The recovery paper has come out

Finally, my "recovery paper" came out:

Recruitment of polysynaptic connections underlies functional recovery of a neural circuit after lesion.

Akira Sakurai, Arianna N. Tamvacakis, Paul S. Katz

DOI: 10.1523/ENEURO.0056-16.2016

This paper shows that, when a neural circuit failed by losing one of its synapses within, functional recovery can occur through reorganization of the remaining neural circuitry. We show that a molluscan neural circuit recruits additional neurons in response to a lesion. The extent of recruitment predicts the extent of behavioral recovery. 

Even in a well-defined (sort of) invertebrate neural circuit, there are indirect, polysynaptic pathways that provide compensatory function or flexibility to the circuit. Such individual variability appears to be hidden under normal conditions but becomes relevant when challenged by neural injury.

This paper is a sequel of two preceding papers:

Functional Recovery after Lesion of a Central Pattern Generator (2009)

Hidden synaptic differences in a neural circuit underlie differential behavioral susceptibility to a neural injury (2014)

A simple half-center network oscillator with a twist

My latest paper came out from J Neurophys:

The central pattern generator underlying swimming in Dendronotus iris: A simple half-center network oscillator with a twist  

by Akira Sakurai and Paul S. Katz

DOI: 10.1152/jn.00150.2016

This paper describes the central oscillatory circuit underlying rhythmic swimming of a nudibranch sea slug, Dendronotus iris.

Dendronotus iris swims by rhythmically flexing its body to left and right.

The Dendronotus brain is a cluster of lobes or "ganglia." The neurons that produce the rhythmic motor output for swimming have their cell bodies in the pedal ganglia. They all project the axons toward the other side of the brain to synapse with their contralateral counterparts.  

The circuit is a typical "half-center oscillator" that consists of only two bilateral pairs of neurons. The paired neurons each inhibit their contralateral counterparts.  The circuit has a “twisted” organization; that is, a neuron in one pair is excitatory-coupled contralaterally to a neuron in the other pair.

The Dendronotus swim CPG is a half-center oscillator.  The left illustration shows actual synaptic connections. The left Si3 (L-Si3) neuron forms an excitatory synapse and electrical connection onto the right Si2 (R-Si2), forming a twisted configuration. A modified version is shown on the right. Coupled Si2 and Si3 neurons form a functional unit that works as a half-center to produce rhythmic bursting.

The half-center oscillator is the simplest design of a network oscillator, in which two neuronal elements with no endogenous rhythmicity form reciprocally inhibititory synapses. The half-center theory was first proposed by T. Graham Brown in 1911 after the historical experiment with a walking cat with transected spinal cord.

In addition to the reciprocal inhibition, each functional unit of the half-center oscillator generally contains the excitatory neurons that provide rhythmic excitatory drive onto the mutually-inhibitory neurons (eg., Clione, tadpole, lamprey, zebrafish, and mice). Because of high complexity with so many neurons involved, the role of the excitatory neurons have not been clearly understood.  Here, we found that the Dendronotus swim circuit consists of only 4 neurons. By using "Dynamic Clamping" technique, we manipulated the strength of the excitatory synapse and found that they play crucial roles for the circuit to function as the half-center oscillator.  To our knowledge, this is probably the simplest half-center oscillator described to date. Because of such simplicity, this circuit is also highly manipulable, and hence may provide a good system to study the fundamental properties of a network oscillator. 

Oil stone vs. sandpaper

For sharpening forceps and spring scissors I use "translucent grade" Arkansas oil stone purchased from Dan's Whetstone Company, Inc.

My friend asked me which is finer, the oil stone or 2000 grit sand paper. Which is better for sharpening forceps and scissors?
So I compared them.

This is 2000 sandpaper with the tips of my favorite spring scissors.
The grain itself seems fine enough for sharpening, but the surface is not very smooth. Looks bumpy. It may be OK for sharpening a fine tip.
When I used it, however, I found it too soft to polish a sharp edge. The paper dents slightly and that blunts the edge. Just a paper, but it felt like a sponge at this scale. It's like sharpening a razor blade with a sheet of mud. The edges got chamfered, which is not good.

Now, this is the oil stone.

It looks like the stone surface is out of focus, but actually it's not.

It turned out that the surface of the extra-fine grade oil stone is smoother than 2000 sandpaper.
And much harder. This is important. The polishing material has to be solid hard to make a good sharp edge.

Sutter P-97 SRAM battery outage

If you are here looking for "how to replace SRAM on Sutter P-97", just get a DS1243Y-120 from anywhere online. It is far cheaper than buying it from Sutter. 

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Well, I noticed that our electrode puller (Sutter P-97) had forgotten everything when I came back from SfN.  Not just those programs I put, it showed weird characters for time and date. I gave a suspicious inquiry to students whether they did something stupid. They did not, of course. It turned out that the battery for internal storage chip failed while no being used for a weak.

The Sutter company personnel told me that the battery on the internal memory chip had failed.  I needed to reinstall a new memory chip (SRAM CO-U688243-P97). The puller maker gave it a long name, but it is just a regular SRAM chip made by Dallas.  

On the mother board inside P-97, it says DS1243Y.

I looked up and found there are several subtypes: 

DALLAS DS1243Y

DALLAS DS1243Y-120

DALLAS DS1243Y-150

DS1243Y-120 is way cheaper than DS1243Y.

"120" means access time (120 nano sec). DS1243Y has 200 ns access time whereas DS1243Y-120 has 120 ns. Of course, the faster the better. I can get DS1243Y-120 online for ~$10 or less. DS1243Y is about $70-$75. The prices must be determined by supply, not specs.

I get a quote from Sutter and they said their DS1243Y costs $60 and $20 for shipping. It sounded reasonable. So I took it. I thought maybe it has to be DS1243Y. Maybe it cannot run with DS1243Y-120. 

Turned out, they sent me DS1243Y-120! Like I said, I could get it at 1/10 price from anywhere online. Don't get it from the puller maker. 

After removing 6 screws, the top cover can sit on the front panel like this. Avoid bumping the sensitive part under the solenoid cage. 

I bent some pins when pulling it out. Who cares.
Insert a small flat-head screwdriver into the slit at the front end. There is no opening to wedge on the side.

The memory test says "THE RAM IS BAD" probably because this is DS1243Y-120. But it works fine. Also, the programed date, day and month, still showing weird characters. Guess it's trying to look like made in Japan.

Two papers accepted on the same day, published on the same day

I have long been working on this paper "Hidden synaptic differences underlie..." which was just published in eLife last week. It describes the mechanism underlying different susceptibility to brain injury among individuals. Individuals, I mean sea slug Tritonia individuals.
   This work was first presented in 2008 at SfN in Chicago and I received a Faculty of 1000 recognition. They interviewed me and the video was uploaded on YouTube.
   It was tough paper to write.  There are so many tedious correlograms. Reviewers disliked them, and I agree because most of them show no significance. Dynamic clamping gave some good taste to this manuscript, although some may point out that I should have called it dynamic current injection rather than "clamping." The variable for presynaptic voltage was fixed and used as a constant parameter. I only used dynamic clamp software. If truly clamped, however, the injected current would not be strong enough to have an effect on membrane activity because of space clamp issue. Reviewers were supportive but literally told me to rewrite the whole manuscript. I agreed. I rewrote it.

Meanwhile, I was doing revision of another paper on the swim CPG of a hooded nudibranch, Melibe leonina. This is totally different topic; it simply re-describes the CPG circuit for Melibe swimming behavior. This paper may be boring for most of readers because it is so descriptive. But, to me, it was such fun to work on; it was like building a CPG from scratch. Very classical and straightforward. I did not have to make it sounds sexy like the other eLife paper. I knew this paper should get accepted easily. It did.

These two  manuscripts were resubmitted on the same day. Both got accepted on the same day. And published on the same day. I am trying to push out 3 more papers this year.

Pin selections

Choosing pins for your preparations is important. Especially for physiological experiments run on a Sylgard-coated dish. Here are some choices:

1) Tungsten needles

   This is standard and classic. I used to make them a lot when I was using isopod heart preps (very small). There are a lot to talk about how to make them.  However, I don't use them any more just because it takes a tedious time in electropolishing. I just don't have time for that. Plus, it needs extra care for a highly corrosive chemical solution.

2) Stainless steel pins

   We mainly use 10 µm diameter ones purchased from FST. These are great pins, but you need to cut them to proper length; 1 cm is too long for our preparations. Don't use a pinching wire cutter, because it crushes the cut end.  Just use regular dissection scissors. I use the fine scissors with serrated edge.

3) Cactus spines

10 µm diameter is too large for smaller brains such as those from Melibe and Flabellina. Some people use the tips of glass microelectrodes, but they are too fragile to handle with forceps. They also mess up the Sylgard floor, because the broken tips will remain there.
   We use cactus spines. Just one cactus stick can provide us enough pins. We harvest pins every time we do experiments. It keeps us running fine as long as we don't forget watering them once a month.
   I don't know their species names. The diameters about the size of my thumb. The white produces finer spines than the yellow.

4) Coffee filter

Do you have an old coffee filter? Check it out. Some coffee filters are made of fine stainless steel mesh. The diameter of the steel fiber is about the same as the FST pins (10 µm). This Zig-zag shape will prevent it from being pushed out from Sylgard resin. Indeed, short FST pins sometimes get repelled and pushed out of a freshly-made Sylgard resin.

May be not just for pins. Such a fine stainless fiber may be useful for some other purposes, too. Any ideas?

Left two, Cactus spines; middle two, coffee filter fibers; right two, FST pins (10 µm diameter).
 

   The FST pins are most commonly used in our lab. This is the hardest among the three, but difficult to handle with once bent.

   Cactus spines are the finest, but not very sturdy enough for reuse. Often crushed by forceps.

   The coffee filter fiber has the same thickness as the FST pins, but much softer. Their zig-zag shape can be useful for secure fix.

Two papers about cellular mechanisms for prepulse inhibition studied in Tritonia

 

A cellular mechanism for prepulse inhibition (2003)

by William N. Frost, Li-Ming Tian, Travis A. Hoppe, Donna L. Mongeluzi, and Jean Wang

This paper shows the first cellular-level evidence for prepulse inhibition in Tritonia. A weak sensory stimulus has both excitatory and inhibitory effects on S-cells. The inhibitory effects shunt the synaptic output of some S-cells.

Prepulse inhibition (PPI):

Strong, unexpected stimuli elicit startle responses in all animals, but it can be markedly attenuated if closely preceded by a weak stimulus. 

Schizophrenics show abnormally low level of PPI.

Tritonia PPI

A prepulse stimulus was given by vibrated stick before an electric shock to elicit the swim

The prepulse acts to hyperpolarize two cell types in the swim circuit

Tactile stimuli hyperpolarized DSI and S-cells, but it also cause a lot of excitatory responses in other neurons.

The prepulse shortens and narrows the S cell action potential

Spike narrowing seen in the S cell AP.

Identification of a neuron, Pl 9, mediating the prepulse-elicited S cell inhibition

Pl 9 appears to receive direct EPSPs from most or all ipsilateral S cells and inturn produces direct IPSPs bilaterally onto the entire S cell population, as well as onto the Tr1, DRI, and VSI-B interneurons.

Pl 9 mediates prepulse-elicited hyperpolarizing inhibition of the S cells.

Evidence that Pl 9 mediates PPI

Intracellular stimulation of a single Pl 9 blocked the swim motor program.

Killing a single Pl 9 reduced PPI

Pl 9 inhibits S cell synaptic efficacy

S cell-evoked EPSPs in the swim network neurons were reduced in size when S cells were activated. 

Pl 9 reduces S cell synaptic efficacy via presynaptic inhibition of S cell transmitter release

Pl 9-evoked IPSPs are mediated by Chloride ion, blocked by d-tubocurarine

Pl 9 caused reduction of presynaptic action potentials of S cells in duration and amplitude

The S cell synaptic efficacy depended on its membrane potential

Identification of a neuron, Pl 10, involved in prespulse-elicite postsynaptic inhibition of the DSIs

PI 9 had no direct contact onto DSI, but Pl 10 does.

Pl 10 can block the swim

This study above looking at change in synaptic efficacy of S cells

The next study then looked at change in spike propagation

Axonal conduction block as a novel mechanism of prepulse inhibition

by Anne H. Lee, Evgenia V. Megalou, Jean Wang, and William N. Frost

Prepulse nerve stimuli produce PPI of the swim motor program, and conduction block of S-cell action potential trains

PdN3 alone (swim) vs PdN3 preceded by CN1,4,5 (prepulse) - in all cases CN1,4,5 stim prevented the swim.

Stim of CN1,4,5 caused conduction failure of S-cells evoked by PdN3 stim

Sometimes saw incomplete blockade

Prepulse-elicited conduction block has the temporal features of behavioral PPI in Tritonia

Conduction failure seen when stim was given with 250-500 ms latency (stim, 400 ms)

Conduction block also occurs in response to skin prepulse

Evidence that interneuron Pl-9 mediates PPI of S-cell conduction block

Peak Pl firing was 43 and 59 Hz in response to the tactile stimulation

photoinnactivation of Pl-9 blocked PPI