Effects of a hydroxy-cinnamoyl conjugate of spermidine in the neuromuscular junction of crayfish /

N'-coumaroyl spermidine (NlCSpd) is a plant derived chemical which is proposed to belong to a class of low molecular weight neuroactive substances called phenolic polyamines. NlCSpd is stnicturally similar to glutamate receptor blocking toxins found in certain spiders and wasps, such as JSTX-3 and NSTX-3 found in Nephila spiders. The goal of the present study was to determine if plant-derived phenolic polyamines act like other structurally related chemicals found in Arthropod venoms, such as JSTX-3, and whether they can be classified in the same pharmacological group as the spider and wasp toxins. A comparison was made to determine the relative potencies of various phenolic polyamines fi-om plants and insect venoms. This comparison was done by measuring the effect of various concentrations ofNlCSpd on the amplitude of excitatory postsynaptic potentials (EPSPs) elicited in muscle of the crayfish Proccanbarus clarkii. NlCSpd was also tested on L-glutamate induced potentials to determine if a postsynaptic component to sj^naptic block occurs. NlCSpd and an analogue with an a longer polyamine chain, NlCSpm, blocked EPSPs in a dose dependent manner, NlCSpd having an IC50 of lOOnM. NlCSpd also blocked L-glutamate induced potentials. The two main components of the NlCSpd molecule alone are insufficient for activity. NlCSpd acts postsynaptically by interfering with crayfish glutamatergic synaptic transmission, likely blocking glutamate receptors by interacting with the same site(s) as other phenolic polyamines. Certain moieties on the polyamines molecule are necessary for activity while others are not.

The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Cholesterol-Independent Effects of Methyl-b- Cyclodextrin on Chemical Synapses

The cholesterol chelating agent, methyl-b-cyclodextrin (MbCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MbCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MbCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MbCD impaired impulse propagation and decreased EJP amplitude by 40% (P,0.05) in preparations from crayfish acclimatized to 14uC but not from those acclimatized to 21uC. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P,0.05). MbCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and coldacclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P,0.05; 50% reduction in warm, P,0.05). MbCD reduced cholesterol in isolated nerve and muscle from cold- and warmacclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P,0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MbCD on glutamatesensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MbCD can affect both presynaptic and postsynaptic properties, and that some effects of MbCD are unrelated to cholesterol chelation.

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

The action of octopamine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10(-5) and 10(-4) M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10(-5) M OA increased synaptically driven contractions by ≈ 1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

Effects of reflection and social isolation on crayfish behaviour /

Visual stimuli and socialization influence exploratory behaviour in crayfish. The predominant components of spontaneous exploratory behaviour were determined by observing the activity of solitary adult crayfish (Procambarus clarkii) in a glass aquarium containing fresh water and no objects. Five distinct behaviours were observed: rearing up (climbing on the wall), turning around, cornering (facing the comer), backward walking, and crossing (crossing the midline of the aquarium). The frequency of rearing up, cornering and turning around decreased when reflection from the glass wall was blocked with black cardboard, black paint or non-reflective transparent plastic. In a tank containing mirrors on one side and non-reflective plastic on the other, crayfish cornered, reared up, and turned around more in front of the mirrors. Socialization was necessary for crayfish to respond to the reflection. Crayfish that were housed in pairs for two weeks exhibited more rearing up, turning around and cornering in front of the mirrors than in the non-reflective side. Crayfish isolated for two weeks did not show these differences. Socialized crayfish also exhibited more rearing up, turning around and cornering than did isolated crayfish. Thus, crayfish respond to visual stimuli provided by a glass tank, but the responds depends on socialization.

Responses of crayfish to a reflective environment /

This thesis examines how dominance status of crayfish alters responses to their own reflection. Crayfish are social animals that fight to develop a dominance hierarchy consisting of dominant and subordinate members. After socialization, crayfish were videotaped in an aquarium with mirrors on one half of the tank and a non-reflective plastic on the other half. Dominants paired for 14 days perform more cornering, turning, crossing and spent more time in a reflective environment versus a non-reflective environment. Subordinate crayfish exhibit more reverse walking in a mirrored environment while isolated crayfish show no preference for reflection. This change in behaviour occurs immediately for dominants paired for 30 min while subordinates require 3 days of pairing to exhibit the same behaviour as subordinate crayfish paired for 14 days. Thus, 30 min of pairing is required to enhance responses to a reflection observed in dominant crayfish while 3 days is required to decrease subordinate responses to a reflection. These findings propose that male socialized crayfish respond to their mirror image as they do a male conspecific. Their responses depend on both their dominance status and the length of socialization which suggests that crayfish are learning to behave in a characteristic manner as a result of their social experience.

A possible role for intrinsic calcium buffers in the long- term adaptation of crayfish neurons

Increasing the impulse activity of neurons in vivo over 3 or more days causes a reduction in transmitter release that persists for days or weeks (eg. Mercier and Atwood, 1989). This effect is usually accompanied by decreased synaptic fatigue. These two changes involve presynaptic mechanisms and indicate "long-term adaptation" (LTA) of nerve terminals. Previous experiments have shown that LTA requires extracellular calcium and protein synthesis (eg. Hong and Lnenicka, Soc. Neurosci. Abstr. 17:1322) and appears to involve communication between the cell body and the nerve terminals. The present study examines the possibility that the reduction in transmitter release is caused by an -increase in the calcium buffering ability within the nerve terminals. It examines the responses of adapted and control nerve terminals to exogenously applied calcium buffer, BAPTA-AM, which decreases transmitter release (Robitialle and Charlton, 1992). If LTA increases intrinsic Ca2+-buffering, the membrane permeant form of BAPTA should have less effect on adapted nerve terminals than on controls. Experiments are performed on the phasic abdominal extensor motor neurons of the crayfish, Procambarns clarkii. BAPTA-AM decreases excitatory postsynaptic potentials (EPSP's) of the phasic extensor muscles in a dosedependent manner between 5 and 50 JLM. LTA is elicited by in vivo stimulation at 2.5 Hz for 2-4 h per day over 3 days, which reduces EPSP's by over 50%. Experiments indicate that BAPTA-AM produces no significant change in EPSP reduction in adapted neurons when compared to controls. These results do not support the hypothesis that increased daily activity alters rapid intrinsic calcium buffers, that are able to reduce transmitter output in the same manner as BAPTA.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

Reproduction in the temperate crayfish, Orconectes rusticus: intermale aggression, copulatary behaviour, and sperm competition

The chelipeds of Orconectes rusticus are sexually dimorphic; males possessing the larger. Males use their chelae in intermale aggressive interactions, both to threaten, and assault opponents. In dyadic interactions males with larger chelae were dominant over otherwise physically similar opponents. A high frequency of attack behaviour, coupled with a low frequency of threats during these interactions indicates that actual physical contact is required for opponent assessment. Large clawed males oriented females into the copulatory position faster than small clawed males. Females more frequently escaped the precopulatory-grasp attempts of small clawed males. Additionally, male-female pairs that included a large clawed male remained in copula longer than pairs that included a small clawed male. Sperm of the second male to mate took precedence over the sperm of the primary male. Sperm precedence was incomplete; about 900/0 paternity accrued to the second male.

Modulation of muscle contraction by a FMRFamide-related peptide

A FMRFamide-like neuropeptide with the sequence "DRNFLRF-NH2" was recently isolated from pericardial organs of crayfish (Mercier et aI., Peptides, 14, 137-143, 1993). This neuropeptide, referred to as "DF2'" has already been shown to elicit cardioexcitation and to enhance synaptic transmission at neuromuscular junctions. Possible effects ofDF2 on muscle were investigated using superficial extensor muscles of the abdomen of the crayfish, Procambarus clar/ai. These muscles are of the tonic type and generate slow contractions that affect posture. DF2, at concentrations of 10-8 M or higher, increased muscle tonus and induced spontaneous, rhythmic contractions. These effects were antagonized by 5 rnM Mn2+ but not by lO-7M tetrodotoxin (TTX). Thus, they represent direct actions on muscle cells (rather than effects on motor neurons) and are likely to involve calcium influx. In contrast, deep abdominal extensor muscles, responsible for rapid swimming movements, and superficial flexor muscles do not generate contractions in response to the peptide. 2 Spontaneous contractions were also induced in the superficial extensor muscles by decreasing the temperature to II-13°C. Such contractions were also TTX-insensitive and they were antagonized by adding calcium channel blockers (Mn2+, Cd2+ or Ni2+) or by removing calcium from the bathing solution. This suggests that the spontaneous contractions depend on an influx of calcium from the extracellular solution. N-type and L-type voltage dependent calcium channel blockers did not reduce the effect of the peptide or the spontaneous contractions suggesting that calcium influx is not through N- or L-type calcium channels.

Neuromuscular adaptations in endurance-trained boys and men

Competitive sports participation in youth is becoming increasingly more common in the Western world. It is widely accepted that sports participation, specifically endurance training, is beneficial for physical, psychomotor, and social development of children. The research on the effect of endurance training in children has focused mainly on healthrelated benefits and physiological adaptations, particularly on maximal oxygen uptake. However, corresponding research on neuromuscular adaptations to endurance training and the latter's possible effects on muscle strength in youth is lacking. In children and adults, resistance training can enhance strength and mcrease muscle activation. However, data on the effect of endurance training on strength and neuromuscular adaptations are limited. While some evidence exists demonstrating increased muscle activation and possibly increased strength in endurance athletes compared with untrained adults, the neuromuscular adaptations to endurance training in children have not been examined. Thus, the purpose of this study was to examine maximal isometric torque and rate of torque development (RID), along with the pattern of muscle activation during elbow and knee flexion and extension in muscle-endurancetrained and untrained men and boys. Subjects included 65 males: untrained boys (n=18), endurance-trained boys (n=12), untrained men (n=20) and endurance-trained men (n=15). Maximal isometric torque and rate of torque development were measured using an isokinetic dynamometer (Biodex III), and neuromuscular activation was assessed using surface electromyography (SEMG). Muscle strength and activation were assessed in the dominant arm and leg, in a cross-balanced fashion during elbow and knee flexion and extension. The main variables included peak torque (T), RTD, rate of muscle activation (Q30), Electro-mechanical delay (EMD), time to peak RTD and co-activation index. Age differences in T, RTD, electro-mechanical delay (EMD) and rate of muscle activation (Q30) were consistently observed in the four contractions tested. Additionally, Q30, nonnalized for peak EMG amplitude, was consistently higher in the endurancetrained men compared with untrained men. Co-activation index was generally low in all contractions. For example, during maximal voluntary isometric knee extension, men were stronger, had higher RTD and Q30, whether absolute or nonnalized values were used. Moreover, boys exhibited longer EMD (64.8 ± 18.5 ms vs. 56.6 ± 15.3 ms, for boys and men respectively) and time to peak RTD (112.4 ± 33.4 ms vs. 100.8 ± 39.1 ms for boys and men, respectively). In addition, endurance-trained men had lower T compared with untrained men, yet they also exhibited significantly higher nonnalized Q30 (1.9 ± 1.2 vs. 1.1 ± 0.7 for endurance-trained men and untrained men, respectively). No training effect was apparent in the boys. In conclusion, the findings demonstrate muscle strength and activation to be lower in children compared with adults, regardless of training status. The higher Q30 of the endurance-trained men suggests neural adaptations, similar to those expected in response to resistance training. The lower peak torque may su9gest a higher relative involvement oftype I muscle fibres in the endurance-trained athletes. Future research is required to better understand the effect of growth and development on muscle strength and activation patterns during dynamic and sub-maximal isometric contractions. Furthennore, training intervention studies could reveal the effects of endurance training during different developmental stages, as well as in different muscle groups.

Second Welland Canal - Book 3, Survey Map 14 - Village of Junction and Crowland

Survey map of the Second Welland Canal created by the Welland Canal Company showing the border area of the townships of Crowland and Humberstone, as well as the Village of Junction. Identified structures associated with the Canal include ditches, guard lock, old canal, new towing path, bridge, feeder to Dunnville, covered drain. Surveyor measurements and notes can be seen in red and black ink and pencil. Local area landmarks include James Turf Tavern and possible marshland. Roads parallel to Canal include western Road Allowance, the new towing road, road to Welland and road to Junction. Roads perpendicular to Canal include Road Allowance between the 5th and 6th Concession. Properties and property owners are noted as Thomas. C. Street, James Tuft, and John Hellems. Lots noted are: Lots Number 26, 27, 6th Concession.