axons passing through it.  Stimulating this brain region could be inhibiting firing, or exciting neurons, or something else entirely. 

So Gradinaru et al. (2009) decided to stimulate specific (genetically identified) classes of neurons to determine which aspect of the electrical stimulation was actually 'curing' the Parkinson's symptoms. 

First they tested the most common hypothesis, that the stimulation inhibited the STN.  However, when they directly inhibited the neurons of the STN, their mice still ran in circles, indicating that the brain was still unbalanced. 

Second they stimulated the glial cells around the STN, but still no luck.

Third they excited the neurons of the STN.... but... still the mouse ran in circles. 

I imagine this was very puzzling to the scientists conducting this research.  If stimulating the STN with a big metal electrode is not exciting, and not inhibiting the STN, what on earth could it be doing?

Finally they tried targeting the axons of the motor cortex which run through the STN.  And! low and behold, the mouse did not run in circles any more. Below, rotations per minute is basically a measure of circle running, and the HFS is the stimulation. You can see that as soon as the stimulation is activated the mice generally stopped running in circles, but when the stimulation was turned off, they ran in circles again. 

Gradinaru et al., 2009

So a new theory has emerged, that the stimulation of the STN might actually be acting on other neurons which are not even located in that brain structure.  It clearly works in mice, but whether this is the way that Deep Brain Stimulation works in humans is still not clear. 



© TheCellularScale



Gradinaru V, Mogri M, Thompson KR, Henderson JM, & Deisseroth K (2009). Optical deconstruction of parkinsonian neural circuitry. Science (New York, N.Y.), 324 (5925), 354-9 PMID: 19299587

Virtual Reality for Worms

How do you build a virtual environment for a worm?


The Nematode C. Elegans with glowing neurons (source)

Using a little optogenetic trickery, you can directly activate specific worm neurons with light.  If you know your worm neurons, you can stimulate ones that make it think it has suddenly touched something with its nose or that the environment is suddenly very salty. 

Before we dive into worm VR, let's back up and discuss this specific worm.

The Magnificent C. Elegans
C. Elegans is a surprisingly popular subject of study in neuroscience. It has a simple and well defined nervous system that contains only 302 neurons (in the hermaphrodite, the rare males have a few extra neurons).  All the neurons and even all the connections between the neurons have been pretty well characterized.  They are small (hundreds can fit on a standard sized petri dish) and they reproduce quickly.  And it that wasn't enough to make C. elegans a desirable subject for study, they can be genetically altered with relative ease, and exhibit rudimentary learning skills. 

A recent technological development has made clever use of genetic tools that allow calcium influx (an indicator of neural activity) to be visualized in neurons and allow neurons to be activated by light.
Faumont et al., (2011) have created a worm tracking system that uses the fluorescence from a genetically altered neuron to locate the worm and recenter the microscope on the worm in real time. This allows for completely non-invasive visualization of neuronal calcium/activity in the awake behaving animal. 

The recent paper in PLoS One, describes exactly how they got the microscope to track the worm in real time without blurring of the signal or messing up the calcium imaging. The paper is open access, so you can go read the details for free.

To see this larger and more clearly, you can download this video and their 4 other supplementary videos here. 
In this video, you can see the animal moving around in the top left, the path it follows in the top right, the calcium fluorescence signal in the bottom left (notice the calcium neuron is always in the field of view), and the activity of this particular neuron when the worm is traveling either forward (blue) or backward (red). 

The "Dedicated Circuit" Hypothesis
The neuron imaged in this video is called AVB, and it is a 'command neuron'.  Faumont et al. show that it increases in activity when the worm is moving forward and decreases when the worm moves backwards.  A similar command neuron, AVA, does just the opposite, increasing when the worm moves backward and decreasing when it moves forward.  These data support what is called the "dedicated circuit hypothesis" which says that the worm uses one set of neurons to go forward and a completely different set of neurons to move backwards.

While Faumont et al. shows that the dedicated circuit hypothesis is supported for command neurons, they find that the activity of the actual motor neurons (the neurons on the body wall that control contraction of the muscles) does not support this hypothesis.  If the dedicated circuit hypothesis was true, the A-type motor neurons should only be active and oscillating during backward movement, and the B-type motor neurons should only be active during forward movement.  They found that this wasn't true, that both were active and oscillating during both forward and backward motion. 

Virtual Reality for Worms
Now back to virtual reality.  This Faumont et al. paper is a showcase of new tools that can be used to study C. Elegans in a simultaneously macroscopic and microscopic way.  One of the new techniques the introduce is the optogenetic stimulation of specific neurons in specific places to create and 'environment' for the worm. 

Faumont et al., 2011 Figure 2

When they genetically express channel rhodopsin, the channel which activates neurons when exposed to blue light in the ASH neuron (a neuron sensitive to osmolarity, or saltiness, changes), they can activate that neuron whenever they want by turning on the blue light.  They create a virtual environment by tracking the worm as it travels in a field, and activating the blue light when it reaches a certain xy coordinate.  In the figure above they activate the neuron when the worm's nose is within the outer ring (traces turn blue).  This makes the worm 'think' that the ring is full of saltier liquid than the rest of the area. 

This virtual environment takes away all the technical difficulties of actually creating a ring of salty water in a pool of less salty water, and the VR environment can be quickly and easily changed into any shape or size, when desired. 
This new tracking method, in combination with calcium imaging and optogenetics, represents a leap forward in cellular scale neuroscience, to non-invasively visualize neuronal activity, activate neurons, and record the coinciding behavior is a combination mammalian neuroscientists can only dream about.

Note: there are ways to image calcium in the neurons of moving mice, but even this requires installing a 'window' into the skull and mounting a mini-microscope on the mouse's head. In addition, the neurons visualized are limited to the ones closest to the surface of the brain.
© TheCellularScale


Faumont S, Rondeau G, Thiele TR, Lawton KJ, McCormick KE, Sottile M, Griesbeck O, Heckscher ES, Roberts WM, Doe CQ, & Lockery SR (2011). An image-free opto-mechanical system for creating virtual environments and imaging neuronal activity in freely moving Caenorhabditis elegans. PloS one, 6 (9) PMID: 21969859

Erasing Memories Cell by Cell

3d glass brain
by Kazuhiko Nakamura

We've discussed recent findings about erasing fears from memories, but today we'll be talking about erasing the fear memory itself. This involves actually inhibiting or killing the individual neurons that encode for a particular memory, so for obvious reasons these experiments are done on mice rather than humans. 

Mice can be trained to associate a mild electrical foot shock with a tone.  The tone plays and then a foot shock is given.  Once the mouse has learned this association, it will freeze in place when the tone is played.  This is called an auditory fear memory. 
Using a fear memory paradigm, Sheena Josselyn in her Toronto lab discovered how to visualize the neurons which are active during fear memory formation. They also developed a way to target and delete them, consequently deleting the memory. 

In Han et al. (2009), some beautiful genetic trickery was used to promote a 'kill switch' only in the neurons which are active during the memory formation.  This kill switch is the diphtheria toxin receptor.  Normally cells do not have this receptor, but when they promote this receptor artificially on the cell surface, an injection of diphtheria toxin will kill that cell, but not neighboring (dtr-free) cells.  The real impressive genetics is in promoting the diphtheria toxin receptor only in neurons active during memory formation.  To do this, the Josselyn lab used a marker for cell activity in amygdala neurons during memory formation, CREB.  Specifically, they used a transgenic mouse that expressed the diphtheria toxin receptor only when CREB activates cre.

So now with the memory encoded and the kill switch in place, they pull the trigger and inject diphtheria toxin into the mice. This kills all the amygdala cells that were active during memory formation (about 250 amygdala cells or so, Han et al., 2009 figure 1B).  They then test the mice again for freezing behavior.

Han et al., 2009 Figure 3

The second set of columns (CREB-cre, DT) is the experiment I have described.  Before any drug is injected the mice freeze in response to the tone, but after the diphtheria toxin (DT) injection, the mice freeze much less in response to the tone. What is really essential to this study is the control experiments that they ran. 

They wanted to make sure that just killing any 250 neurons in the amygdala didn't causes memory loss.  So instead of using the CREB promoter to activate cre (and thus the diphtheria toxin receptor) they used a control promoter (cntrl-cre, DT above) to promote cre in about the same number of neurons, but not dependent on neural activity.  In this case, there is no statistical difference in how much the mouse freezes in response to the tone. (compare the first two columns to each other.) 

Similarly, they wanted to make sure that the diphtheria toxin (DT) itself didn't erase the memories. They injected CREB that did not promote cre, and thus did not cause any diphtheria receptors to be expressed (CREB, DT). In this case, there was again no difference between pre and post DT injection.  Finally, they wanted to make sure it wasn't the CREB-cre construct itself, so they added the CREB-cre like normal, but did not inject the diphtheria toxin, so the receptors were expressed on these cells, but were not activated. In this case again, not difference in the amount of freezing. 

Because none of these control groups showed a difference in freezing, Josselyn could be confident that she had really shown that the specific neurons that encoded the memory were necessary for recalling the memory. 

They are also clear that the amygdala is not seriously damaged in this study, as the mice can re-learn the task after the specific neurons have been deleted.

One particularly interesting aspect of this study, which the authors do not discuss, is the number of neurons necessary for encoding a memory.  They delete hundreds of neurons.  I wonder if deleting half of them or even a quarter would result in the same erasure of the memory. How many neurons does it take to encode a memory?

Recently this concept of targeting proteins to only the active cells has been extended to include channel rhodopsin, the protein which allows cells to be activated by light.  Liu et al., (2012) was able to reactivate the neurons that were specifically active during the learning of a fear response. Stimulating these neurons caused the mouse to freeze, suggesting that stimulating these neurons reactivates the memory. This paper is covered thoroughly by Mo Costandi at Neurophilosophy.


© TheCellularScale

Han JH, Kushner SA, Yiu AP, Hsiang HL, Buch T, Waisman A, Bontempi B, Neve RL, Frankland PW, & Josselyn SA (2009). Selective erasure of a fear memory. Science (New York, N.Y.), 323 (5920), 1492-6 PMID: 19286560

The cells that make us eat: Part 2

In the last post, we discussed the finding that stimulating the AgRP neurons in the hypothalamus directly causes mice to eat.  You can see the video of the mouse eating with the light stimulation here.

Today we will look at a follow up paper by the same group.  This paper looks at the mechanisms that might naturally stimulate these neurons.  As the authors mention in the discussion, the origin of the pathways that naturally cause these neurons to fire is not known. (as in, the part of the brain that sends the main signals to these neurons is still a mystery)  However, they can investigate what is happening at the junction between these pathways and the AgRP neurons, that is, at the synapse.

Using brains from mice that are either hungry or full, the researchers found that in hungry mice, the AgRP neurons recieve more synaptic input. 

But why?

There are a few ways this could happen.  One possibility is that the upstream neurons are firing more frequently because they are receiving more input from other pathways. But since it is not known where these neurons are, that is difficult to test.  Another possibility is that the output ends of these neurons, the part which arrives at the synapse, releases neurotransmitter more easily in hungry mice than in full mice. 

The research in this paper supports option number 2, that the hunger signal modulation occurs right at the synapse.

The researchers find that through a complex molecular pathway, involving ghrelin, leptin, and opioid signalling, the neurotransmitter release at the synapse is regulated by the animal's hunger state. Although too detailed to fully summarize here, the paper presents a satisfying and thorough explanation for how the AgRP neurons (the ones that cause eating when stimulated) could be modulated by hunger signals from the body. 

One of the nice parts about this explanation is that it avoids the 'never ending chain of neurons' problem, where activity in one neuron is caused by a neuron stimulating it, which in turn is caused by a neuron stimulate that one, which is caused by... you get the point. 
This paper strongly suggests that dynamic modulation of eating behavior can happen right at the synapse.

Yang Y, Atasoy D, Su HH, & Sternson SM (2011). Hunger states switch a flip-flop memory circuit via a synaptic AMPK-dependent positive feedback loop. Cell, 146 (6), 992-1003 PMID: 21925320

The cells that make us eat: Part 1

It is always exciting when a specific behavior can be directly linked to particular neurons. 

In this case, eating.  In March 2011, a paper came out from the Sternson lab at Janelia Farm explaining that when certain neurons (AgRP) in the mouse hypothalamus were stimulated with light, the mouse would spontaneously start eating.  The mouse would pretty much keep eating (except for water breaks) until the stimulation stopped.  What's even more interesting is that the neurons right next to these (POMC) had pretty much the opposite effect.  When they were stimulated, the mouse didn't eat much and over time lost weight.

This may seem like the basis for the next miracle diet,  find a way to stimulate POMC neurons and suppress AgRP neurons, right?

Unfortunately it's not that easy.  There is a lot more work to be done.  In fact this is just the beginning of understanding the hunger circuit.  Sure stimulating the neurons directly with light causes eating, but what naturally stimulates those neurons? 

Neurons fire when they recieve signals from other neurons which in turn fire when they receive signals from other neurons.... and so forth in a never ending chain.

So, where does the 'must eat' signal ultimately come from?
where is the beginning of that neural chain?

We will investigate this a little more in Part 2, when we look at a follow up paper.

Aponte Y, Atasoy D, & Sternson SM (2011). AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nature neuroscience, 14 (3), 351-5 PMID: 21209617

Neurons activated by light

Optogenetics is a relatively new technique that inserts a light-sensitive channel or pump into a cell that would normally not be light sensitive. This channel can be inserted into the neurons of a mouse, worm, fly, rat, or whatever (has not been tried on humans yet to my knowledge).
Then, when a light is flashed onto that neuron, it activates, much like it would if it had just been stimulated by electricity or the normal neuronal pathways leading to it. 

What is so great about this is that with tricks of genetics, a researcher can get this channel to express only in one kind of cell, making it so that cell1 is activated when the light comes on, but cell2 is not. 

Previously if you wanted to stimulate neurons in a living brain you had to insert metal electrodes which would stimulate all the cells within a certain radius.  Optogenetics gives cellular specificity, where metal electrodes don't. 

Some great discoveries are already being made using this technique:

Which neurons cure the effects of Parkinson's Disease during deep brain stimulation (Gradinaru et al., 2009)?

Which neurons make a mouse eat (Aponte et al., 2011)?

And many more.

Though this is not a technique that is meant to be used on humans for disease treatment, metal stimulating electrodes are already being used to treat some disorders (parkinson's see here and here. and depression).  How would the ethics of optogenetic treatments differ? Instead of a metal electrode, a genetic alteration would have to take place in some neurons and a fiber optic cable would have to be implanted. 
Thoughts?