|Back to General Research|
|Back to Home Page||For the lastest movie clips of research projects (go to)|
(click on topic to jump to section on this page)
|Integration of Sensory|
Whole Animal Perception
Muscle Receptor Organ (Proprioception)
|Whole Animal Projets|
|General Behavior ..... Physiological Responses...... Regulation of Behavior|
1. General Behavior:
There are various subprojects that are being tactled in this theme of behavior. Here we just mention projects strictly focused on observational behavior.
ABSTRACT -This study examines behaviour of cave-adapted blind crayfish to novel territories of various sizes. Cave crayfish, Orconectes australis packardi , were kept in a dark room within individual aquaria for 7 days. Crayfish are then placed into a small tank (33 X 28 X 23 cm, 10-15 cm water depth), and a large tank (54 x 37 x 30 cm, 10-15 cm water depth). Time, distance of movements, and length of pauses are recorded. Upon initial placement in a new setting, crayfish will walk around the perimeter where their antenna can contact the side of the tank. The animals will subsequently move around the tank away from the perimeter, or they will begin to dig a burrow. Familiarity within the environment for an individual crayfish is defined as when the animal initiates digging or remains in one place for over 5 minutes. So far this study demonstrates that the time required to become familiar to a new setting depends on the size of the setting. This suggests that a balance between sensory input and inner processing, termed 'familiarity' can be reached.
ABSTRACT- The purpose of this study is to investigate the repertoire of social behaviors that cave adapted blind crayfish have as compared to those previously reported with interacting sighted crayfish. Since cave crayfish display phototactic behavior, presumably mediated by the caudal photoreceptors in the 6th abdominal ganglion, we test if light, an environmental disturbance for the cave crayfish, alters the normal social behaviors. Observations made in order to quantify social interactions are carried out under infrared or dim, white light. The results reveal that following the paring of same sized individuals, previously housed in isolated conditions for 2 weeks, both tended to tail flip or rapidly escape from each other upon initial antennae contact. This behavior replicates the observations made within the natural cave environment. The rapid tail flip behavior becomes less frequent over time following repetitive interactions. Under white light, many behavioral acts shifted from socially contacting to no contact or retreating. The blind crayfish did not exhibit behaviors usually associated with visual displays and posturing (i.e., the raised meral spread was absent). Ethograms are being constructed of the behavioral repertoires used in the social interactions between aggressive and submissive partners.
ABSTRACT- We examine the probability of the crayfish, P. clarkii, to tail flip in response to a touch on the dorsal tail fan related to the size and the behavioral state of the animal. Alterations in the animal's internal physical state, such as when the animal autotomizes its chelipeds, will cause the larger sized animals to tail flip, when previously they would not. Altering the external environment by removal of water, causes small crayfish, which normally habituate slowly, to rapidly habituate. Observation of large adult crayfish in a species that is adapted to live in total cave darkness, O. australis packardi, revealed that they are more likely to tail flip than are the sighted large P. clarkii. Results indicate that the behavioral state of the crayfish can result in rapid and long term alterations in the tail flip response and in habituation rates to repetitive stimuli. This ability to show plasticity in gain setting may be regulated by neuromodulators and can occur in large adults of the sighted crayfish. Differences between the two species indicate that size may not be the sole contributing factor to account for tail flip behaviors.
|To View More Cave Critters|
Agonistic encounter between two crayfish
O. australis packardi
2. Physiological Responses:
ABSTRACT- Most animals assess the environment in which they live and alter their behavior according to various stimuli. As an observer, one looks for changes in the behavior of the animal which indicates if an animal is responsive to an event. When the animal does not make significant behavioral changes as measured by bodily movements, the animal may be characterized as unresponsive to a given stimulus. This study demonstrates that when behavioral movements of crayfish cannot be observed, physiological measures of heart rate (HR) and ventilatory rate (VR) show dramatic changes following presentation of a defined sensory stimuli. In the majority of cases, upon anticipating a social interaction with another crayfish both HR and VR will increase. During an agonistic encounter between two crayfish, the level of HR and VR correlate to the intensity of the interaction. Such rapid responses in cardiac and respiratory systems to environmental disturbances and anticipation of a social interaction suggest an autonomic-like regulation commonly associated with mammals. Since behavioral observations do not allow an internal status to be assessed, it is suggested that HR and VR may serve as useful bioindices in crustaceans to their internal drive or possibly an awareness level to environmental cues.
[This project is in colaboration with Dr. Thomas Breithaupt (Fakultät für Biologie, Universität Konstanz, Konstanz, Germany), Dr. Zhanna Shuranova (Inst. of Higher Nervous Activity and Neurophysiology, Moscow, Russia), and Dr. Yuri Burmistrov (Inst. for Information Transmission Problems, Moscow, Russia)].
ABSTRACT- The activity of visual systems is known to affect development of the neural tissue associated with vision in both vertebrates and invertebrates. Three species of crayfish were compared for variations in the gross structures of the eye, and of the underlying neural tissue of the optic system associated with environmental adaptation. The troglobitic crayfish, Orconectes australis packardi Rhoades, and two epigean crayfish, Cambarus tenebrosus and Procambarus clarkii, were used. C. tenebrosus raised in the cave are functionally blind although ommatidia do develop, indicating that the primary sensory structures still develop without normal input. Troglobitic crayfish have lost the genomic ability to form a functional visual system. Electrophysiological records from neurons within the optic stalk of O. australis packardi showed no response to light. The neuronal ganglia within the eye stalk of C. tenebrosus is disorganized which could be the reason for the lack of a behavioral response related to sight. Second order neurons associated with olfaction arise in the central brain and send processes to lobula within the eye stalk via the protocerebral tract. Cross sections of this tract revealed that the troglobitic crayfish have more olfactory projection neurons and fewer large axon profiles than the other two crayfish, suggesting that O. australis packardi has more neural processing devoted to olfaction, as an adaptation to cave life.
3. Regulation of Behavior:
ABSTRACT- In vertebrates the regulation of internal organs is primarily driven by the autonomic nervous system. In addition whole body status to environmental cues tune the body to a 'fight or flight' mode or to a relaxed state which are both controlled by sympathetic and para sympathetic components of the autonomic nervous system. Traditionally, many internal organs in crustaceans are treated as "autonomic" ones able to function a long time after partial or even complete isolation from the CNS (the best examples are the cardiac ganglion and the stomatogastric ganglion). Recent evidence suggests however that an ample and sophisticated neural control of these internal organs is typical for freely behaving crustaceans. To prepare for rapid escape or confrontation, or maintenance of a resting state during a relaxed period, these animals appear to use certain neural structures which can be compared to an autonomic nervous system, which is best described in higher vertebrates. The purpose of this review is to draw upon the similarities and examine evidence for an analog to the autonomic nervous system in the higher invertebrates - the crayfish and other decapod crustaceans. The conclusion was made that an obvious functional similarity takes place in both vertebrates and crustaceans. The existence of intrinsic nerve nets inside the heart and digestive system as well as an inhibitory or acceleratory extrinsic control of their functions by specialized neurotransmitters is one common feature. Additionally, the release of neuromodulators and neurohormones into the circulation is observed in invertebrates mimicking that of the autonomic, sympatoadrenal system of vertebrates.
[This project is in colaboration with Dr. Zhanna Shuranova (Inst. of Higher Nervous Activity and Neurophysiology, Moscow, Russia), and Dr. Yuri Burmistrov (Inst. for Information Transmission Problems, Moscow, Russia)].
|Synaptic Differentiation...Neuromodulation...Plasticity...Muscle Phenotype/Regeneration|
1. Synaptic Differentiation:
ABSTRACT- In the crayfish opener neuromuscular preparation, regional differences in synaptic transmission are observed among the terminals of a single motor neuron. With a single stimulus, the high-output terminals of the proximal region of the muscle produce a larger excitatory post-synaptic potential than the low-output terminals of the central region of the muscle. We tested the hypothesis is that the low-output terminals exhibit more facilitation than high-output terminals for twin pulse, train, and continuous stimulation paradigms. Previous studies have not employed several stimulation paradigms to induce facilitation among high- and low-output terminals of a single motor neuron. We found that the high-output terminals on the proximal fibers facilitate more than the low-output terminals on the central muscle fibers, in contrast with previous studies on similar muscles. The difference in measured facilitation is dependent on the stimulation paradigm. These results are important since ultrastructural differences between these high- and low-output terminals are known and can be used to correlate to physiological measures. Short term facilitation is a form of short term memory at the synaptic level and the processes understood at the crayfish neuromuscular junction may well be applicable to all chemical synapses.
The long-term objective is to first determine if acute application of neuromodulators effect synaptic sites differently, depending on the neurons intrinsic differences in synaptic efficacy and secondly to determine the long-term physiological and anatomical effects neuromodulators have on these distinct synaptic junctions. The nerve cell is chemically integrated with other cells at morphologically-identified locations called synapses. The characteristics of the synapse are affected by its activity level which modulates long-term alterations in both structure and performance. Neuromodulators that are endogenously released either enhance or suppress synaptic efficacy, in turn affecting the behavioral state of an animal. Neuromodulators can affect cellular process by various intracellular messenger cascades that are dependent on the type of neuromodulator and its associated receptor. Such changes are believed to play a role in learning and memory, as well as in the behavioral state of an animal.
We wish to determine the degree neuromodulators (i.e. serotonin, octopamine and 20-hydroxyecdysone), that circulate in the hemolymph, affect cellular activity among cells that have intrinsic differences in their metabolic rates and synaptic efficacies. This will be pursued with the use of three model neuromuscular junction (NMJ) preparations in the crayfish. The leg opener NMJ preparation allows one to address neuromodulators role in regional influences of synaptic transmission of a single motor neuron that innervates both fast and slow muscle fibers. The leg extensor NMJ preparation, with its phasic and tonic motor neurons innervating the same muscle fibers. These preparations provide the opportunity to examine intrinsic differences in high- and low-output terminals in the absence and presence of neuromodulators. Measurements of synaptic efficacy with quantal analysis from visually, identifiable NMJs are obtainable with focal macropatch recordings. The precise area recorded is able to be marked for anatomical reconstruction at the synaptic level, which provides the ability to directly correlate structure to function. The central hypothesis is to determine first if acute application of neuromodulators effect synaptic sites differently depending on the neurons intrinsic differences in synaptic efficacy and secondly the long-term physiological and anatomical effects neuromodulators have on these distinct synaptic junctions. In determining the mechanism responsible for modulator differentiation between regions of a single motor neuron and among different motor neurons utilization of second-messenger cascades will be addressed.
ABSTRACT- The mechanisms by which synaptic vesicles are transported and
primed to fuse with the presynaptic membrane are important to all chemical
synapses. Processes of signal transduction that affect vesicular dynamics,
such as the second- messenger cascades induced by neuromodulators, are
more readily addressed in assessable synaptic preparations of neuromuscular
junctions in the crayfish. We assessed the effects of serotonin (5-HT)
through the analysis of the latency jitter and the quantal parameters:
n & p in the opener muscle of the walking leg in crayfish.
There is an increase in the size of the postsynaptic currents due to more
vesicles being released. Quantal analysis reveals a presynaptic mechanism
by an increase in the number of vesicles being released. Latency measures
show more events occur with a short latency in the presence of 5-HT. No
effect on the frequency or size of spontaneous release was detected. Thus,
the influence of 5-HT is presynaptic, leading to a release of more vesicles
at a faster rate.
The hypothesis to be tested is that the intrinsic differences of release mechanisms in tonic motor neurons will be more susceptible to neuromodulation than those found in phasic motor neurons. We will have to resolve if the differences in synaptic currents measured among phasic and tonic terminals in the absence of neuromodulation are in part due to synaptic structural differences or to the intrinsic differences in the ability to activate second-messenger systems or differences in both.
We want to quantitate the differences in short-term facilitation (STF) and long-term facilitation (LTF) among two motor neurons, phasic and tonic, that are innervating, in parallel, a single muscle fiber. STF has been shown to be very dependent on the availability of free calcium ions in the nerve terminal and LTF is substantially attenuated upon blockage of cAMP.
We wish to examine the intrinsic differences in the response to neuromodulators among these two motor neurons in order to determine if second-messenger systems are differentially regulated.
We plan toexamine if neuromodulator effects on quantal release parameters (m, n and p), within a region of the nerve terminal of two parallel motor neurons, phasic and tonic, having varying degrees of synaptic efficacy, are due to differences in synaptic physiology, metabolic properties, and synaptic structure.
ABSTRACT- Serotonin (5-HT) induces a variety of physiological and behavioral effects in crustaceans. However, the mechanisms employed by 5?HT to effect behavorial changes are not fully understood. Among the mechanisms by which these changes might occur are alterations in synaptic drive and efficacy of sensory, interneurons and motor neurons, as well as direct effects on muscles. We investigated these aspects with the use of a defined sensory-motor system, which is entirely contained within a single abdominal segment and consists of a `cuticular sensory neurons-segmental ganglia-abdominal superficial flexor motor neurons- muscles,' circuit. Our studies address the role of 5-HT in altering (1) the activity of motor neurons induced by sensory stimulation, (2) the inherent excitability of superficial flexor motor neurons, (3) transmitter release properties of the motor nerve terminal, and (4) input resistance of the muscle. Using en passant recordings from the motor nerve, with and without sensory stimulation, and intracellular recordings from the muscle, we show that 5-HT enhances sensory drive and output from the ventral nerve cord resulting in an increase in the firing frequency of the motor neurons. Also, 5-HT increases transmitter release at the neuromuscular junction, and alters input resistance of the muscle fibers.
ABSTRACT- Long-term adaptation can be induced in crayfish, Procambarus
clarkii, phasic motor neurons by daily low-frequency stimulation (Lnenicka
and Atwood, J. Neurobiol., 1985). Biochemical , ultrastructural, and physiological
changes result in a new 'tonic-like' state. We hypothesized that, compared
to control motoneurons, motoneurons undergoing adaptation would show an
increased response to the neuromodulator serotonin, 5-HT. To test the
hypothesis, phasic motoneurons innervating the deep abdominal extensor
muscles of crayfish were stimulated at 2.5 Hz, 2 h/day, for 7 days. On
day eight, control and conditioned motoneurons from the same segment were
stimulated at 1 Hz and the excitatory post-synaptic potentials (EPSPs)
were recorded from DEL1 muscle fibers innervated by each motor neuron
type. Recordings were made in crayfish saline without and with 100 nM
5-HT. The percent change in the EPSP amplitudes after exposure to 5-HT
for the conditioned preparations were all greater than for shams. The
mean percent increase is ~99%. Thus, the conditioned tonic-like motor
neurons behaved like intrinsically tonic motoneurons regarding their sensitivity
to 5-HT. Although the demonstrated synaptic responses show neuronal phenotype
transformation, muscle protein isoform profiles, including those of myosin
heavy chains, do not indicate muscle phenotype alterations after one week
of conditioning their innervating motoneurons.
ABSTRACT- Serotonin (5-HT) levels in the hemolymph of crustaceans is
implied to alter aggressiveness which impacts social interactions. The
activation of IP3 as a second messenger cascade within crayfish motor
neurons in response to application of 5-HT, suggests that the 5-HT receptor
subtypes on the motor neurons are analogous to the vertebrate 5-HT2A receptors.
Based on evidence in other systems, it would be expected that chronically
sustained 5-HT levels of aggressive individuals would result in a compensatory
negative feed-back regulation and/or that target tissues would diminish
their sensitivity to high levels of circulating, free 5-HT. We addressed
the issue of up- and down-regulation in the sensitivity of the responsiveness
to exogenously applied 5-HT at the NMJs of crayfish in which the animals
have altered endogenous 5-HT levels. Injections of the 5-HT1 and 5-HT2
vertebrate receptor agonist, m-CPP, for 1 week resulted in a decreased
responsiveness to application of 5-HT. The compound p-CPA blocks the enzymatic
synthesis of 5-HT and following 7days of p-CPA injections, a super-sensitivity
to exogenous application of 5-HT for both tonic and phasic NMJs was observed.
However, acute applications of p-CPA and m-CPP, followed by extensive
saline washing, did not reveal any altered receptivity to 5-HT application.
4. Muscle Phenotype & Regeneration :
ABSTRACT- Differential expression of multiple myosin heavy chain (MyHC) genes largely determines the diversity of critical physiological, histochemical, and enzymatic properties characteristic of skeletal muscle. Hypotheses to explain myofiber diversity range from intrinsic control of expression based on myoblast lineage to extrinsic control by innervation, hormones, and usage. The unique innervation and specialized function of crayfish (Procambarus clarkii) appendicular and abdominal musculature provide a model to test these hypotheses. The leg opener and superficial abdominal extensor muscles are innervated by tonic excitatory motoneurons. High resolution SDS-PAGE revealed that these two muscles express the same MyHC profile. In contrast, the deep abdominal extensor muscles, innervated by phasic motoneurons, express MyHC profiles different from the tonic profiles. The claw closer muscles are dually innervated by tonic and phasic motoneurons and a mixed phenotype was observed, albeit biased towards the phasic profile seen in the closer muscle. These results indicate that multiple MyHC isoforms are present in the crayfish and that differential expression is associated with diversity of muscle type and function.
[This project is in collaboration with Dr. William A. LaFramboise (Pittsburgh, PA) and Dr. Philip Bonner (Univ. of KY)]
ABSTRACT- The anatomy, physiology, and biochemistry of the dorsal membrane
muscle (DMA) and the superficial extensor muscle accessory head (SEAcc)
in the abdomen of the crayfish, Procambarus clarkii and lobster, Homarus
americanus, is reported. These muscles have not been previously characterized
physiologically or biochemically. The anatomy was originally described
by Pilgrim and Wiersma (J. Morph.113:453-587, 1963). The arrangement of
these muscles varies depending on the abdominal segment. The function
of the dorsal membrane muscle is to retract the thin articulating membrane
joining the cuticular segments so that the dorsal membrane does not evert
during extension of the abdomen. Consequently, the articular membrane
does not protrude, and thus potential damage to the membrane is minimized.
Examination of nerve terminal morphology revealed strings of varicosities,
usually only associated with tonic terminals. The electrophysiological
data indicate that there are at least four tonic excitatory and one inhibitory
motor neuron innervating these muscles. Facilitation indices and fatigue?resistance
indicate physiologically the tonic nature of innervation. Anti-GABA antibodies
demonstrate the anatomical presence of an inhibitor motor neuron. The
SDS-electrophoretic analysis of myofibrillar proteins and Western blots
of key protein isoforms for these muscles in crayfish and lobsters also
indicate that the DMA and SEAcc muscles are tonic phenotype.
[This project is in colaboration with Dr. Donald L. Mykles, Department of Biology, Colorado State University, Fort Collins, CO 80523, USA]
ABSTRACT- Loss of chelipeds in crustaceans results in severe atrophy
of the major muscle responsible for lifting the limb, the anterior levator.
We decided to test if this loss of mechanical load altered muscle phenotype
as measured by SDS-PAGE analysis of levator total protein and actomyosin
fractions. Levator muscles of adult crayfish, Procambarus clarkii,
with either functional regenerate limbs or lack of limb buds (papilla
stage) were compared with those from normal contralateral limbs and those
from pristine animals. We find that there is no difference in protein
profiles among the three conditions. However, the total protein profile
for the dually excited levator muscle is unique compared to those of fast
or slow muscles of the abdomen (L and SEL, respectively), which receive
only phasic or tonic excitatory innervation. The levator myosin heavy
chain profile is similar to that of slow phenotype muscles such as the
SEL and opener. We conclude that load does not influence levator phenotype.
This is likely due to either the intact innervation and continued activation
of the levator during atrophy, or the maintainance of passive tension
on the muscle.
[This project is in colaboration with Dr. Stacia B. Moffett, Washington State University, Department of Zoology, Pullman, WA.]
|Quantal Analysis Projects|
|Fluctuation in Quantal Size.......Variance over Time.....|
1. Fluctuation in Quantal Size:
ABSTRACT- Investigating the function of individual synapses is essential
to understanding the mechanisms that influence the efficacy of chemical
synaptic transmission. The known simplicity of the synaptic structure
at the crayfish neuromuscular junction (NMJ) and its quantal nature of
release allow an assessment of discrete synapses within the motor nerve
terminals. By analyzing areas of the current trace (a measure of charge)
using modern statistical techniques in density estimation and mixture
modeling, we illustrate that the "gold standard" of directly
counting quantal events is unstable in determining the number of release
sites (n) and the probability of release at these sites (p)
when the probability of release is low. In particular, the classical approach
estimates one site even when there are clearly multiple sites operating.
In addition, these modern methods were used to estimate the probability
of release at each site (p) and to determine whether new sites
are recruited as the firing frequency is increased. We find that new sites
are recruited, thus increasing n, and that the probability of release
for individual sites (p) increases with the firing frequency.
[This project is in colaboration with Drs. Kert Viele and Arnold J. Stromberg, Department of Statistics, Univ. of Kentucky, Lexington, KY, USA]
2. Variance over Time
ABSTRACT- Rapid synaptic transmission occurs in response to a Ca2+ transient generated in presynaptic nerve terminal by depolarization. There is substantial inherent variation among various types of synapses in the magnitude of the synaptic currents which leads in part to the variation of the evoked excitatory postsynaptic potentials (EPSPs). As a model system, crayfish and Drosophila melanogaster provides experimental advantages for such studies because of the genetic manipulability of particular proteins known to play a role in synaptic transmission. This implies that these findings can be directly correlated to the underlying structural entities, which are responsible for efficacy in chemical synaptic transmission in general. The EPSPs have nonlinear trends, which were summarized using non-parametric smoothing techniques. Using the residuals from smoothing analysis, the variability is able to be rapidly assessed over time with the help of control charts. Control limits were derived for median and interquartile range charts based on resampling techniques. These control limits provide an index to the fluctuations that can be used to determine how experimental manipulation or various therapies alter synaptic properties over time. There is promise that understanding the fundamental basics in analysis of synaptic transmission will be directly relevant to all neural systems, including humans.
[This project is in colaboration with Dr. Arnold J. Stromberg, Department of Statistics, Univ. of Kentucky, Lexington, KY, USA]
|Synaptic Structure Projects|
|Anatomical Correlation.....True vesicle size..|
Since the tonic terminals show many more synapses structurally per length of terminal then the phasic counterpart but display a lower synaptic output, I postulate that the tonic terminals would show a greater effect to excitatory neuromodulation then phasic terminals. We aim to test the hypothesis that silent synaptic sites may be recruited during neuromodulation and during enhanced electrical activity.
The hypothesis to be tested is that duration-dependence of neuromodulation will be observed to alter the synaptic structure, localization of docked vesicles (within 50 nm of the synapse), and the readily-releasable vesicle populations (within 150 nm of the synapse). The aim is to directly correlate observed physiological parameters that have been altered through modulation to synaptic changes that can account for the short- and long-term effects associated with neuromodulation at the neuromuscular junction. The ability to record synaptic events from visualized varicosities, while applying neuromodulators and marking the site for serial reconstruction at the electron microscopic level, is a very valuable asset offered by the crayfish neuromuscular junction for the direct structure-function relationship at the synaptic sites.
This aim has pertinate implications to account for long-term synaptic
changes that may occur due to maintained behavioral social status such
as domanince or submissivness known to be present in crayfish (Huber et
ABSTRACT- Chemical synaptic transmission occurs when vesicles within
a presynaptic neuron fuse to the membrane and release their contents into
the synaptic cleft to which a postsynaptic cell may respond. The volume
of the vesicles can determine how much transmitter can be released. In
this study we are working on an approach to characterize the dimensions
of synaptic vesicles within a population containing varied sizes of vesicles.
The foundation of the methodical procedures are fundamental for a wide
range of applications of sterological based problems. The approach used
in this study allows investigators to readily index the potential distribution
in vesicle sizes within a population and to assess experimental manipulations
that may effect vesicle dimensions. This is obtained by calculating the
relative percentages of vesicle sizes. The mathematical treatments involved
a continuous removal of the observed distribution form the next smallest
population of vesicle diameters of the overall distribution. We have also
provided a CC-program for others to use.
[This project is in colaboration with Dr. S. Kim (Department of Mathematics, Univ. of Kentucky, Lexington, KY) and Dr. H.L. Atwood (Department of Physiology, Univ. of Toronto, Ontario, Canada)]
|Back to General Research|
|Back to Home Page|