physiological and anatomical assessment of synapses at the crayfish neuromuscular junction

207 Pages·2016·3.88 MB·English

UUnniivveerrssiittyy ooff KKeennttuucckkyy
UUKKnnoowwlleeddggee
University of Kentucky Doctoral Dissertations Graduate School
2006
PPHHYYSSIIOOLLOOGGIICCAALL AANNDD AANNAATTOOMMIICCAALL AASSSSEESSSSMMEENNTT OOFF
SSYYNNAAPPSSEESS AATT TTHHEE CCRRAAYYFFIISSHH NNEEUURROOMMUUSSCCUULLAARR JJUUNNCCTTIIOONN
Andrew Fredericks Moser Johnstone
University of Kentucky, [email protected]
RRiigghhtt cclliicckk ttoo ooppeenn aa ffeeeeddbbaacckk ffoorrmm iinn aa nneeww ttaabb ttoo lleett uuss kknnooww hhooww tthhiiss ddooccuummeenntt bbeenneefifittss yyoouu..
RReeccoommmmeennddeedd CCiittaattiioonn
Johnstone, Andrew Fredericks Moser, "PHYSIOLOGICAL AND ANATOMICAL ASSESSMENT OF SYNAPSES
AT THE CRAYFISH NEUROMUSCULAR JUNCTION" (2006). University of Kentucky Doctoral Dissertations.
274.
https://uknowledge.uky.edu/gradschool_diss/274
This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been
accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of
UKnowledge. For more information, please contact [email protected].
ABSTRACT OF DISSERTATION

Andrew Fredericks Moser Johnstone

The Graduate School

University of Kentucky

2006
PHYSIOLOGICAL AND ANATOMICAL ASSESSMENT OF
SYNAPSES AT THE CRAYFISH NEUROMUSCULAR JUNCTION

__________________________________________

ABSTRACT OF DISSERTATION
_____________________________________

A dissertation submitted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in the
College of Arts and Sciences at the
University of Kentucky

By
Andrew Fredericks Moser Johnstone

Lexington, Kentucky

Director: Dr. Robin Lewis Cooper, Associate Professor of Biology

Lexington, Kentucky

2006

Copyright © Andrew Fredericks Moser Johnstone 2006
ABSTRACT OF DISSERTATION

PHYSIOLOGICAL AND ANATOMICAL ASSESMENT OF SYNAPSES AT THE
CRAYFISH NEUROMUSCULAR JUNCTION

The crayfish, Procambarus clarkii, has a multitude of ideal sites in which
synaptic transmission may be studied. Its opener muscle, being innervated by a
single excitatory neuron is a good model for studying the structure/function of
neuromuscular junctions since the preparation is identifiable from animal to
animal and the nerve terminals are visible using a vital dye. This allows ease in
finding a suitable site to record from in each preparation and offers the ability to
relocate it anatomically. Marking a recorded site and rebuilding it through
electron microscopy gives good detail of synaptic struture for assesment.
In the first of these studies, low output sites known as stems (which lie
between varicosities) were used to reduce n (number of release sites) in order to
minimize synaptic complexity so individual quantal events could be analyzed by
their unique parameters (area, peak, tau, rise time and latency). This was in
attempt to uncover specific quantal signatures that could be traced back to the
structure of the area recorded. It was found that even at stem regions synaptic
structure is still complex having multiple synapses each of which could harbor a
number of AZs. This gives insight as to how quantal analysis should be treated.
Even low output synapses n must be treated at the AZ level.
Synaptic depression was studied at the crayfish extensor muscle. By
depressing the phasic neuron and recording from the muscle it appears that
depression is a presynaptic phenomenon. The use of 5-HT gave insight to
vesicular dynamics within the nerve terminal, by delaying depression and
increasing maximum EPSP amplitude. TEM of phasic nerve terminals reveals no
change in numbers of dock or RRP vesicles. Short term facilitation and vesicular
dynamics were studied with the use of 5-HT and a neurotoxin TBOA, which
blocks the glutamate transporter. In this study I showed differential mechanisms
that control RRP and RP vesicles. By blocking glutamate reuptake, the RRP is
depleted as shown by reduced EPSPs, but recovered with 5-HT application. The
understanding of vesicle dynamics in any system has relevance for all chemical
synapses.

KEY WORDS: Active zones, quantal analysis, synaptic
ultrastructure, synaptic plasticity.

Andrew F. M. Johnstone

Dr. Robin L. Cooper (Advisor)

Dr. Brian C. Rymond (Director of Graduate Studies)
PHYSIOLOGICAL AND ANATOMICAL ASSESSMENT OF
SYNAPSES AT THE CRAYFISH NEUROMUSCULAR JUNCTION

By

Andrew Fredericks Moser Johnstone

Dr. Robin L. Cooper
Director or Dissertation

Dr. Brian C. Rymond
Director of Graduate Studies

November 30, 2006
RULES FOR THE USE OF DISSERTATIONS

Unpublished dissertations submitted for the Doctor's degree and deposited
in the University of Kentucky Library are as a rule open for inspection, but
are to be used only with due regard to the rights of the authors.

Bibliographical references may be noted, but quotations or summaries of
parts may be published only with the permission of the author, and with the
usual scholarly acknowledgments.

Extensive copying or publication of the dissertation in whole or in part also
requires the consent of the Dean of the Graduate School of the University
of Kentucky.

A library that borrows this dissertation for use by its patrons is expected to
secure the signature of each user.
DISSERTATION

Andrew Fredericks Moser Johnstone

The Graduate School

University of Kentucky

2006
PHYSIOLOGICAL AND ANATOMICAL ASSESSMENT OF
SYNAPSES AT THE CRAYFISH NEUROMUSCULAR JUNCTION

__________________________________________

DISSERTATION
__________________________________________

A dissertation submitted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in the
College of Arts and Sciences at the
University of Kentucky

By
Andrew Fredericks Moser Johnstone

Lexington, Kentucky

Director: Dr. Robin Lewis Cooper, Associate Professor

Lexington, Kentucky

2006

Copyright © Andrew Fredericks Moser Johnstone 2006
ACKNOWLEDGEMENTS

I acknowledge all those that helped in making this dissertation possible.
First and foremost I thank my family for their love and support through this
process, especially my wife Jessica, for her love and making sure I stayed on
track. My parents, Sandy and John, who were nothing but encouraging and to
my grandmother, Mary Ann (Pete), who was caring enough to edit my work.
In addition, I would like to thank my advisor, Robin L. Cooper, for his
guidance and patience for the last four years. Without him, my skills and
knowledge of this field would not be possible. Also, thanks to my committee
members who were willing to give their time to help guide me to the completion
of this dissertation. Also, I am grateful for the guidance that Mary-Gayle Engle
gave me through my electron microscopy training, her constant help allowed me
to have the skills to make the projects possible.
I also, must acknowledge and thank my graduate lab mates, Sameera
Dasari, Mohati Desai and Sonya Bierbower, who were always available for
advice and/or moral support for which I am indebted to. Thanks also to the
numerous undergraduates in the lab who were a constant source of
entertainment for the last four years. Special thanks to an undergraduate at the
time, Stephanie Logsdon, who I assisted in her Beckman scholar project, which
is part of this dissertation.
Also, to all the co investigators to these projects, Dr. Kert Viele and Mark
Lancaster, whose statistical advice and work were invaluable to these projects.
iii