NeurophysiologyLaboratoryProjectAbstracts2006

Biological Sciences 331 | Smith College

Emily Stackpole, Catherine Peo, Kristen Hokenson

Increasing carbachol concentration increases the burst frequency and decreases lag

time between adjacent crayfish swimmerets

The crayfish swimmeret system is innervated by the first roots of the abdominal

ganglia in segments two through five. The swimmeret movements are controlled by

central pattern generators in each ganglion that cause the swimmerets to beat in pairs to

produce a return and power stroke. A metachronal wave is produced in which the

swimmerets beat in the posterior to anterior direction. This metachronal wave can be

observed between adjacent ganglia as coordinated bursts created by ascending and

descending interneurons (Mulloney et al., 1993). Previous studies show that carbachol, a

cholinergic agonist, can induce the intersegmental bursting pattern in a dose-dependent

manner (Braun and Mulloney, 1993). We studied the effects of carbachol on the

frequency of intersegmental bursting and the latency between bursts of adjacent ganglia.

Extracellular recordings were simultaneously measured using pin electrodes from

the N1 roots of the third and fourth abdominal ganglia in an isolated crayfish nerve cord.

The nerve cord was washed and saturated in varying concentrations of carbachol (0µM,

20µM, 30µM, 40µM, 50µM, 60µM, 70µM, 80µM, 100µM).

There was an overall increase in intersegmental burst frequency as the

concentration of carbachol increased. The pattern activity was first observed at 30µM

concentration. The root of the fourth ganglion fired before the root of third ganglion and

was consistently coordinated. This shows the posterior to anterior movement that causes

the metachronal wave (Figure 1). The lag time between the bursts of the fourth and third

ganglion showed an overall decrease as the concentration of carbachol increased, but the

fraction of lag time did not decrease (Figure 2).

Inconsistencies in the data may be a result of using different specimens,

variability of time before activity measurements, and switching between hemiganglia N1

roots. These results agree with previous studies (Mulloney et al., 1993; Braun and

Mulloney, 1993) with regard to the effects of carbachol on intersegmental bursting.

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Figure 1: Burst pattern at 40µM carbachol. Figure 2: Fraction of lag time does not change

Top trace ganglion 3, bottom ganglion 4. with increasing carbachol concentration.

The Role of Coordinating Interneurons in the Propagation of

Swimmeret Motor Patterns in Adjacent Ganglia.

Elizabeth Snide, Jen Enman and Kerst Nelson

In the current study, the role of coordinating interneurons was examined by stimulating the

swimmeret motor pattern using carbachol and measuring the response of a neighboring ganglion’s first root

using pin electrodes. Carbachol was administered at concentrations of 10 microM, 20 microM, 50 microM

and 100 microM to the isolated fourth ganglion of a crayfish swimmeret motor system and later into the

general saline bath. The fourth ganglion was isolated in a vaseline well to ensure carbachol was only

absorbed at the desired location.

Figure 1:Carbachol was administered to the isolated fourth ganglion (red well). Swimmeret motor pattern was recorded from the first root of the adjacent second ganglion (blue well). Repeated experiment with carbachol administered in general saline bath.

Carbachol, a cholinergic agonist, acts on both

nicotinic and muscarinic acetylcholinergic receptors

allowing it to both initiate and modulate the swimmeret

motor generator pattern. If the addition of carbachol to

an isolated ganglion (AB4) produces a bursting pattern

response in the first root of the second ganglion, it can

be concluded that carbachol has an excitatory effect on

the coordinating interneurons of the crayfish

swimmeret system.

Figure 2: 50 microM carbachol was added to the fourth ganglion isolated in a vaseline well, and

triggered activity in the first root of a neighboring ganglion.

<-- Addition of carbachol

Activity recorded from first root of a neighboring ganglion

Carbachol does appear to induce the swimmeret motor pattern through ascending coordinating

interneurons. The addition of varying concentrations of carbachol (10, 20, 50 and 100 microM) to an

isolated ganglion was able to cause a response in a neighboring ganglion. This provides evidence that the

swimmeret motor pattern depends on internal regulation, neighboring ganglia influence the actions of one

another. Each ganglion is responsible for coordinating the timing and consistency of the motor pattern in its

neighbors through interneuronal mechanisms.

Recording from the first root of both the isolated ganglion and the neighboring ganglion would allow

for a better understanding of the influence of coordinating interneurons. This would allow us to examine the

time differences between the addition of carbachol and the responses at the two different ganglia. A

comparison between ascending and descending interneuronal connections could help increase understanding

of differences in internal control of the swimmeret motor pattern.

Shauna Gordon-McKeon & Joanie Davis

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Utilized for swimming, swimmerets are paired appendages located on the abdominal segments of crayfish. The crayfish swimmeret pattern controls the swimmerets to generate the rhythmic movement pattern of swimming. Muscles are necessary for the crayfish swimmeret pattern generator because they control the swimmerets. Two sets of muscles, the resturn stroke (RS) muslces and power stroke (PS) muscles, are needed for locomotion. The RS and PS muscles are innervated by four abdominal ganglia, A2-A5. From this crayfish pattern generator, a bursting pattern of alternating return and power strokes is produced, allowing locomation of the crayfish. The swimmeret pattern generator system of the crayfish is similar to most other neurological systems in that it can by modulated by neurotransmitters. By isolating the chain of abdominal ganglia from a crayfish, the bursting pattern can be pharmacologically induced. In an experiment by Braun and Mulloney (1993), it was demonstrated that the agonist peptide proctolin will induce bursting activity from an isolated crayfish nerve cord. In addition, uninterrupted proctolin activity in the presence of scopolamine, a muscarinic receptor antagonist, was demonstrated, suggesting that cholinerigic neurotransmission is not involved in the proctolin-induced swimmeret motor pattern. In our experiment, the goal was to replicate and expand on Braun and Mulloney’s observations that the proctolin-induced swimmeret motor pattern is independent of cholinergic neurotransmission. Our hypothesis was that there would be either no interaction between scopolamine and proctolin or an interaction that increased the bursting pattern of proctolin. To test this hypothesis, proctolin was used to induce the swimmeret pattern generator. Proctolin successfully generated a bursting pattern from the spontaneous baseline firing activity. Once the bursting pattern was induced by proctolin, scopolamine was co-applied to the nerve cord with proctolin. Unexpectedly, scopolaine eliminated all firing activity of the nerve cord (Figure 1), eventually including the spontaneous baseline activity. To demonstrate that the scopolamine was the reason for the elimination, scopolamine-proctolin solution was washed off of the nerve cord and proctolin was reapplied. The bursting pattern returned once the proctolin was reapplied in isolation. To verify these results, the experiment was replicated and the same results were observed. Contrary to our own hypothesis and to the results from Braun and Mulloney, scopolamine does interact with proctolin-induced bursting pattern. Therefore, it is plausible that the proctolin-induced bursting pattern is dependent upon cholinergic neurotransmission. It is important to determine the interactions of the crayfish swimmeret system because the mechanims of central pattern generators are still not understood. Therefore, the ultimate goal is to better understand central pattern generators.

Figure 1 Co-applied with proctolin, scopolamine eliminated the bursting activity of proctolin.

Observed Effects of Proctolin and Carbachol on the Crayfish Swimmeret System

Erin Watt & Elizabeth Kelly

Central pattern generators in the central nervous system responsible for producing rhythmic

movement and behavior have been studied extensively in crustaceans, including the swimmeret

system of the crayfish. On the ventral section of the crayfish abdomen, four pairs of appendages,

known as swimmerets, beat in a metachronal wave cycle from posterior to anterior (Cattaert &

Le Ray, 2000). This rhythmic motor pattern is responsible for behaviors such as swimming,

walking, burrowing and egg aeration. The central pattern generator responsible for the rhythmic

motor pattern can be studied in an isolated crayfish nerve cord. Until recently, the application of

the peptide proctolin was the only pharmacological means of stimulating this swimmeret system

in vitro. Braun and Mulloney (1993) have explored many different nicotinic and muscarinic

agonists. One compound tested in this study, the cholinergic analogue carbachol, is an agonist of

both muscarinic and nicotinic acetylcholine receptors. This compound was able to generate

bursts of firing without activating the nerve cord and it increased active nerve cord bursts in a

dose dependant manner (Braun & Mulloney, 1993).

Cholinergic agonists display a dose dependant effect not found previously with proctolin and

some are able to generate bursts of firing without the activation by proctolin, carbachol being the

most potent of these pharmacological agents. In the present study, increasing doses of proctolin

(50 *M, 100 *M) and then carbachol (10 *M, 100 *M) were applied to an isolated crayfish nerve

cord to elicit the swimmeret firing. Records were made from the N1 root of the nerve cord.

Dissection of the nerve cord to obtain recordings of the central pattern generator pattern proved

difficult. Careful dissection and de-sheathing of the nerve cord finally elicited a central pattern

generator response with 100 *M Proctolin (Figure 1). No response was observed at 50 !M

proctolin or 10!M carbachol. When 100!M carbachol was applied to the nerve cord, a large

response was observed (not shown), resembling the response of a similar preparation to nicotine.

This response was particularly interesting as both are nicotinic agonists.

It was concluded that the swimmeret motor pattern could be obtained with the application of

proctolin. De-sheathing the nerve cord drastically improved results. One problem with the

approach to this experiment was attempting to keep a rigid experimental design which did not

allow for extended periods of drug application. This may explain why no response was noted for

50 !M proctolin or 10!M carbachol. Patience and flexibility in future experimental designs

should be considered.

Figure 1: Swimmeret motor pattern consisting of the power stroke and return stroke from an

isolated nerve cord.

Frequency of Carbachol-Induced Bursting Patterns Is Concentration Dependent Megumi Sasaki, Heather Burnham and Catherine Engström

Driven by a central pattern generator, the crayfish swimmeret motor system produces a

rhythmic pattern that moves metachronously through phase-locked swimmeret pairs, posterior to

anterior. Swimmeret action assists in posture, forward motion and other motor behaviors

(Cattaert and Ray, 2001). The four swimmeret pairs, located ventrally on the second through

fifth abdominal sections, are innervated by segmental ganglia located on the ventral nerve cord.

The nerve root that projects from each ganglion, N1, divides into anterior and posterior branches.

These branches contain the axons for the return-stroke (RS) and the power-stroke (PS) neurons,

respectively.

In this experiment carbachol, an acetylcholine analogue, was applied to an isolated nerve

cord in step wise increments and resulting bursting patterns were measured extracellularly. It was

hypothesized that bursting patterns would increase in frequency when carbachol concentrations

increased. Previous studies have indicated that applications of carbachol to isolated preparations

will induce, increase and alter bursting patterns in the swimmeret motor system (Braun and

Mulloney, 1993).

Recordings in this experiment were taken from nerve root N1 on segmental ganglia 3 and

5. The isolated nerve cord was bathed in increasing concentrations of carbachol (6.25µM, 12.5

µM, 25 µM, 40 µM, 50 µM, 60 µM, 80 µM and 100 µM). Bursting patterns were measured by

means of extracellular pin electrodes with the nerve isolated in a Vaseline well. Initially, the

nerve cord was washed with saline, including one five minute soak, before changing

concentrations to check reversibility of carbachol. It was concluded that the reversibility effect of

a saline soak was only apparent in significantly longer soak times; hence, it was eliminated due

to time constraints. Identifiable bursting patterns could not be discerned during the first trial.

Only the third trial, after partial nerve cord de-sheathing, was successful in generating a bursting

pattern as seen in figure 1.

The third trial illustrates that increasing concentrations of carbachol increased the

bursting frequency. It was also observed that bursting patterns degraded into irregular firing of

spikes or absence of spikes in the presence of carbachol concentrations above 50µM. However,

the change in bursting pattern in higher concentrations may be due to possible nerve death or

coating in Vaseline. Results from trial three demonstrate increased bursting frequencies and is

consistent with the results shown by Braun and Mulloney’s 1993 experiment.

Bursting Pattern Dependency on Carbachol Concentrations

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Figure1. Increase in Carbachol concentration increases the bursting frequencies of the RS and PS motor neurons.

Identifiable bursting patterns were not evident below 12.5 µM.

Carbachol-Induced Degeneration of the Swimmeret Central Pattern Generator

Macha O’Brien, Jeanne McKeon, and Arlene Ellis

Crayfish swimmerets are bilaterally paired appendages that facilitate forward

locomotion. The swimmeret central pattern generator (CPG) is characterized by alternating

bursts of power stroke and return stroke activity in the first roots of ganglia A2-A5. The CPG

can be induced by application of carbachol, an acetylcholine (ACh) analogue. The rhythm is

activated by muscarinic ACh (mACh) receptors, while nicotinic ACh receptors (nACh) play

a modulatory role, but are incapable of inducing the pattern on their own.

In the present study, the effects of carbachol on the swimmeret CPG were examined

by recording from the first root of ganglia A3-A5 in isolated nerve cord preparations (n = 3).

Standard recording techniques with pin electrodes were used. Carbachol doses were tested in

a step-wise manner (3 µM, 10 µM, 30 µM, 50 µM, 75 µM, 100 µM), with a saline wash

between each dose. The lowest dose found to generate the swimmeret pattern was 30 µM, a

result not observable until after 5-10 minutes of treatment (Fig. 1). The frequency of the

swimmeret rhythm was increased by 50 µM carbachol, but this effect was only observed in

one specimen, with another specimen showing degeneration of the CPG. Additionally, in

two specimens, higher concentrations of carbachol (75 µM, 100 µM) were found to

degenerate the swimmeret rhythm (See figures 2 and 3).

Because carbachol acts on both nACh and mACh receptors, we propose that

degeneration of the rhythm may have resulted from a chronic state of depolarization (via

cation influx at nACh receptors) that may have led to sodium ion channel inactivation in

swimmeret motoneurons. A future experiment could investigate this possibility by

hyperpolarizing the preparation prior to application of high doses of carbachol.

Figure 3. Swimmeret pattern degeneration at higher doses of

carbachol in two separate specimens. The number of swimmeret

pattern bursts were counted from a representative 10 second trace.

Figure 1. Swimmeret pattern observed over time under the presence of 30 uM carbachol. Periods of high and

low spike frequencies were observed after the application of carbachol that were consistent with the bursting pattern of the crayfish swimmeret CPG.

Figure 2. Swimmeret pattern degenerated at higher doses of carbachol. At carbachol concentrations of 75 uM and 100 uM the swimmeret CPG began to degenerate. Above the representative trace of 100 uM shows the degeneration of the bursts.

The Combined Affects of the Acetylcholine Analogue, Carbachol, and Proctolin on

Modulation of the Swimmeret Bursting Pattern in the Crayfish

Leslie Christensen, Marissa Simms and Jennifer Smar

Swimming behaviour in the crayfish is modulated by the swimmeret motor system.

Swimmerets exist in four bilateral pairs along the 2nd, 3rd, 4th and 5th abdominal

segments; each abdominal segment is innervated by root N1 motor neurons (Ia, Ib). N1

drives swimmeret movement. Swimmeret pairs move synchronously from the posterior

to anterior segments. The posterior swimmeret pair begins the swimmeret motor cycle

with a power-stroke which propagates the crayfish forward followed by a smooth return

stroke. A slight phase delay between swimmeret pair movement creates a metachronal

cycle, or wave-like movement. Swimmeret motor neuron burst frequency in the resting

crayfish swimmeret system is evoked by individual applications of the peptide proctolin

within a narrow concentration range (Mulloney et al., 1987) as well as proctolin and the

acetylcholine agonist carbachol in combined graded concentrations (1 !M- 10 !M)

(Braun and Mulloney, 1993).

The purpose of this investigation was to examine the effects of proctolin, as well as

the combination of proctolin and carbachol, on motor neuron burst frequency modulation

in the crayfish swimmeret system.

Recordings were made from N1 of the 2nd abdominal segment of an isolated crayfish

nerve cord. Extracellular recording techniques, with electrodes placed inside and outside

a Vaseline well, were employed; chart recording speed was set at 25 mm/s. Nerve cords

were incubated with proctolin (50 !M), as well as proctolin (50 !M) combined with

carbachol mixtures (50!M). The nerve cord was incubated with saline washes in between

proctolin and carbachol applications.

Spontaneous activity was elicited from the nerve cord in Saline. Application of

proctolin induced a regular swimmeret motor pattern (0.9425 bursts/s) (Fig. 1). Proctolin

and carbachol combined produced an irregular swimmeret motor pattern (2.6675

bursts/s).

Proctolin alone elicited a more regular and robust swimmeret motor pattern than did

proctolin and carbachol combined; these results support existing evidence that proctolin

elicits a regular swimmeret motor pattern in the crayfish (Braun and Mulloney, 1993).

The proctolin and carbachol combination, however, produced a higher bursting frequency

than did proctolin alone, supporting the notion that proctolin and carbachol each act on

distinct pathways in the crayfish swimmeret system.

Figure 1. Swimmeret bursting pattern elicited by 50 !M proctolin.

The Addition of Carbachol Induces Swimmeret Patterns

Selam Nida Amelia Keller Samri Gebre

The crayfish swimmeret motor system is involved in various behaviors, including

locomotion and burrowing. Each beat of the swimmeret involves a forward stroke, called a

return-stroke, and the backward stroke, called the power-stroke. The swimmerets are arranged in

bilateral pairs on the second, third, fourth and fifth abdominal segments (Cattaert and Le Ray,

2001). Each pair beats after the one behind it has beaten, so that all together they beat in a

pattern called a metachronal wave. A central pattern generator in the abdominal ganglion

controls the rhythmic activity of the swimmeret. Previous experiments have shown stimulation

and modulation of the swimmeret pattern by cholinergic neurotransmitters and their analogues

such as carbachol. In this experiment, we attempted to find out if carbachol, acytocholine

agonist, elicited a swimmeret pattern. Different concentrations of carbachol were used to

examine increase in burst frequency with increase in concentration.

We began by dissecting the nerve cord out of the abdominal segment of the crayfish. We

recorded from the first root of the second ganglion using an extracellular electrode. The baseline

pattern of spontaneous activity was recorded while the nerve cord was in saline solution. We

then applied incrementally increasing concentrations of carbachol to the nerve cord and recorded

the results.

Unfortunately, at concentrations of 2, 10, 50 and 80 µM of carbachol, we were unable to

observe any swimmeret pattern. We observed only spontaneous non-rhythmic activity.

Therefore, we were unable to observe the dose dependent stimulation of the swimmeret by

carbachol. For the 100 µM preparation, we desheathed the nerve cord to make it absorb the

carbachol more effectively. In this preparation, we observed a pattern that was suggestive of the

swimmeret pattern.

Fig.1.Baseline spontaneous activity Fig. 2. Swimmeret pattern with 100µM carbachol

From the above results, we have concluded that acytocholinergic neurotransmitters are

involved in stimulating the motor neurons. However, since only one of our carbachol

concentrations was effective, we were not able to conclude the effective ranges of carbachol

concentration to activate the swimmeret system. We believe for future experiments, the nerve

cord should be desheathed for each trial to get effective results.

The Effect of Increasing Carbachol Concentration on the Crayfish Swimmeret

Motor Pattern

The crayfish swimmeret system is composed of four pairs of limbs that extend on the

ventral side of each abdominal segment in the crayfish (Mulloney et al., 1993). In nature,

these swimmerets allow the crayfish to achieve a variety of motor behaviors including:

burrowing, walking, egg aeration in females, as well as defensive actions and postures

(Cattaert et al., 2001). These motor behaviors are triggered and modulated through

specific patterns of neuronal activity; this activity can be induced in laboratory conditions

by adding cholinergic agonists and analogues

The purpose of our experiment was to investigate the effects of varying concentrations of

the acetylcholine analogue, carbachol, on the crayfish swimmeret bursting patterns.

Using suction electrodes and standard physiological techniques we measured the bursting

rate and pattern from the first nerve root of the second abdominal ganglia after exposure

to increasing concentrations of carbachol (saline, 25 !M, 50 !M, and 75 !M). Between

each concentration increase the preparation was washed twice in saline to establish the

change in activity as a direct result of the carbachol. The return of the spontaneous firing

activity to near baseline demonstrated that what we observed was a drug effect.

Previous literature has shown that increased concentrations of carbachol leads to

increased burst frequency and changes to burst shape in the swimmeret motor pattern

(Braun and Mulloney 1993). Additionally, this response becomes saturated at around 50

!M carbachol (Mulloney 1997). In our experiments, we observed increased burst

frequency during the application of carbachol but did not notice any saturation or

concentration dependency. At 25 !M, we observed two prolonged bursts, interrupted by

a period of fast-swimming bursts. At 50 !M, we again observed the two prolonged

bursts, but this time they were not interrupted by the fast swimming pattern. At our final

concentration of 75 !M we observed two periods of the prolonged bursts followed by an

irregular fast-swimming pattern. We do not know whether or not the pattern of two

prolonged bursts after the carbachol application is real or coincidental.

After each increasing concentration of carbachol, the baseline spontaneous firing

observed during the saline washes slowed. This effect was also noticed by Mulloney,

who attributed it to the high concentrations of carbachol depolarizing the motor neurons,

effectively inactivating them until they repolarize. Another possible contributing factor is

that the preparation could have been reacting to the stress of the experiment.

To improve upon this experiment in the future, students might consider removing the

nerve sheath in hopes that the preparation will respond to lower concentrations of

carbachol. Additionally, the order in which the different concentrations were added

could be reversed from week to week in order to see if stress was actually a factor.

Authors: Virginia Frontiero, Grace Pokela, and Mar Todd-Brown

Effects of Proctolin & Carbachol on Motor Unit Recruitment

Danielle Ricciardi, Emily Tyner, & Kaitlyn Webster

The crayfish swimmeret system has been the focus of extensive research from which it was

discovered that swimmeret movement is controlled using a central pattern generator (CPG).

Command neurons, interneurons, and motor neurons contribute to the CPG. Command neurons

initiate activity, motor neurons drive the actual movement, and coordinating interneurons (true to

their name) coordinate the modules and the system as a whole. By isolating the crayfish nerve cord,

scientists have been able to eliminate the effects of sensory feedback. Several experiments have

observed the various effects of different cholinergic agonists on initiation and modulation of the

swimmeret motor pattern (Braun & Mulloney, 1993).

The objective of this experiment was to determine whether a combination of proctolin plus

carbachol would recruit more motor units in the crayfish swimmeret system than either proctolin or

carbachol alone. Previous research shows that proctolin, pilocarpine, and carbachol elicit the

swimmeret pattern. A combination of proctolin and pilocarpine recruits more motor units than

either drug alone (Braun & Mulloney, 1993). Therefore, it was logical to propose that proctolin plus

another muscarinic agonist would result in additional recruitment of motor neurons.

Adapting the procedure from previous experiments (Braun & Mulloney, 1993), we utilized standard

extracellular recording techniques of the crayfish nerve cord. The nerve cords were submerged in

the following concentrations/combinations of drug(s) at different times, with the ganglion sheath

intact unless otherwise noted: 100!M proctolin; 50!M carbachol, ganglion unsheathed; 50!M

carbachol; 60!M carbachol; 20!M carbachol + 20!M proctolin; 20!M proctolin + 40!M

carbachol.

Since experiments never elicited sustained swimmeret patterns, the effects of proctolin versus

carbachol on motor unit recruitment could not be observed. However, variations in non-rhythmic

firing for sheathed and unsheathed ganglia were observed. Carbachol (50!M) elicited non-rhythmic

firing from the unsheathed third ganglion. We hypothesize that for an unsheathed ganglion, 50M is

too high a concentration for direct contact. Other research shows (Braun & Malloney, 1993) that

high concentrations of nicotine have an inhibitory effect on firing. A similar event could have

occurred in our experiment. The combinations of 20!M carbachol + 20!M proctolin, and 40!M

carbachol + 20!M proctolin did not elicit the swimmeret pattern, and results were further obscured

by electrical noise. Switching to N1 of the second sheathed ganglion and adding 50!M carbachol

generated a short but unsustained pattern. This suggests that 50!M carbachol can initiate patterns

in sheathed ganglia but may be too strong a concentration for unsheathed ganglia.

It is possible that we may have damaged the ganglion when removing the sheath, inhibiting drug

uptake. Also, reducing background noise would eliminate interfering signals. Finally, a better

understanding of agonist concentrations and effects on sheathed versus unsheathed ganglia, in

addition to further research on agonists interactions is recommend in further experimentation.

Aida Manu, Jessica Bossé, and Courtney Liebling BIO 331: Neurophysiology Lab

Evaluation of Spike Histogram Module for Analyzing Spike Recordings from Crayfish

Crayfish have paired limbs (swimmerets) on their abdomens that are capable of

propelling the segmented animals through the water. Crayfish have four pairs of

swimmerets, and each swimmeret is innervated by approximately 85 motor neurons that

are driven by a local central pattern-generating circuit. One way to study how the central

pattern generator might work is to compare the effects of different neurotransmitters and

modulators on the swimmeret motor system. The cholinergic analogue carbachol, which

acts on both nicotinic and muscarinic receptors, was applied to the isolated nerve cord. A

concentration of 50µl carbachol elicited the swimmeret pattern, and recordings were

taken from the first root of the third ganglion of the crayfish nerve cord. Spontaneous

activity was also recorded.

The Spike Histogram Module is a software add-on for Chart (a recording

program) that adds the capability to discriminate and analyze extracellular neural spike

recordings. The purpose of this project was to use recordings of the crayfish swimmeret

system to explore the functions of Spike Histogram and provide insight as to its possible

use in the BIO 331 Lab section. The program was able to discern three distinct spike

populations in the recording of spontaneous activity. Amplitude histograms, interspike

interval histograms, and rate meter histograms were made for each population. Because

the swimmeret pattern was recorded with different settings, it was difficult to analyze the

carbachol-elicited recording.

The Spike Histogram software would be an effective program for use in the BIO

331 Lab. Using this software, it is possible to obtain more informative data from the

experiments, for example, the number of spikes in the discriminated populations. The

software also provides students with more control over the display of their recordings,

enhancing their understanding of the different components of their data.

The main disadvantage of using this software in lab is that students will require a

lot of lab time to familiarize themselves with all of its features and to solve technical

difficulties that they may face. Time-consuming problems were encountered during this

test run of the software, such as figuring out whether or not inverting the data was

necessary, and setting appropriate detection thresholds. Having to invert data may not be

desirable in some experiments. However, this can be resolved by spending more time

becoming familiar with the software and providing the students with a simplified manual

based on the checklist that was produced in this lab. This would allow them to

concentrate on the neurophysiology concepts that they are interested in, and not worry

about the technicalities of the software. The software could also be introduced early in

the semester, so students can become familiar with it by the time they are ready to

undertake their final lab projects. Although the software is costly, its use in lab is

recommended because the students will be more efficient at displaying and working with

the results of their recordings.

Neha Bhargava

Annie Tanenhaus

Sara Royston

May 2, 2006

Abstract: The Spike Histogram Software

In the context of this undergraduate neurophysiology lab, the recording and

analysis of neural activity is essential in conveying an understanding of the mechanisms

involved in producing such activity. This recording is especially important in analyzing

the crayfish swimmeret system, in which identifying patterns of neural activity are

important to making generalizations about the system, which is one of the ultimate goals

of the independent projects. Traditionally, the oscilloscope is used in recording such

data. However, this current form of recording only allows us to analyze oscilloscope data

through direct observations of the recording as a printed record, or through Sound Studio.

Ideally, we would use software which could discriminate information from the recordings

that would not be immediately obvious by simply viewing the recordings.

We examined the Spike Histogram Software as a candidate for a possible new

method of analyzing crayfish swimmeret recordings. The Spike Histogram Module,

which functions within the Chart program, allows analysis of both individual spikes, and

larger patterns of spikes obtained through extracellular recording techniques.

There are several advantages to using this software. First, Spike Histogram

provides a powerful tool in classifying spikes based on characteristics such as amplitude

and time, and allows for easy visualization of populations of similar spikes through the

“Discriminator” function. The program, through the “Amplitude Histogram” also

condenses information about the distribution of spike amplitudes by organizing them in a

histogram format. Information about the frequency of spikes can also be easily

quantified with the “Interspike Interval” histogram. Furthermore, information about he

rhythmicity of the spike can be visualized with the “Autocorrelation” function, which

creates an autocorrelogram.

Though the program is able to perform a number of powerful and abstract

functions, problems in practicality and applicability exist. For example, the amount of

time that it takes for one to familiarize themselves with the software is considerable.

Furthermore, a number of the advanced features of the program would only make it more

difficult to grasp the concepts that the class is trying to convey. Though adopting the

software would provide the advantage of a powerful tool for data analysis, the purposes

of this lab class would not justify the use of this software.