How to Build a Neuron: Step 2

Recently we've discussed the first step in how to build a neuron. Today we will discuss step 2: reconstructing that stained cell.

Hippocampus CA1 Pyramidal neuron (from Neuromorpho.org)

There are a couple of ways that you turn an image (or image stack) of a neuron into a digital neuron file like the one pictured above.  Basically there is an easy way and a hard way.  The hard way is to reconstruct the neuron manually, where you literally trace the neuron by hand.  The easy way is to auto-trace the neuron.

In a recent Frontier's in Neuroinformatics article, Myatt et al. (2012) explain the hard to easy gradient in reconstruction methods.

• "Manual (Camera lucida). Prisms are employed to visually overlay the microscope image onto a piece of paper, and the neuron is then traced by hand. Although primarily used for 2D tracings, 3D reconstructions can be derived from these with time consuming post-processing (Ropireddy et al., 2011).
• Semi-manual (e.g., Neuron_Morpho, Neurolucida). Digital segments are added by hand through a software interface, typically sequentially, beginning at the soma, and working down the dendritic tree.
• Semi-automatic [e.g., NeuronJ (Meijering et al., 2004; 2D reconstruction only) and Imaris (3D reconstruction)]. User interaction defines the basic morphology, such as identifying the tree root and terminations, but branch paths are traced by the computer
• Fully automatic (e.g., Imaris, NeuronStudio; Rodriguez et al., 2003, AutoNeuron add-on for Neurolucida). The entire morphology is extracted with minimal user-input. " (Myatt et al., 2012)

You may ask: "Why not just do it the easy way?" Good question.  It is actually surprisingly difficult to make a versatile program that can accurately reconstruct neurons.  So difficult in fact that in 2010 an open challenge was issued with a monetary prize for the best automatic reconstruction algorithm. Five teams competed in this DIADEM challenge and the results and process are explained in detail in a special issue of Neuroinformatics. (And in less detail in this HHMI press release)

automatic reconstructions of neurons (source)

Advances in automatic reconstruction are being made at an astounding pace, but most neural reconstructions are still being done in a semi-manual or semi-automatic way. 

If you are interested in reconstructing some neurons, you can download Neuromantic for free or Neurolucida for money. There is other reconstruction software available, summarized nicely in Myatt et al. 2012, but these are the two I am most familiar with. 

In the next edition of "How to Build a Neuron" I will tell you how you can completely skip step 1 (the staining of the neuron) and step 2 (the reconstruction of the neuron). 

For ease of access, the whole "How to Build a Neuron" series is archived.


© TheCellularScale


Myatt DR, Hadlington T, Ascoli GA, & Nasuto SJ (2012). Neuromantic - from semi-manual to semi-automatic reconstruction of neuron morphology. Frontiers in neuroinformatics, 6 PMID: 22438842

LMAYQ: Safety First

Welcome to part 3 of the million part series "Let Me Answer Your Questions" (LMAYQ).  Here I address questions that people have asked The Internet.  The Internet, in turn, directed these people to The Cellular Scale, where their questions were... not answered. 

Today our topic is:

 

These 3 questions inspire me to warn you readers about a couple dangerous things.


1. "Will Parkinson's doctor let me have DBS?"

This is a really really important question because I absolutely do not know the answer to this. It must have brought someone to my post lauding the amazing power of Deep Brain Stimulation and its ability to stop Parkinson's Disease symptoms in their tracks. This is such an important question because it has prompted me to make this point:

This blog is not giving medical advice.

 

 

I am not a clinical doctor and more importantly The Internet is not your doctor and you should be careful when you ask The Internet medical questions. Yes DBS has shown amazing results, but its long term side effects are not even known yet and it requires a very serious surgical procedure. Just because I think its an amazing step forward, doesn't mean I think you should get it.  I don't know any thing about you or your condition (and neither does anyone else on The Internet). 


2. "Is DBS a cure for Parkinson's Disease?"

Following up on question 1, I titled my blog post "How does DBS cure Parkinson' Disease?", and focused on some new research looking into its mechanism of action. It was somewhat careless of me to use the word 'cure' all throughout the post, when really it should have been 'treatment'.  The most accurate answer to your question is "DBS treats Parkinson's Disease symptoms"


3. "What happens when you give a mouse cocaine?"
 
This is a good and interesting question, but also inspires me to make a warning statement:

Do not try this at home. 

Do NOT give your pet mice random drugs (even legal ones). It is not healthy for them.

This question brought someone to the post "If you give a mouse a placebo..." which discusses how to trick a mouse into thinking it is getting cocaine. This is really important research isolating the effect of the actual cocaine directly acting on the brain compared to the effect of the inactive cocaine acting indirectly on the brain.

The answer is: If you give a mouse cocaine, it can get addicted to it, which is why mice are used to study the effects of cocaine addiction and the efficacy of possible treatments.

But really I want to use this question as an opportunity to explain how research on animals is different from just giving animals drugs.

All the research I report on here is peer-reviewed.  This means other scientists besides the authors have read it carefully, and had an opportunity to point out any flaws or weak points in the research design or execution. In addition, research that uses animals adheres to extensive animal usage and handling regulations. It's not like someone just thinks 'dude, I wonder what would happen if you gave a mouse this or that' and then does and calls it research. Before scientists uses a single animal for any research, they have to write a thorough (usually 6 pages or so) explanation for exactly how many animals they are using and why they have to use that many and what they are going to use them for.  They have to list the steps they will take to minimize pain and explain how they will ensure that the animals are treated humanely. Then that document is read and discussed by a whole committee whose job it is to make sure that animals are being treated well at that institute. The committee then can either approve the animal use, or reject it and ask the scientist to only use this many instead of that many or use this kind of surgery instead of that kind of surgery. 

So there you have it, some warnings about what you should and shouldn't draw from this blog (and The Internet in general). Stay tuned for more fantastic answers to your fantastic questions.

© TheCellularScale

 

Twists and turns on smell's evolutionary road

Smell is a complicated sense and its evolutionary path is a convoluted one. Olfactory receptor cells developed different shapes and different chemical receptors and were sometimes divided into separate organs and sometimes not.

Rainbow Goldfish: experimental animal (source)

A research group from the Rocky Mountain Taste and Smell Center (not affiliated with Coors) decided to research the olfactory cells of the noble goldfish. Goldfish are an interesting vertebrate because they, like humans, do not have the pheromone-sensing vomeronasal organ (though rats, a much closer evolutionary relative, do have it).

This group published a paper analyzing the morphology and chemical signature of the different types of smell cells in the goldfish olfactory epithelium (basically the back of the nose). Since Form and Function is one of my favorite topics, this paper sparked my interest. 

Hansen et al. (2004) show that there are three main shapes for the goldfish smell cells.

Hansen et al., 2004 Figure 3 (3 types of cells in the goldfish olfactory epithelium)

There are the Ciliated, the microvillous, and the crypt cells. 

"Ciliated ORNs are tall cells, with their nuclei usually located in the lower half of the OE. The cells possess a narrow dendrite and long apical processes radiating from an olfactory knob at the distal end (Fig. 3). Crypt receptor cells are obvious because of their typical ovoid shape and location in the upper half of the OE (Fig. 3). These ORNs possess microvilli that border the apical rim of the cell. At the same time, they possess cilia that are located in a “crypt”-like invagination." Hansen et al., 2004

Hansen et al. wanted to see whether these morphological characteristics correlated with the chemical signature of the cell. More specifically, they wanted to see which type of receptors these cells had and which g protein they expressed. 

They found that there was a direct correlation between the shape of the neuron and the type of smells it was sensitive to (as indicated by the receptors and g proteins it expresses). 

The most interesting finding was that the microvillous and crypt cells in the goldfish have very similar characteristics to the cells in the rat vomeronasal organ, and probably also serve the function of sensing pheromones. The paper inspires questions about why rats might have evolved a separate organ to house their pheromone receptors, while goldfish have all their receptors packed into one organ. Why would a separate organ be necessary if a range of informative odors can be sensed using one organ?

Eisthen (2004)

In her commentary on the paper, Eisthen presents an evolutionary tree showing the animals that have the vomeronasal organ and those that do not.  (I've blogged about her work on the olfactory sense of the axolotl here)


Even though goldfish have all these cells in one organ, the cell types aren't evenly intermixed.  The microvillous and crypt cells are concentrated closer to one end. The authors speculate that the differential location of these cells within the goldfish olfactory epithelium might be an intermediate evolutionary step towards an actual separate organ.


© TheCellularScale


Hansen A, Anderson KT, & Finger TE (2004). Differential distribution of olfactory receptor neurons in goldfish: structural and molecular correlates. The Journal of comparative neurology, 477 (4), 347-59 PMID: 15329885


Eisthen HL (2004). The goldfish knows: olfactory receptor cell morphology predicts receptor gene expression. The Journal of comparative neurology, 477 (4), 341-6 PMID: 15329884

How to Build a Neuron: Step 1

There are many reasons to try to build a neuron, but fully building a model neuron is an extensive process with many steps.  Today we will discuss the very first step in the neuron-building process: determining the activity and  shape of the neuron.

Biocytin filled cortical neurons (source)

To determine the shape of neuron, you have to stain it somehow.  There are several ways to do this, but we will focus on the biocytin filling method.
To determine the activity of a neuron, you have to use electrophysiology to record its electrical activity. The biocytin filling method makes use of the same patch clamp electrode to record the electrical activity of the neuron and to fill it with the biocytin molecule that can be later dyed.  So this method is perfect for building a neuron because with it you can correlate the shape of the neuron directly with its activity patterns. 

Neural activity correlated with neural morphology (source)

A recent Nature Protocols paper by Marx et al. (2012) provides step by step details for how to fill and dye a neuron using the biocytin method. 

The basic biocytin staining protocol is as follows:

1. make brain slices
2. fill the neuron with biocytin while recording its electrical activity
3. fix the brain slice in paraformaldehyde
4. quench the endogenous peroxidase
5. connect the biocytin to avidin (using the vectastain ABC kit)
6. colorize the avidin (using DAB and nickel)
7. mount the slices on gelatin subbed slides
8. dehydrate the slices SLOWLY through very small steps of ethanol concentration
9. clear with xylene and coverslip

Marx et al. provide some excellent specifics in the paper that make the whole process understandable and more importantly, doable. They even have a troubleshooting section which explains what might have gone wrong under several conditions.

Marx et al., 2012 Figure 2

One of their best tips in the paper is to dehydrate the slices very slowly.  They show that when you dehydrate the tissue quickly, you get a cork-screw artifact (A) that is not physiologically meaningful, but when you dehydrate slowly, you get a more accurate morphology. 

So there you have it, Step 1 of neuron building.  Step 2 will be coming soon, can be found here.
And all the "build a neuron" steps will be indexed here.

© TheCellularScale


Marx M, Günter RH, Hucko W, Radnikow G, & Feldmeyer D (2012). Improved biocytin labeling and neuronal 3D reconstruction. Nature protocols, 7 (2), 394-407 PMID: 22301777

LMAYQ: Can Odor be recorded?

Let Me Answer Your Questions: part 2, in which I answer your very important questions via google search terms. Part 1 and all subsequent LMAYQ posts will be archived in the LMAYQ index.

by Likarious

So let's get to it, what fascinating questions are you asking google?


1. "Can odor be recorded?"  

This likely brought someone to my post "You can't trust your receptors:smell" in which I discuss the EOG (electrolfactogram) where you can record the electrical activity of a smell receptor while certain smells are presented.  But it does not answer the question of whether a smell itself can be recorded.

So I looked into it a little bit and surprisingly, the answer is yes!

Nakamoto and others have created an "odor recorder"

Nakamoto 2005 figure 1

Unlike visual recording, which only need red, green, and blue to make essentially all the colors, odor recording requires a few more components. For example, the authors created an apple smell using 8 components.

I would love to say that this odor recorder is going to appear in every living room and plug into the TV so that restaurant and perfume marketing can be truly effective, I just don't see the demand being strong enough to make it worth mass producing. Though, I think it would be pretty amazing. 

I also had doubts as to whether the odor recorder could accurately transmit the scent of a really nice perfume which is not static, but develops over time. But The 2005 Nakamoto paper shows that they can actually record the changes of an odor over time!

While there is always the fact that a perfume reacts differently with every one's skin, the odor recorder actually seems like a promising device and might find a market in die hard perfume fans.

or..."odor recorder prevents murder"

The quest to permanently record the scent of a woman drives a man to murder in the mediocre movie "Perfume: the Story of a Murderer."  If only he was in possession of an odor recorder.

© TheCellularScale

Nakamoto T (2005). Study of odor recorder for dynamical change of odor. Chemical senses, 30 Suppl 1 PMID: 15738143

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

A new look at light

You might know that your retina senses light primarily through its rods and cones which are sensory cells specialized in converting photons into electrical signals.


Pisa at Sunset (I took this picture)


What you might not know is that there is a third light-sensitive cell in the mammalian eye. These cells are retinal ganglion cells (RGCs), but not all RGCs are directly sensitive to light.

But what you really probably don't know is that these RGCs sense light using the same protein that allows a toad's (Xenopus Laevis) skin to sense light (melanopsin). 


"It's true, I tell ya!"

These cells (the non-rod, non-cone light sensors) react to light directly, but they aren't exactly good at it. Their sensitivity is lower than the rods and cones, and they don't seem to transmit shape or color information.  So what is their purpose? Why have a secondary set of cells that sense light in a poor and unfocused way when you already have highly specialized rods and cones? 

To make it even more confusing, the rods and cones actually connect to these cells, adding their light-sensing information to theirs. 

weird, right? In a recent review paper, Pickard and Sollars (2012) explain that these cells likely play a role in controlling the sleep-wake cycle (circadian rhythm). Rats and mice with strongly degenerated rods and cones still set their circadian clock by the light cycle they are exposed to.  These cells send strong projections to the hypothalamus which controls everything sleep-wake cycle.


In addition, these cells or at least the melanopsin gene, may play a role in Seasonal Affective Disorder (SAD) by modulating the light-dependent cycles of the suprachiasmatic nucleus (a part of the hypothalamus).



SAD (source)

Their vague ability to sense 'brightness' makes these cells nicely suited to regulating the body's response to daily and seasonal changes in light. But whether these cells need to be light-sensitive to perform these functions or whether their sensitivity to light is just an evolutionary remnant is unclear. 



© TheCellularScale



 Pickard GE, & Sollars PJ (2012). Intrinsically photosensitive retinal ganglion cells. Reviews of physiology, biochemistry and pharmacology, 162, 59-90 PMID: 22160822



Let me answer your questions: Part 1

One of the most entertaining things about writing this blog, is seeing the search terms that people have used to find it. Some of them leave me completely befuddled ("Giraffe eating man"), and some are amusing combinations of words that happened to be in a particular post. But my favorite ones take the form of questions.

(by Nanecakes)

I am pretty sure that most of the questions people have used to find my blog are not answered in my blog, and I hate the thought of disappointing people. So I thought I would take some time and answer a few of my favorites.

(All of these are actual 'search terms' that showed up on my blogger stats, I am not making these up.)



1. "Do neurons make you smart?"


This is a surprisingly interesting question. My answer is probably. All the evidence points to us needing neurons to think.  When neurons get damaged in certain parts of the brain, things start going badly in the 'smart' department. If you had no neurons, you probably couldn't think. 
There are two caveats that make this question interesting. 

1. Sometimes things that do not have neurons act 'smart' (see my post about the almost-neurons of the Venus Fly trap)

2. If neurons make you smart, they also make you stupid. You need neurons to perform a 'stupid' action just as much as you need neurons to perform a 'smart' action. An example here is when I am trying to drive somewhere I don't often go, I might accidentally find myself on my way to work.  I might think "how stupid of me, I wasn't even thinking" And it would be true.  The stupid action of turning the wrong way is because my striatal neurons have encoded the drive to work really strongly. 



2. "Does Shrek wear pants?"

This question directed someone to my post "How animals, Shrek, and Yoda stimulate your neurons."

And yes, in fact, Shrek does wear pants, though I had to google that term myself to find out. The issue here is that Shrek's pants are a dark olive color, close in hue to his skin tone, making it hard to tell and remember that he is wearing pants under that short tunic thing. 

UPDATE: 8/6/12 I have been informed by an astute reader that Shrek does not in fact wear 'pants' but rather 'tights'.  I suppose this is a more accurate description of his attire, so I formally apologize for spreading Shrek-related misinformation.

3. "Why is it better to play female Mass Effect?"


This is sort of answered by "4 reasons all women should play Mass Effect" But the question is specifically referring to playing the game as a woman rather than as a man.

A lot of people play Mass Effect as a woman because they think the voice-acting is better. (I agree with this)

I imagine most women play Mass Effect as a woman for the same reason most men play Mass Effect as a man. It's more fun to be a character when you can relate yourself to the character. I think it would be great if everyone played Mass Effect as a woman in a perspective taking experiment.

And I think Commander Shepard makes an excellent role model for young girls aspiring to one day save the galaxy.

FemShep Barbie (pure genius from Introverted Wife)

(FemShep Barbie would be such great friends with my Computational Neuroscientist Barbie!)


Readers, I hope you have found this post informative. I plan to continue answering these important questions for you in the future. 

© TheCellularScale

The effect of familiar male voices on neurons

male zebra finch trying to impress female (Max-Planck)

Zebra finches are a popular model for language learning because unlike most research animals which may have instinctual vocalizations, zebra finches (the male ones at least) learn their signature song from experience.

The importance of social experience in male song learning is clear, but what about the effect of social experience on the female response to the male voice?

Menardy et al., 2012 (figure 2C)

Menardy et al., (2012) have recently analyzed the neural response in females to the male distance calls (not songs).  They tested the  response in anesthetized birds, but also in awake, alert birds. To do so, they used a nifty little recording device that they mounted on the back of the females.


They tested the response of the neurons in the caudomedial nidopallium (NCM) to the calls of the female's mate (4 months spent together), a non-mate familiar male (3 days spent together), and an unfamiliar male (complete stranger).

In general, the neurons in the female NCM responded more strongly to the calls of the males that they knew than to the stranger's call. 

Menardy et al., 2012 (figure 5A)

So why is this and what does it mean? The authors point out that this change in neural response could be a result of extensive social interactions (the female bird spent some quality time with the mate and the familiar male), or it could be a result of having heard the call before. 

In other words, Is the NCM encoding a recognition signal ('ah, that's Nick's voice') or a familiarity signal ('I've heard this sound before')?

It is likely that some form of neuroplasticity is taking place during the male-female interactions, but the mechanisms and the meaning behind it are not clear yet. Some interesting experiments might be to test the effect of traditional 'learning disruptors' (such as protein synthesis inhibitors) on this neural preference for familiarity. 

© TheCellularScale


Menardy F, Touiki K, Dutrieux G, Bozon B, Vignal C, Mathevon N, & Del Negro C (2012). Social experience affects neuronal responses to male calls in adult female zebra finches. The European journal of neuroscience, 35 (8), 1322-36 PMID: 22512260