NEW NEUROPHYSIOLOGY LABORTORY COURSE TO START AT UK SPRING 2013
R.L. Cooper , J. Titlow, Dept of Biology, Univ. of Kentucky;
Z. R. Majeed, Dept .of Biology, Univ. of Kentucky & Dept .of Biology, College of Sci, Univ. of Salahaddin, Erbil, Iraq
Overview of course:
Extracellular experiments:
Short term facilitation at the NMJ :
Joint proprioceptive organ in a walking leg
Anatomical
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Fatt and Katz in 1951 showed that acetylcholine was the transmitter
that depolarized the frog motor endplate and that voltages would
summate.
The undergraduate students are to be exposed to the marvels
of Neuroscience and to experience the future of the field by
learning "Hands-On" neurophysiology, analysis, and
neuropharmacology in a laboratory based atmosphere.
Relative amplitude
Introduction
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Both the frog and crustacean NMJs were the models of choice for
investigating the frontiers of facilitation for many years. This was most
likely due to the robust nature of these experimental preparations.
Goal of course:
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The goal is to train future students in various science
disciplines to the integrative nature of science so that they can
better prepare themselves with the appropriate training during
the remaining years of undergraduate schooling and help to
direct their efforts and thus competitiveness towards particular
graduate programs.
By the end of this course, one should:
1. Have a conceptual understanding of the information processing in
the nervous system.
2. Understand the molecular mechanisms that enable signal
transmission in the nervous system in terms of receptor potentials,
synaptic potentials and action potentials.
3. Know the cellular specializations and the molecular machinery
involved in neuron-neuron communication at the state of the art level.
4. Develop a basic knowledge of sensory processing.
5. Be able to understand and critically analyze research papers in the
field of Neuroscience.
6. Be able to develop new ideas and suggest future research directions
in the field of Neuroscience.
A typical intracellular response of STF in the opener muscle of the
crayfish walking leg is shown here:
Recordings can be made with extracelluar electrodes. The signals from
the cut nerve endings will be small in amplitude. To be able to visualize
the signals, one needs to amplify the electrical response with either a
preamplifier and/or the gain on the oscilloscope. The preamplifier can be
set at a gain of 1,000 times. If audio amplifiers are available, they can
be used to help hear the signals. The suction electrodes consists of a
syringe with a hypodermic needle attached, a replaceable tip made of
polyethylene tubing, and two insulated silver wires.
Methylene blue of motor nerve
terminals for the superficial flexor
muscles of the crayfish
Tonic and phasic NMJs :
Crayfish muscle
Anatomy
Outline of course:
Week 3. Measure facilitation and depression in tonic and phasic
neuromuscular junctions in crayfish abdomen muscles.
Week 4. Learn to record from proprioceptors (extracellular) in the
crab leg and relate to joint positions.
Week 5. Learn to record from tension receptors in the crab leg related
to muscle length and force.
Week 6. Learn how to forward fill neurons from the crab leg
proprioceptors (CoCl2, 4-Di-2 ASP) as well as stain with methylene
blue.
Intracellular experiments:
Measuring membrane potentials in crayfish muscle fibers:
The students will learn how to properly record the potential across
a membrane, with glass electrodes, in the DEL1 and DEL2
muscles in a crayfish. The students furthered their investigation of
membrane potentials by determining the effects of increased
extracellular K+ levels. Using several solutions of increasing K+
levels, the cells were covered and allowed to soak for 5 min. The
resting membrane potential was then recorded again. The
students will graph their values for the resting potential against
the log potassium concentration and compare these values to the
theoretical values determined by using the Nernst equation.
EPSP
mEPSP
Leech ganglion preparation :
Leech Ganglion- identified neuron cell body
Changing Extracellular K+
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Rp (mV)
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•A square impulse will be applied to the
Rz cell.
•The initial hyperpolarization and final
depolarization indicates the capacitance
artifact.
•The artifact hyperpolarization and
depolarization also indicate the length of
the square impulse applied to the Rz
cell.
•Length of square impulse stimulus was
approximately 300 ms.
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Week 8. Mapping skin receptive fields on the leech while recording
from neurons. Dye fills.
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[K+]o
Experimental
Theoretical
Week 9. Learn how to remove and culture leech neurons for forming
synapses in culture.
Week 10. Vision: crayfish & fruit fly eyes and caudal photo receptor
in crayfish.
Week 11. Quantal analysis of synaptic transmission: Crayfish NMJ
record quantal responses.
Week 12. Plot I V curves and use Ohms law to determine Rm:
Crayfish skeletal muscle. Pharmacology of glutamate receptors
Week 13. Student presentations.
Intracellular approach
Quantal Analysis
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Week 7. Learn how to dissect the leech ventral nerve cord and obtain
intracellular recordings from identified neurons. Current injections
and threshold measures. Potentially two intracellular electrodes and
record in situ synaptic connections. Investigate the ionic currents
making up the action potentials.
Muscle tension and tension receptors
from a crab leg
Lucifer yellow staining of a Rz and P cell in the ganglion
by intracellular pressure injection.
Week 1. Learn about equipment (extracellular & intracellular amps,
microscopes, electrode puller). Solutions and laboratory tools.
Animal care. Lab notebooks & reports.
Week 2. Measure membrane potentials in crayfish abdomen muscles
and plot Rp vs [K]o graphs. Also learn how to stimulate motor nerves
and record EPSPs/IPSPs.
One can see individual axons to correlate to the different size
extracellular spikes that are recorded. Also a goal can be to
correlate innervation patterns on the muscle for the different types of
terminals and the physiological responses.
These are traces of individual spontaneous events
that can be recorded and used for measures. Students will compared
these types of traces to examine if subsets of events are present. The
purpose is to look for quantal events that fall into distinct groupings.
Measuring synaptic potentials in crayfish muscle fibers:
Record excitatory and inhibitory junctional potentials (EJP's and IJP's) will be a goal
fro the students. Recording action potentials extracellularly from the superficial
branch of the third root using a fine-tipped suction electrode applied to the side of
the nerve, and match different sized spikes in the nerve with junctional potentials in
the muscle fibers is another goal. By penetrating several muscle fibers, one can
see that not all fibers are innervated by all the efferent neurons, and that the same
neuron may elicit different-sized junctional potentials in different fibers. Make a map
to show distribution of the EJP's and IJP's in the muscle. We also induced reflex
firing of some motor neurons by stroking the sides of the abdomen with a fine
brush.
Leech ganglion shown
below the patch of skin.
One can map receptive
fields on the skin while
recording intracellularly
from different sensory
neurons.
Synaptic field potentials can be measured with focal
macropatch electrodes to assess presynaptic vesicular
events. The synaptic potentials can be obtained using the
loose patch technique by lightly placing a 10-20 m firepolished glass electrode directly over various regions on a
muscle fiber. The evoked field excitatory postsynaptic
potentials (fEPSPs) and field miniature excitatory
postsynaptic potentials (fmEPSPs) can be recorded and
analyzed to determine the mean quantal content (m).
Direct counts of the number of evoked quantal events and
failures in evoked release are to be used as an index of
altering synaptic function.
In addition to the direct quantal counts, the area of the
evoked and spontaneous events will be measured over
time in each preparation for comparison. The area of the
evoked and spontaneous events will be determined by the
Simpson's method.
One can record extracellular
spikes from the tension nerve
while monitoring the
development of force for direct
correlations
Computational
Swimmy is a virtual neural circuit simulator designed by Frank
Krasne and his colleagues at UCLA. The free software and
supporting materials are part of an inquiry-based project that
covers basic neurophysiological principles. Students learn how
central pattern generators function and correlate neural circuit
physiology to animal behavior. Cells in the circuit are silenced
or activated by simulating current injections. Then based on
activity at the cellular level (below) and the behavioral level
(above) students derive the circuit’s connectivity.