Assignment On Brine Shrimp

Brine Shrimp

The common name of brine shrimp is Artemia that seldom reaches more than 1/2 in. (1.3 cm) in length and is commonly used for fish food in aquariums. Brine shrimp, which are not closely related to true shrimp, can be found almost everywhere in the world in inland saltwaters, although they are completely absent from oceans. They can live in water having several times the salinity of seawater, but they can also tolerate water having only one tenth the marine salt concentration. Brine shrimp usually occur in huge numbers and can be seen in vast windblown lines in the Great Salt Lake. Their absence from the sea has been explained by their vulnerability to attack by predators and the absence of the latter in their inland saline habitat.

Artemia is a well known genus as one member, sometimes identified as a hybrid species Artemia salina × nyos, is sold as a novelty gift, most often under the marketing name Sea-Monkeys.

Scientific classification

Kingdom:	Animalia
Phylum:	Arthropoda
Subphylum:	Crustacea
Class:	Branchiopoda
Order:	Anostraca
Family:	Artemiidae Grochowski, 1895
Genus:	Artemia Leach, 1819

Species: Artemia franciscana

Fig: Brine shrimp

Life cycle

Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours. Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average.

Ecology

Wild brine shrimp eat microscopic planktonic algae. Cultured brine shrimp can also be fed particulate foods including yeast, wheat flour, soybean powder or egg yolk. Brine shrimp can tolerate varying levels of salinity. A common biology experiment in school is to investigate the effect of salinity levels on the growth of these creatures. The preferred level of salinity is about 30–35 ppt (parts per thousand).

Nutritional benefits

The nutritional properties of newly hatched brine shrimp make them particularly suitable to be sold as aquarium food as they are high in lipids and unsaturated fatty acids (but low in calcium)..

Threatened

Artemia monica, the variety commonly known as Mono Lake brine shrimp, are found only in Mono Lake, Mono County, California.Despite there being trillions of these creatures in Mono Lake, it was felt that rising levels of salinity and sodium hydroxide concentration of the lake would endanger them because of the increase in pH. Sea-Monkeys® are in no way harmful to humans or the environment. If they somehow find their way into natural water ways or sewer lines, they simply will not be able to survive outside of the formula

Lethality assay

The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity.

Preparation of the bioassays

The tests were conducted in multiwell plates in filtered (0.45 μm pore diameter) and sterilized seawater (final volume 5 ml). Each of the extracts for each species was tested at 1000, 100 and 10 μg of extract per ml. The concentrations were obtained by transferring the corresponding volume from the stock solution to different wells for evaporation. After evaporation, 5 ml of seawater were added to each well with gentle shaking to ensure that the compounds diffused adequately in the aqueous solution. Four replicates were used for each treatment and control. The control was performed by adding the solvent used to dissolve the extracts in the assays, and it was allowed to evaporate. Before the assays, the average time of appearance of the first free nauplii and the subsequent developmental stages was calculated. The first cysts hatched approximately after 12 h of incubation (average time); the maximum percentage of instar II (55.33%) appeared 12 h later (24 h after the start of incubation). All the tests were performed in a temperature-controlled room at 28°C, under a continuous light.

Brine shrimp lethality bioassay

The brine shrimp lethality bioassay was used to predict the cytotoxic activity ^[15],[19] of the n-hexane, carbon tetrachloride, chloroform, and aqueous fractions from methanolic crude extracts. For the experiment, 4 mg of each of the extracts was dissolved in dimethylsulfoxide (DMSO) and solutions of varying concentrations (400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 µg/ ml) were obtained by the serial dilution technique using simulated seawater. The solutions were then added to the pre-marked vials containing 10 live brine shrimp nauplii in 5 ml simulated seawater. After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. The mortality endpoint of this bioassay was defined as the absence of controlled forward motion during 30 s of observation. ^[20] From this data, the percent of lethality of the brine shrimp nauplii for each concentration and control was calculated. An approximate linear correlation was observed when logarithm of concentration versus percentage of mortality ^[21] was plotted on the graph paper and the values of LC ₅₀ were calculated using Microsoft Excel 2003 [Figure 1]. Vincristine sulphate was used as positive control.

Result and Discussion

With the exception of aqueous fraction, all the other fractions of D. indica leaves were active against most of the tested organisms Table 1. The average zone of inhibition produced by the n-hexane, carbon tetrachloride, and chloroform fraction was ranged from 6-8 mm, 7-8 mm, and 6-7 mm, respectively, at a concentration of 400 µg/ disc. Against the Escherichia ^{More Details} coli, only chloroform fraction was active (zone of inhibition was 7 mm) and carbon tetrachloride fraction exhibited highest antimicrobial activity compared to other solvent fractions. In both the cases of bacteria and fungi, the zone of inhibition was found to be 6-8 mm.
The LC ₅₀ values obtained from brine shrimp lethality bioassay [Table 2] and [Table 3] were 1.94, 4.46, 2.13, and 5.13 µg/ml for n-hexane (HX), carbon tetrachloride (CT), chloroform (CF), and aqueous (AQ) fraction, respectively. Compared to positive control (vincristine sulphate, VS, LC ₅₀ 0.52 µg/ml), all the fractions tested showed good brine shrimp larvicidal activity. Again the crude extracts resulting in LC ₅₀ values less than 250 µg/ml were considered significantly active and had the potential for further investigation. ^[22] The cytotoxic activity exhibited by the solvent fractions was promising and this clearly indicates the presence of potent bioactive compounds.

Table: 01

Table: 02

Table: 03

Conclusion

The antimicrobial and cytotoxic activities of various fractions of D. indica leaves, found in this study, may explain some of the traditional medicinal uses of this plant. These could be of particular interest in relation to find out its unexplored efficacy and can be a potential source of chemically interesting and biologically important drug candidates.

Assignment On scope of Microbiology

Introduction:

Microbiology is the specific - study of the microorganisms’

Microbiology is the specific branch of biology that essentially deals with the elaborated investigation of microscope organism termed as microbes that are composed of only one cell. These are typically either unicellular or multi cellular microscopic organisms that are distributed abundantly both in the living bodies of plants and animals and also in the air, water, soil, and marine kingdom.

One day also define microbiology as – ‘the study of living organisms of microscope size, that include essentially bacteria, fungi, algae, protojozoa and the infectious agents at the very borderline of life which are broadly known as viruses.

It is mainly concerned with a variety of vital and important aspects, such as: typical form, inherent structure reproduction, Physiological characteristic, metabolic pathways (viz., anabolism, and catabolism) and logical classification. Besides, it includes the study of their:

• Distribution in nature,
• Relationship to each other and to other living organisms,
• Specific effects on humans, plants, and animals, and
• Reactions to various physical and chemical agents.

Microbiology

Microbiology (from Greek μῑκρος, mīkros, "small"; βίος, bios, "life"; and -λογία, -logia) is the study of microorganisms, which are unicellular or cell-cluster microscopic organisms. This includes eukaryotes such as fungi and protists, and prokaryotes, which are bacteria and archaea. Viruses, though not strictly classed as living organisms, are also studied. In short; microbiology refers to the study of life and organisms that are too small to be seen with the naked eye.

Fig: An agar plate streaked with microorganisms

Microbiology is a peer-reviewed academic journal that covers research in all aspects of microbiology, including the pharmacy, biochemistry, cell biology, molecular biology, developmental biology, physiology, pathogenicity, biodiversity, evolution and genetics of microorganisms and viruses of microorganisms. It also covers plant–microbe interactions, and environmental and theoretical microbiology.

Pharmacy and Microbiology:

Pharmacy is the health profession that links the health sciences with the chemical sciences, and it is charged with ensuring the safe and effective use of medication. The scope of pharmacy practice includes more traditional roles such as compounding and dispensing medications, and it also includes more modern services related to patient care, including clinical services, reviewing medications for safety and efficacy, and providing drug information. Pharmacists, therefore, are the experts on drug therapy and are the primary health professionals who optimize medication use to provide patients with positive health outcomes. Pharmacists are highly-trained and skilled healthcare professionals who perform various roles to ensure optimal health outcomes for their patients.

The field of microbiology is also vast. For this reason the field has been funder divided into various types. If we illustrate the names and the subjected area it will be easy to find out the relationship between pharmacy and microbiology.

Rapid advances in the biomedical sciences are radically redefining the types of educational experiences that evidence-based clinicians need. Biomedical science is becoming more indispensable, and the role of genetics is key to our understanding of physiology and pathogenesis, and is vital in determining the interplay of genes in therapeutic outcomes.

In microbiology we can study organism in great detail and observe their life process while they are actively metabolizing, growing reproducing, and aging, and dying. By modifying their environment we can alter metabolic activities, regulate growth, fid even change some details of their genetic pattern.

Scope of Microbiology:

Microbiology is not an industry in itself, but an important technology that will have a large impact on many different industrial sectors in the future. The advantageous fields of microbiology are essentially the ones enumerated below:

Antibiotics-penicillin:

Fungi secrete antibiotics to kill all the things around it so that it has all of the nutrients for itself. The fermentation process of penicillin is showed below.

Fig: Biosynthesis of penicillin

Fig: Chemical structure of penicillin G

Production of human hormones:

Bacteria have been inserted with human genes that control the production of insulin, Human growth hormone and interferon. The bacteria can produce these in mass quantities that human cannot. Some of these are discussed here.

Human insulin

Amongst the earliest uses of biotechnology in pharmaceutical manufacturing is the use of recombinant DNA technology to modify Escherichia coli bacteria to produce human insulin, which was performed at Genentech in 1978. Prior to the development of this technique, insulin was extracted from the pancreas glands of cattle, pigs, and other farm animals, While generally efficacious in the treatment of diabetes, animal-derived insulin is not indistinguishable from human insulin, and may therefore produce allergic reactions. Genentech researchers produced artificial genes for each of the two protein chains that comprise the insulin molecule. The artificial genes were “then inserted. Into plasmids … among a group of genes that: Are activated by lactose. Thus, the insulin producing genes were also activated by lactose. The recombinant plasmids were inserted into Escherichia coli bacteria, which were “induced to produce 100,000 molecules of either chain A or chain B human insulin.” The two protein chains were then combined to produce insulin molecules.\

Fig: Insulin

Production of vitamin:

Bacteria like E. Coli present in human colon are involved in synthesis of vitamins like vitamin b12, folic acid, biotin and K, which may be used by the host. Such bacteria are often used for commercial preparation of vitamins like riboflavin.

Fig: The E. coli bacterium Fig: Chemical structure of Riboflavin

Antibiotics From fungi

Fungi are well known as a source of antibiotics but new therapeutic compounds with novel pharmacological activities have also been developed in recent years. One such example are the cyclosporins and later shown to possess immunosuppressive activity.

Cyclosporin A (C₆₂H₁₁₁N₁₁O₁₂) is currently the most widely used drug for preventing rejection of human organ transplants.

Fig: Chemical structure of Cyclosporin A

Production of Antiseptic:

Antisepticsare antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis or putrefaction. They should generally be distinguished from antibiotics that destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects. Some antiseptics are true germicides, capable of destroying microbes (bactericidal) whilst others are bacteriostatic and only prevent or inhibit their growth. Antibacterial are antiseptics that only act against bacteria. Microbicides which kill virus particles are called viricides. Some common examples of antiseptics are alcohols, Quaternary ammonium compounds, Boric acid, Chlorhexidine Gluconate, Hydrogen peroxide, Iodine, phenol etc.

For the growth of bacteria there must be a food supply, moisture, in most cases oxygen, and a certain minimum temperature. These conditions have been studied and applied in preserving of food and the ancient practice of embalming the dead, which is the earliest known systemic use of antiseptics. In early inquiries, there was much emphasis on the prevention of putrefaction, and procedures were carried out to find how much of an agent must be added to a given solution in order prevent development of undesirable bacteria. However, for various reasons, this method was inaccurate, and today an antiseptic is judged by its effect on pure cultures of defined pathogenic celicular single helix microbes and their vegetative and spore forms. The standardization of antiseptics has been implemented in many instances, and a water solution of phenol of a certain fixed strength is now used as the standard to which other antiseptics are compared.

Medical Microbiology:

Medical Microbiology helps in the diagonostic protocol for identification of causative agents of various human aliments, alimentes & subsepuents preventive measures. Some are stated blew:

Infection control:

Inaction control using knowledge to control infection, or prevent the spread of disease.

Genetic Engineering:

Microorganisms can now be genetically engineered to manufacture large amounts of human hormones and other urgently needed substance. It is the study of how genes are organized and regulated in microbes in relation to their cellular functions, closely related to the field of molecular biology. Microorganisms play a central role in recombinant DNA technology and genetic engineering. Important tools of biotechnology are microbial cells, microbial genes, and microbial enzymes. This information is a key step for the pharmacists in their research and general preparation of various medications. This opens the possibility for microbial production of foods, fuels, hormones, diagnostics agents, medicines, antibiotics, antibodies, vaccines that is useful in our society and civilization.

Fig: Microbial genetics

Other scope of microbiology:

Pharmaceutical Microbiology:

Pharmaceutical Microbiology – is the part of industrial microbiology that is responsible for creating medications. The making of life-saving drugs, antibiotics e.g. Penicillin’s, ampicillin, chloramphenicol, ciprofloxacin, tetracyclines, and streptomycin belong to the sector of pharmaceutical microbiology. There are many useful products made by microbes.

Industrial microbiology:

Industrial microbiology making of ethanol, acetic acid lactic acid, citric acid, glucose, syrup, high-fructose syrup.

Waste-Treatment Microbiology:

Waste-Treatment Microbiology treatment of domestic and industrial effluents or wastes by lowering the COD. Without microbes we would have an over abundance of dead things. We would run out of space on earth. Microbes are also used to clean up oil, toxic waste, and dynamite and sewage treatment.

Food Microbiology:

Food Microbiology ­­­­- Food chain are an important component of the food chain. They allow for recycling of nutrients and some microbes are actual parts of food chain. They allow for recycling of nutrients and some microbes are actual parts of food chain. Bacteria in ocean vents are the source of energy for ocean vents ecosystems. Brewing and backing have been carried out for thousands of years and both are depend on the conversion of sugar into alcohol and carbon dioxide by yeasts. In bread making carbon dioxide causes the dough to rise giving lightness to the bread whilst the alcohol is driven off during baking. Early processes were depending on contamination by ‘wild’ yeast .Today pure strains are normally employed and some 1.5 million tons of baker’s yeast (Saccharomyces cerevisiae) are produced worldwide every year. Cheese production also has ancient origins.

Beverage Microbiology:

Beverage Microbiology making of beer, sandy, wine, and a variety of alcoholic beverage e.g. whisky, brandy, rum, gin, vodka, etc.

Aero microbiology:

Aero microbiology helps in the overall presentation and preparation of food, food prone dieses, and their ultimate prevention.

Exomicrobiology:

Exomicrobiology to help in the exploration of life in the outer space.

Geochemical microbiology:

Geochemical microbiology to help in the study of coal, mineral deposits, and gas formation; prospecting the deposits of gas and oil, coal, recovery of minerals from low –grade ores .Some bacteria have the ability to produce methane, one of the cleanest burning fuels.

Agricultural Microbiology:

Agricultural Microbiology Use of microbes in agriculture and farming symbiotically associated bacteria are able to biologically convert nitrogen gas present in the atmosphere into ammonia which helps in enriching the soil and promotes optimal growth of plants. Such bacteria are useful in the areas of agriculture and farming and increasing crop yield without use of chemical fertilizer. The use of fungi as biocontrol agents to kill insects (mycoinsecticides) and weeds (mycoherbicides) has the potential to replace many of the toxic chemicals currently in use. Several species of fungi have now been commercially formulated as mycoinsecticides.

Conclusion:

Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding.

Microbiology and metabolic engineering offer and alternative approach that are becoming more accessible as the tools for engineering microbes are further developed. Microbiology is researched actively, and the field is advancing continually. We have probably only studied about one percent of all of the microbe species on earth.

Neuromuscular Transmission

Introduction:

All multicellular organisms have a nervous system, which may be defined as assemblages of cells specialized by their shape and function to act as the major coordinating organ of the body. Nervous tissue underlies the ability to sense the environment, to move and react to stimuli, and to generate and control all behavior of the organism. Compared to vertebrate nervous systems, invertebrate systems are somewhat simpler and can be more easily analyzed. Invertebrate nerve cells tend to be much larger and fewer in number than those of vertebrates. They are also easily accessible and less complexly organized; and they are hardy and amenable to revealing experimental manipulations. However, the rules governing the structure, chemistry, organization, and function of nervous tissue have been strongly conserved phylogenetically. Therefore, although humans and the higher vertebrates have unique behavioral and intellectual capabilities, the underlying physical-chemical principles of nerve cell activity and the strategies for organizing higher nervous systems are already present in the lower forms. Thus neuroscientists have taken advantage of the simpler nervous systems of invertebrates to acquire further understanding of those processes by which all brains function.

Nerve:

A nerve is an enclosed, cable-like bundle of peripheral axons (the long, slender projections of neurons). A nerve provides a common pathway for the electrochemical nerve impulses that are transmitted along each of the axons. Nerves are found only in the peripheral nervous system. In the central nervous system, the analogous structures are known as tracts. Neurons are sometimes called nerve cells, though this term is technically inaccurate since many neurons do not form nerves, and nerves also include non-neuronal Schwann cells that coat the axons in myelin. Nerves are categorized into three groups based on the direction that signals are conducted: Nerves can be categorized into two groups based on where they connect to the central nervous system: Within the endoneurium, the individual nerve fibers are surrounded by a low protein liquid called endoneurial fluid. The endoneurium has properties analogous to the blood-brain barrier, in that it prevents certain molecules

The Nerve Message :

The plasma membrane of neurons, like all other cells, has an unequal distribution of ions and electrical charges between the two sides of the membrane. The outside of the membrane has a positive charge, inside has a negative charge. This charge difference is a resting potential and is measured in millivolts. Passage of ions across the cell membrane passes the electrical charge along the cell. The voltage potential is -65mV (millivolts) of a cell at rest (resting potential). Resting potential results from differences between sodium and potassium positively charged ions and negatively charged ions in the cytoplasm. Sodium ions are more concentrated outside the membrane, while potassium ions are more concentrated inside the membrane. This imbalance is maintained by the active transport of ions to reset the membrane known as the sodium potassium pump. The sodium-potassium pump maintains this unequal concentration by actively transporting ions against their concentration gradients.

Fig: Transmission of an action potential..

Changed polarity of the membrane, the action potential, results in propagation of the nerve impulse along the membrane. An action potential is a temporary reversal of the electrical potential along the membrane for a few milliseconds. Sodium gates and potassium gates open in the membrane to allow their respective ions to cross. Sodium and potassium ions reverse positions by passing through membrane protein channel gates that can be opened or closed to control ion passage. Sodium crosses first. At the height of the membrane potential reversal, potassium channels open to allow potassium ions to pass to the outside of the membrane. Potassium crosses second, resulting in changed ionic distributions, which must be reset by the continuously running sodium-potassium pump. Eventually enough potassium ions pass to the outside to restore the membrane charges to those of the original resting potential.The cell begins then to pump the ions back to their original sides of the membrane.

The action potential begins at one spot on the membrane, but spreads to adjacent areas of the membrane, propagating the message along the length of the cell membrane. After passage of the action potential, there is a brief period, the refractory period, during which the membrane cannot be stimulated. This prevents the message from being transmitted backward along the membrane

Steps in an Action Potential

1. At rest the outside of the membrane is more positive than the inside.
2. Sodium moves inside the cell causing an action potential, the influx of positive sodium ions makes the inside of the membrane more positive than the outside.
3. Potassium ions flow out of the cell, restoring the resting potential net charges.
4. Sodium ions are pumped out of the cell and potassium ions are pumped into the cell, restoring the original distribution of ions.

Synapses :

The junction between a nerve cell and another cell is called a synapse. Messages travel within the neuron as an electrical action potential. The space between two cells is known as the synaptic cleft. To cross the synaptic cleft requires the actions of neurotransmitters. Neurotransmitters are stored in small synaptic vessicles clustered at the tip of the axon.

Fig: Synapes

Neuron:

The neuron is the functional unit of the nervous system. Humans have about 100 billion neurons in their brain alone! While variable in size and shape, all neurons have three parts. Dendrites receive information from another cell and transmit the message to the cell body. The cell body contains the nucleus, mitochondria and other organelles typical of eukaryotic cells. The axon conducts messages away from the cell body.

What is Neuromuscular Transmission (NMT)?

Neuromuscular Transmission (NMT) is the transfer of an impulse between a nerve and a muscle in the neuromuscular junction. NMT can be blocked by neuromuscular blocking agents -drugs which cause transient muscle paralysis and prevent the patient from moving and breathing spontaneously.

Muscle relaxation is used during general anesthesia to enable endotracheal intubation and to provide the surgeon with optimal working conditions. In critical care muscle relaxation is used during mechanical ventilation to minimize the patient ’s work of breathing and to improve oxygenation.

A) Overview of Neuromuscular Transmission

An action potential originating at the axon hillock of a motor neuron is conducted to the nerve terminal

↓

Nerve terminal is depolarized opening Ca channels

↓

Vesicles fuse with nerve terminal membrane and release Acetylcholine (Ach)

↓

ACh binds to receptors on the muscle end-plate → Depolarization of the muscle membrane

↓

Threshold of Voltage-sensitive Na channels in muscle membrane is reached, and muscle AP fires

↓

AP spreads over surface of muscle and muscle contracts

B) Structure of the Neuromuscular Junction

1) At the neuromuscular junction an alpha motor neuron loses its myelin sheath and forms numerous nerve terminal branches. Each of these branches ends in a terminal bulb called a “bouton.”

2) The terminal boutons remain close and form synaptic contacts on a specialized area of muscle cell membrane called the “end-plate.”

3) Between the “pre-synaptic” nerve terminal and the “post-synaptic” muscle end-plate is 20 nm cleft containing a basal lamina sheath consisting of collagen and extracellular matrix proteins. An enzyme “acetyl cholinesterase” is also present in the synaptic cleft. This enzyme hydrolyzes Acetylcholine into choline plus acetate.

4) Structures in the Nerve Terminal

a) All nerve terminals in chemical synapses contain: vesicles with transmitter, mitochondria and an “active zone” where vesicles bind and release their contents into the synaptic cleft

b) Nerve terminals also contain Ca channels near the active zones. Ca facilitates the fusion of vesicles with the nerve terminal membrane. In the absence of Ca, neuromuscular transmission cannot occur.

5) Structures in the Synaptic Cleft

a) The 20 nm cleft is filled with collagen and extracellular matrix proteins. Acetylcholine esterase (AChE) is also located in the cleft attached to the collagen.

b) During development molecules are expressed that enable the growing nerve terminal to form a synapse on the correct target.

c) When ACh is released from nerve terminal vesicles, the transmitter diffuses across the cleft and binds to ACh receptors on the muscle end-plate. However, because the enzyme AChE is so effective, most of the Ach is hydrolyzed before it reaches the receptors.

6)Structures at the Muscle End-plate

a)The muscle end-plate is thrown into a highly convoluted series of folds that are characteristic of mature neuromuscular junctions.

b)ACh receptors are localized in clusters at the crests of the folds, strategically located across from the vesicle binding/release sites.

c)When ACh binds to the receptors, the end-plate membrane is “punctured” for cations, allowing Na+ and K+ to diffuse down their chemical gradients. Thus Na moves in and K moves out leading to a depolarization of the end-plate membrane.

d)Voltage-sensitive Na channels are located at the bottoms of the folds and in all the rest of the muscle membrane. If the end-plate depolarization is sufficient to reach threshold, a muscle AP will fire, and the muscle will contract.

C) Steps in Neuromuscular Transmission – Pre-synaptic

1)Nerve Terminal Action Potential Opens Ca Channels

a)Voltage-sensitive Ca channels are especially concentrated in nerve terminal endings near vesicle release sites. These Ca channels have activation and inactivation gates, but are slow to open and close.

b)Because they are slow, the Ca channels open during the falling phase of the nerve terminal AP. The Ca channels are open long enough to allow a rise in intracellular Ca of 1000 times normal (~ 10^-4 M, whereas normal is ~ 10^-7 M).

c)The rise in Ca++ near vesicle binding sites allows fusion of vesicle membranes with the nerve terminal membrane.

d)Vesicle Docking – A fusion pore develops beneath a “docked” vesicle. A channel forms through the fusion pore allowing some ACh to be released. In the final state an “omega figure” is formed, and the ACh is released from the vesicle. The vesicle membrane becomes incorporated into the nerve terminal membrane.

e)Vesicle Recycling

1)Slow to Moderate rates of stimulation – “Kiss and Run Docking.”

2)Fast Stimulation – Fusion and Recycling of Membrane.

a)Vesicles fuse with nerve terminal membrane, form Omega figures, and combine with the membrane

b)Clathrin forms around an invaginated region of membrane. Clathrin proteins form a basket around a pinched off piece of nerve terminal membrane, and transport it to the endosome.

a) New vesicles are formed from the endosome and move to the terminal along cytoskeletal elements.

b) Newly formed vesicles are refilled with ACh by the reaction of choline + acetate catalyzed by the enzyme Acetylcholine transferase.

D) Steps in Neuromuscular Transmission – Post Synaptic

1)Acetylcholine is released into the synaptic cleft directly opposite the ACh Receptors. These are “nicotinic” acetylcholine receptors because they have a high affinity for nicotine as apposed to “muscarine” typical of the autonomic nervous system.

2)Most acetylcholine never reaches the Ach receptors. That is because the synaptic cleft is filled with the enzyme acetylcholine esterase (AChE) that hydrolyzes most of the Ach. The released Ach must pass through this enzyme “gauntlet.”

Ach + Ach esterase → acetate + choline

3)The choline is taken back into the nerve terminal where it is combined with Acetyl CoA in the presence of the enzyme CAT (choline acetyl transferase). The Ach is then repackaged in vesicles for future release.

Choline + AcetylCoA + CAT → Acetylcholine

4)When ACh binds to the receptors, it opens a special channel that “chemically punctures” the end-plate membrane to both Na+ and K+.

5)The opening of the Ach-controlled channel leads to a depolarization of the end-plate region called the “end-plate potential.”

6) The EPP is recorded by placing a microelectrode into the muscle cell near the end-plate.

a) A typical EPP is about 50-60 mV in amplitude. It is usually not seen because as the muscle membrane is depolarized to threshold, voltage-sensitive Na channels on the muscle membrane are activated leading to an all-or-none Action Potential.

b) But it is possible to reduce the amplitude of the EPP to below threshold using curare, a South American alkaloid used by natives for arrow poison.

c) Curare binds to the ACh receptor, and competes with ACh. Although curare binds to Ach receptors, it cannot open the receptor channel, and thus cannot depolarize the end-plate membrane. Therefore, the ACh released from vesicles produces less depolarization of the end-plate membrane.

d) Curare was used in the past as an agent for muscle paralysis to permit surgery especially around joints. It has since been replaced by faster acting and more controllable agents.

7) Characterization of the Acetylcholine Receptor

1) Karlin and Changeux are primarily responsible for our understanding of the structure and function of the nicotinic ACh receptor (AChR).

2) The Nicotinic AChR consists of five subunits: two α’s, β, γ and δ. For maximum effect two ACh molecules must bind to the receptor, one at each alpha site.

3) Once ACh is bound the channel opens, and Na+ flow into the cell down its electrical and chemical gradient; K+ flows out down its chemical gradient. Bernard Katz termed this “a chemical puncture” of the end-plate membrane.

* How does the EPP lead to a Muscle Action Potential?

a) The end-plate is usually found in the center of a muscle fiber.

b) Recall the discussion about conduction of a nerve action potential. Depolarization of the end-plate membrane causes a discharge of the capacitance in the surrounding muscle membrane. Eventually enough charge is drawn off of this capacitance to bring the muscle membrane to – 60 mV, the threshold for voltage sensitive Na-chann.

c) The voltage-sensitive Na channels are located in the depths of the end-plate folds and in the muscle membrane away from the end-plate.

d) As these voltage-gated Na channels reach threshold, the activation gates open and (just as in nerve axons) a regenerative muscle action potential occurs.

e) The AP is conducted from the center of the muscle fiber outward.This eventually leads to muscle contraction (discussed later ).

Disorders of Neuromuscular Transmission

Disorders of neuromuscular transmission affect the neuromuscular junction. They may involve

• Postsynaptic receptors (eg, in myasthenia gravis)
• Presynaptic release of acetylcholine (eg, in botulism)
• Breakdown of acetylcholine within the synapse (eg, due to drugs or neurotoxic chemicals)

Common features of these disorders include fluctuating fatigue and muscle weakness with no sensory deficits.

Eaton-Lambert syndrome: This disorder is due to impaired acetylcholine release from presynaptic nerve.

Botulism: Also due to impaired release of acetylcholine from presynaptic nerve terminals, botulism develops when toxin produced by Clostridium botulinum spores irreversibly binds to the terminal cholinergic nerve endings. The result is severe weakness, sometimes with respiratory compromise. Other systemic symptoms may include mydriasis, dry mouth, constipation, urinary retention, and tachycardia due to unopposed sympathetic nervous system activity (anticholinergic syndrome). These systemic findings are absent in myasthenia gravis..

Drugs or toxic chemicals: Cholinergic drugs, organophosphate insecticides, and most nerve gases block neuromuscular transmission by excessive acetylcholine action that depolarizes postsynaptic receptors. Miosis, bronchorrhea, and myasthenic-like weakness (cholinergic syndrome) result.

Aminoglycoside and polypeptide antibiotics decrease presynaptic acetylcholine release and sensitivity of the postsynaptic membrane to acetylcholine. At high serum levels, these antibiotics may increase neuromuscular block in patients with latent myasthenia gravis.

Other Disorder:

1.Myasthenia gravis

2.Neuromyotonia

3.Lambert-Eaton myasthenic symptoms

Treatment :

1. Myasthenia gravis

· Acetylcholine esterase inhibitors

· Short-term plasma exchange treatment

· Intravenous immunoglobulin (IvIg)

· Thymectomy

· Oral corticosteroids (with a bisphosphonate and antacid)

· Azathioprine

· Other immunosuppressants

2. Lambert-Eaton myasthenic syndrome

· 3,4-diaminopyridine with or without pyridostigmine

· IvIg

· Treatment of the underlying tumor

· Immunosupressive treatment

3. Neuromyotonia

· Antiepileptic drugs

· Treatment of the underlying tumor

· Immunomodulatory therapiedrome

Conclusion :

To identify and investigate any dysfunction of neuromuscular transmission in episodic cluster headache. Abnormal neuromuscular transmission has been shown in migraine with aura and in migraine without aura by using single fiber electromyography. Especially for migraine with aura, a genetic cause has been postulated. Episodic cluster headache is a primary headache disorder in which genetic factors may, at times, play a strong role. Methods.-Single fiber electromyography during voluntary contraction of the extensor digitorum communis muscle, nerve conduction studies of upper and lower extremities.