Evaluating the Effectiveness of Geant4 Software in Measuring the Damage Caused by Ti48 Ion Radiation on Nerve Cells, in Comparison to the Biophysical Model and Empirical Data.

A Major Role for Intracortical Circuits in the Strength and Tuning of Odor-Evoked Excitation in Olfactory Cortex

The brain is a vast collection of excitable nerve cells called neurones separated by a mass of supporting cells called glial cells. Each neurone has a cell body and a large number of cell processes, which reach out to ‘connect’ with other nerve cells in the brain and other cells outside the central nervous system. A good deal is known about how individual nerve cell function because they can be studied in the peripheral nervous system, and in the primitive nervous systems of invertebrate animals, e.g. squid where there are only a few cells in simple arrangement. The ‘connections’ between nerve cells are not electrical but chemical. Neurones are excited to transmit electrical stimuli when specialized receptor areas on their cell surfaces are stimulated by a specific neurotransmitter released by another nerve cell. Both inhibitory and excitory neurotransmitters have been identified, and the state of excitation of any given nerve cell is thought to depend on the balance of inhibition and excitation at the many thousands of different interconnections or synapses on the neuronal surface. At individual synapses the rate of release and disposal of the different neurotransmitters is critical. The firing of an individual neurone is complex enough, but each neurone makes only a tiny contribution to the overall activity of the whole organ. How the integration of activity takes place is largely unknown, although the analogy of a huge computer is an attractive one. For the simpler functions, like control of body movement pathways have been defined and a lot is known about them, at least anatomically. However, when more complex functions such as thinking or memory are considered, it must be admitted that we are almost totally ignorant as to how these functions are conducted. Is a memory stored in the brain as a complex molecule, a sophisticated version of a knot in a handkerchief, or is it stored as an electrical charge in an individual nerve cell? We simply do not know the answer to these very basic questions.

Response Properties of Motion-Sensitive Visual Interneurons in the Lobula Plate of Drosophila melanogaster

In the past 15 years, there has been renewed interest in the detailed spatial analyses of signalling in individual neurons. The behaviour of many nerve cells is difficult to understand on the basis of microelectrode measurements from the soma. Regional electrical properties of neurons have been studied using sharp microelectrode and patch-electrode recordings from neuronal processes, high-resolution multisite optical recordings of Ca2+ concentration changes and by using models to predict the distribution of membrane potential in the entire neuronal arborization. Additional, direct evidence about electrical signalling in neuronal processes of individual cells in situ can now be obtained by recording of membrane potential changes using voltage-sensitive dyes. A number of recent studies have shown that active regional electrical properties of individual neurons are extraordinarily complex, dynamic and, in the general case, impossible to predict by present models. This places a great significance on measuring capabilities in experiments studying the detailed functional organization of individual neurons. The main difficulty in obtaining a more accurate description was that experimental techniques for studying regional electrical properties of neurons were not available. With this motivation, we worked on the development of multisite voltage-sensitive dye recording as a potentially powerful approach. The results described here demonstrate that the sensitivity of voltage-sensitive dye recording from branches of individual neurons was brought to a level at which it can be used routinely in physiologically relevant experiments. The crucial figure-of-merit in this approach, the signal-to-noise ratio from neuronal processes in intact ganglia, has been improved by a factor of roughly 150 over previously available signals. The improvement in the sensitivity allowed, for the first time, direct investigation of several important aspects of the functional organization of an individual neuron: (1) the direction and the velocity of action potential propagation in different neuronal processes in the neuropile was determined; and (2) the interaction of two independent action potentials (spike collision) was monitored directly in a neurite in the neuropile; (3) it was demonstrated that several action potentials are initiated in the same neuron at different sites (multiple spike trigger zones) by a single stimulus; (4) the exact location and the size of one of the remote spike trigger zones was determined; (5) the spread of passive subthreshold signals was followed in the neurites in the neuropile. This kind of information was not previously available. Preliminary experiments on vertebrate neurons indicate partial success in the effort to use intracellularly applied voltage-sensitive dyes to record from neurons in a mammalian brain slice preparation. The results suggest that, with further improvements, it may be possible to follow optically synaptic integration and spike conduction in the dendrites of vertebrate nerve cells. The main impact of these results is a demonstration of a new way of analysing how individual neurons are functionally organized. Limitations and prospects for the further refinement of the technique are discussed mostly in terms of the signal-to-noise ratio; both improvements in the apparatus and design of more sensitive dyes are addressed.

The relationship between urodele regeneration and possible regeneration in mammalian prospects is hard to evidence, but the idea of possible regeneration of neural elements in people is an area of potential clinical importance that is under investigation. One of the great challenges of the future is to understand enough about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is well established that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibres connected to their cell bodies. The nerve fibres severed at the limb amputation level, regrow and invade the blastema, providing blastema cells with indispensable factors. These factors are elaborated within the neuron perikarya and transported via their axons to the blastema. Numerous studies have been so far performed and have elucidated the quantitative relationships between nerve fibres and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of the present investigation was to analyse the quantitative participation and qualitative distinction of nerve cells innervating regenerating parts of the urodele limb and their possible interrelationship with the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral aspect of the spinal cord grey matter (motor neurons). As a means of visualizing the direct implication of these neurons during various regeneration periods, the enzyme horseradish peroxidase was chosen. A total of 34 animals were used, 21 experimental and 13 controls, in order to study labeled nerve cell fluctuations. The results are summarized as follows: (a) The first nerve cells incorporating HRP within 5 days post amputation are found in the dorsal ganglia. Motor neurons in the grey matter are labeled within 7 days. (b) The number of labeled perikarya increases during the nerve-dependent regeneration period (0-21 dpa). The percentage of implicated sensory neurons exceeds that found in the control series. (c) During the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration from the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.

Animal behaviour and movement emerges from the stimulation of nerve cells that are connected together like a circuit. Researchers use various tools to investigate these neural networks in model organisms such as roundworms, fruit flies and zebrafish. The trick is to activate some nerve cells, but not others, so as to isolate their specific role within the neural circuit. One way to do this is to switch genes on or off in individual cells as a way to control their neuronal activity. This can be achieved by building a photocaged version of the enzyme Cre recombinase which is designed to target specific genes. The modified Cre recombinase contains an amino acid (the building blocks of proteins) that inactivates the enzyme. When the cell is illuminated with UV light, a part of the amino acid gets removed allowing Cre recombinase to turn on its target gene. However, cells do not naturally produce these photocaged amino acids. To overcome this, researchers can use a technology called genetic code expansion which provides cells with the tools they need to build proteins containing these synthetic amino acids. Although this technique has been used in live animals, its application has been limited due to the small amount of proteins it produces. Davis et al. therefore set out to improve the efficiency of genetic code expansion so that it can be used to study single nerve cells in freely moving roundworms. In the new system, named LaserTAC, individual cells are targeted with UV light that ‘uncages’ the Cre recombinase enzyme so it can switch on a gene for a protein that controls neuronal activity. Davis et al. used this approach to stimulate a pair of neurons sensitive to touch to see how this impacted the roundworm’s behaviour. This revealed that individual neurons within this pair contribute to the touch response in different ways. However, input from both neurons is required to produce a robust reaction. These findings show that the LaserTAC system can be used to manipulate gene activity in single cells, such as neurons, using light. It allows researchers to precisely control in which cells and when a given gene is switched on or off. Also, with the improved efficiency of the genetic code expansion, this technology could be used to modify proteins other than Cre recombinase and be applied to other artificial amino acids that have been developed in recent years.

As mentioned in Chapter 1, the prime function of nervous systems is one of communication: communication within the organism itself and with the external environment, including communication with other organisms. Furthermore, the individual nerve cells that make up the nervous system communicate with one another and with a variety of cells (effector cells) that carry out the nervous system’s commands. These effector cells include, muscle cells (striated, smooth and cardiac) and a variety of secretory (gland) cells. The nerve impulse is a mechanism whereby these commands may be sent quickly from one end of a nerve cell to the other. In general, there is no cytoplasmic continuity between individual nerve cells or between nerve cells and effector cells. This is the neuron doctrine of Ramon y Cajal, which, in turn, is the cell theory of Schleiden and Schwann applied to the nervous system. Neurons contact one another at synapses (a term coined by Sherrington) or they contact muscle cells at neuromuscular junctions and gland cells at neuro-glandular junctions. It is a convenient shorthand, which has become common usage, to call all these contacts “synapses”.

The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

The central nervous system of the leech has been used for the study of the formation of new synaptic connections by regenerating neurons. In control leeches, individual nerve cells in adjacent ganglia are connected in an orderly and stereotyped manner, with only little variation. In the present experiments, a bundle of axons running between two of the segmental ganglia has been severed and allowed to regenerate. Subsequently, the axons reestablish synaptic connections between certain identified nerve cells in the adjacent ganglia, selectively and accurately. Thus, individual sensory cells in one ganglion show a high degree of neural specificity in reestablishing cell to cell connections with a motor cell in the next ganglion. The performance of the regenerated synapses, however, is significantly altered in a consistent manner. The normal balance between the effects of inhibitory and excitatory innervation in leeches with regenerated synapses is different from that seen in normal leeches, with marked overemphasis on inhibition. Similar alterations have also been seen in a series of ganglia at a distance from the site of the lesion. After the operation, therefore, a widespread modification of synapses occurs along the length of the nerve cord.

A modification of the analytical method employing the appropriate 4-methylumbelliferyl-β-d-glycoside as substrate and measuring the fluorescence of the 4-methylumbelliferone freed by hydrolysis permitted the measurement of β-galactosidase, β-glucuronidase, and β-glucosidase in individual anterior horn cell bodies, in individual dorsal ganglion cell bodies, and in pieces of anterior horn neuropil approximately equivalent in weight (0.01 µg) to one individual cell body. Incubation for 18 hours was necessary to measure the two latter enzymes in a single cell, whereas it was necessary to incubate β-galactosidase for only 4 hours because of its higher activity. When incubation was for 18 hours, the fall-off of activity for β-glucuronidase was negligible, but for β-galactosidase the fall-off in activity was about 57%. The results for β-glucosidase were somewhat anomalous in that the fall-off activity with time for cerebellar molecular layer was very different (over 10-fold) from the fall-off for nerve cells, neuropil, or other cerebellar layers (average of 2.5-fold). The concentration of each of these three enzymes was extremely rich in nerve cell bodies when compared with the neuropil in the cord, varying from 14- to 27-fold for dorsal ganglion cell bodies, and from 8- to 10-fold for anterior horn cell bodies. These results contrast strikingly with those found by others and ourselves in which levels of carbohydrate-metabolizing enzymes, glutamate-metabolizing enzymes, and a dipeptidase in nerve cell bodies were found to be equal to or lower than the levels in neuropil and in other parts of the nervous system.

Spike activity and histofluorescence correlated in the giant dopamine neuron of Planorbis corneus

Beneficial Effects of Combined Antiangiogenesis and Heavy Ion Radiation Therapy

Nerve cells of the human striatum were investigated with the use of a newly developed technique that reveals the pattern of pigmentation of individual nerve cells by means of transparent Golgi impregnations of their cell bodies and processes. Five types of neurons are distinguished: Type I is a medium-sized spine-laden neuron with an axon giving off a great number of collateral branches. The vast majority of the cells in the striatum belong to this type. Numerous intensely stained lipofuscin granules are contained in one pole of the cell body and may also extend into adjacent portions of a dendrite. Type II is a medium-sized to large neuron with long intertwining dendrites decorated with spines of uncommon shape. A distinguishing feature of this cell type is the presence of somal spines. This cell type is devoid of pigment or contains only a few tiny lipofuscin granules. Type III is a large multipolar neuron. The cell body generates a few rather extended dendrites that are very sparsely spined. The finely granulated pigment is evenly dispersed within a large portion of the cytoplasm. Type IV is a large aspiny neuron with rounded cell body and richly branching tortuous dendrites. The axon branches frequently in the vicinity of the parent soma. Large pigment granules are concentrated within a circumscribed part of the cell body close to the cell membrane. Type V is a small to medium-sized aspiny neuron. The dendrites break up into a swirling mass of thin branches. More than one axon may be given off from the soma. The axons branch close to the soma into terminal twigs. Cells of this type contain numerous large and well-stained lipofuscin granules. Each of the cell types has a characteristic pattern of pigmentation. The different varieties of nerve cells in the striatum can therefore be distinguished not only in Golgi impregnations but also in pigment-Nissl preparations.

The nerve fibres of the tractus opticus consist in their greatest majority in minute fibres, and only an extremely small minority in thick fibres. In all probability, the former originate in the small nerve cells of the nerve cell layer of the retina and the latter in the giant cells there. Among the optic fibres and especially among the small bundles of such fibres are found glial cells of two size classes, often ranged in long rosaries.The tractus opticus, upon reaching the lateral geniculate body, divides in two parts, the smaller of which enwraps the body to form a capsule around it, and sending out minute bundles of fibres into the body in their further courses, enters the pulvinar thalami. The greater part of the optic fibres runs into the body from its proximal end in numerous small bundles. Besides, a rather thick branch of the tractus opticus runs right through the cranio-medial part of the geniculate body, which gives out many small bundles into the body and receives a smaller number of axons from the nerve cells in the body, finally to pass over into the brachium corp. quadrig. ant.There is nothing to make the so-called griseum praegeniculatum a singular entity. It is nothing but a cranio-medial invagination of the nucleus corp. genicul. lat., bounded off by the above mentioned branch of the tractus opticus on the lateral side. It is rather to be called the accessory nucleus of the corpus than a praegeniculatum. A part of the axons of the nerve cells in the accessory nucleus goes over into the brachium corp. quandrig. ant., while the other part runs into the radiatio optica through the WERNICKE's field.The zona incerta originates in the vegetative minute fibres making up a part of the pes pedunculi or the capsula interna. These fibres run around the pes pedunculi in an arcuate course, then along the medial side of the accessory nucleus and the corpus geniculatum mediale to appear in the front of the corpus Luysi, and finally to the nucleus ruber probably to come into a close relation with it. Some vegetative fibres emerging at the medial side of the pes pedunculi also partake in the formation of the zona incerta.At the proximal part of the brachium corp. quadrig. ant. there are frequently found groups of nerve cells similar in nature to those in the corpus genicul. lat. This fact makes it all the more indubitable that this brachium is not only an immediate extension of the optic fibres, but also formed in part by the axons of the nerve cells belonging to the nucleus corp. genicul. lat.We have to make further and more detailed examination before settling the problem of the doubtful existence of the BALADO's so-called radiatio cellularum giganticum.The nerve cells in the lateral geniculate body may be classified into the two types of large and small. The large cells are far smaller in number and are chiefly found in the fifth nerve cell layer. This layer is divisible into the inner and the outer sublayers and the large cells are found in the latter in greater abundance. Between these two sublayers I have often found a thin intermediate layer of small nerve cells. Large cells are also found in sporadic arrangement in the basal part of the third nerve cell layer. The small cells are found in all the nerve cell layers and comprise cells of many variable sizes. Both the large and the small cells are pear-shaped, oval, fusiform or spherical in form and have rounded and feminine contours as is common with the sensory nerve cells in the brain stem in general.The large cells are predominantly pear-shaped with their bases toward the periphery of the corpus genicul, lat. An axon from such a cell mostly emerges from its basal face, is thin but dark-staining and often runs into the capsule of optic fibres around the corpus. The short processes, 1-4 in number per cell, generally emerge from the apex of the cell, are lighter-staining but stout and branch out into numerous rami which end in sharp points.

Remarkable methodologic advances in recent years make it possible to study with high precision, sensitivity and accuracy the activities of most enzymes and the levels of most substrates and metabolites in individual cells. The limits and the value of quantitative chemical studies in individual nerve cells are critically evaluated and discussed.