The development of isolated central nervous system preparations

1. Use of invertebrate isolated CNS preparations showed that identified neurons have distinct specific repeatable pharmacological properties. 2. Isolated CNS preparations have been developed from invertebrates to include fish, amphibian, reptile and mammal CNS preparations. 3. The isolated mammalian (mouse, hamster, rat, guinea pig) CNS preparation can remain alive for up to 72 hr and show cells with good resting potentials, action potentials; good functional pathways and tracts; and good responses to drug application. 4. Some possible future developments of isolated mammalian CNS preparations are described.     

Molecular cloning of G protein alpha subunits from the central nervous system of the mollusc Lymnaea stagnalis.

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein alpha subunits that are expressed in the Lymnaea CNS, i.e. G alpha o, G alpha s and G alpha i. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation of G protein alpha subunits suggests that functional insights into G protein-mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.     

Contact inhibition in the failure of mammalian CNS axonal regeneration

Anamniote animals, such as fish and amphibians, are able to regenerate damaged CNS nerves following injury, but regeneration in the mammalian CNS tracts, such as the optic nerve, does not occur. However, severed adult mammalian retinal axons can regenerate into peripheral nerve segments grafted into the brain and this finding has emphasized the importance of the environment in explaining regenerative failure in the adult mammalian CNS. Following lesions, regenerating axons encounter the glial cells, oligodendrocytes and astro-cytes, and their derivatives, respectively myelin and the astrocytic scar. Experiments to investigate the influence of these components on axon growth in culture have revealed cell-surface and extracellular matrix molecules that inhibit axon extension and growth cone motility. Structural and functional characterization of these ligands and their receptors is underway, and may solve the interesting neurobiological conundrum posed by the failure of mammalian CNS regeneration. Simultaneously, this might allow new possibilities for treatment of the severe clinical disabilities resulting from injury to the brain and spinal cord.     

Growth of axons through a lesion in the intact CNS of fetal rat maintained in long-term culture.

The ability of neurons in the central nervous system (CNS) to grow through a lesion and restore conduction has been analysed in developing spinal cord in vitro. The preparation consists of the entire CNS of embryonic rat, isolated and maintained in culture. Conduction of electrical activity and normal morphological appearance (light microscopical and electron microscopical) were maintained in the spinal cord of such preparations for up to 7 d in culture. A complete transverse crush of the spinal cord abolished all conduction for 2 d. After 3-5 d, clear recovery had occurred: electrical conduction across the crush was comparable with that in uninjured preparations. Furthermore, the spinal cord had largely regained its gross normal appearance at the crush site. Axons stained in vivo by carbocyanine dyes had, by 5 d, grown in profusion through the lesion and several millimetres beyond it. These experiments, like those made in neonatal opossum (Treherne et al. 1992) demonstrate that central neurons of immature mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth of nerve fibres in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors. However, unlike the opossum, in which outgrowth occurred at 24 degrees C, although there was prolonged survival of rat spinal cords at this temperature, outgrowth of axons across the lesion required a temperature of 29 degrees C. With rapid and reliable regeneration in vitro it becomes practicable to assay the effects of molecules that promote or inhibit restoration of functional connections.     

Reorganization of the human central nervous system

The key strategies on which the discovery of the functional organization of the central nervous system (CNS) under physiologic and pathophysiologic conditions have been based included (1) our measurements of phase and frequency coordination between the firings of alpha- and gamma-motoneurons and secondary muscle spindle afferents in the human spinal cord, (2) knowledge on CNS reorganization derived upon the improvement of the functions of the lesioned CNS in our patients in the short-term memory and the long-term memory (reorganization), and (3) the dynamic pattern approach for re-learning rhythmic coordinated behavior. The theory of self-organization and pattern formation in nonequilibrium systems is explicitly related to our measurements of the natural firing patterns of sets of identified single neurons in the human spinal premotor network and re-learned coordinated movements following spinal cord and brain lesions. Therapy induced cell proliferation, and maybe, neurogenesis seem to contribute to the host of structural changes during the process of re-learning of the lesioned CNS. So far, coordinated functions like movements could substantially be improved in every of the more than 100 patients with a CNS lesion by applying coordination dynamic therapy. As suggested by the data of our patients on re-learning, the human CNS seems to have a second integrative strategy for learning, re-learning, storing and recalling, which makes an essential contribution of the functional plasticity following a CNS lesion. A method has been developed by us for the simultaneous recording with wire electrodes of extracellular action potentials from single human afferent and efferent nerve fibres of undamaged sacral nerve roots. A classification scheme of the nerve fibres in the human peripheral nervous system (PNS) could be set up in which the individual classes of nerve fibres are characterized by group conduction velocities and group nerve fibre diameters. Natural impulse patterns of several identified single afferent and efferent nerve fibres (motoneuron axons) were extracted from multi-unit impulse patterns, and human CNS functions could be analyzed under physiologic and pathophysiologic conditions. With our discovery of premotor spinal oscillators it became possible to judge upon CNS neuronal network organization based on the firing patterns of these spinal oscillators and their driving afferents. Since motoneurons fire occasionally for low activation and oscillatory for high activation, the coherent organization of subnetworks to generate macroscopic function is very complex and for the time being, may be best described by the theory of coordination dynamics. Since oscillatory firing has also been observed by us in single motor unit firing patterns measured electromyographically, it seems possible to follow up therapeutic intervention in patients with spinal cord and brain lesions not only based on the activity levels and phases of motor programs during locomotion but also based on the physiologic and pathophysiologic firing patterns and recruitment of spinal oscillators. The improvement of the coordination dynamics of the CNS can be partly measured directly by rhythmicity upon the patient performing rhythmic movements coordinated up to milliseconds. Since rhythmic dynamic, coordinated, stereotyped movements are mainly located in the spinal cord and only little supraspinal drive is necessary to initiate, maintain, and terminate them, rhythmic, dynamic, coordinated movements were used in therapy to enforce reorganization of the lesioned CNS by improving the self-organization and relative coordination of spinal oscillators (and their interactions with occasionally firing motoneurons) which became pathologic in their firing following CNS lesion. Paraparetic, tetraparetic spinal cord and brain-lesioned patients re-learned running and other movements by an oscillator formation and coordination dynamic therapy. Our development in neurorehabilitation is in accordance with those of theoretical and computational neurosciences which deal with the self-organization of neuronal networks. In particular, jumping on a springboard 'in-phase' and in 'anti-phase' to re-learn phase relations of oscillator coupling can be understood in the framework of the Haken-Kelso-Bunz coordination dynamic model. By introducing broken symmetry, intention, learning and spasticity in the landscape of the potential function of the integrated CNS activity, the change in self-organization becomes understandable. Movement patterns re-learned by oscillator formation and coordination dynamic therapy evolve from reorganization and regeneration of the lesioned CNS by cooperative and competitive interplay between intrinsic coordination dynamics, extrinsic therapy related inputs with physiologic re-afferent input, including intention, motivation, supervised learning, interpersonal coordination, and genetic constraints including neurogenesis. (ABSTRACT TRUNCATED)     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

Imaging Brain Activity With Voltage- and Calcium-Sensitive Dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.     

Vimentin in the Central Nervous System

Intermediate filament proteins were identified by two-dimensional gel electrophoresis in urea extracts of rat optic nerves undergoing Wallerian degeneration and in cytoskeletal preparations of rat brain and spinal cord during postnatal development. The glial fibrillary acidic (GFA) protein and vimentin were the major optic nerve proteins following Wallerian degeneration. Vimentin was a major cytoskeletal component of newborn central nervous system (CNS) and then progressively decreased until it became barely identifiable in mature brain and spinal cord. The decrease of vimentin occurred concomitantly with an increase in GFA protein. A protein with the apparent molecular weight of 61,000 and isoelectric point of 5.6 was identified in both cytoskeletal preparations of brain and spinal cord, and in urea extracts of normal optic nerves. The protein disappeared together with the polypeptides forming the neurofilament triplet in degenerated optic nerves.     

The dorsal root reflex in isolated mammalian spinal cord

1. The dorsal root reflex has been investigated in an isolated preparation of adult mammalian spinal cord. 2. Both evoked and spontaneous activity can be recorded from the cord in the dorsal spinal roots. 3. The spontaneous activity has a characteristic pattern of firing in bursts of action potentials. Spontaneous and evoked activity are optimum at temperatures between 25 and 27 degrees C; little activity can be detected above 35 degrees C. 4. The spontaneous dorsal root activity has been shown to be correlated with negative potentials in the dorsal horn of the cord, and intracellular recordings made from primary afferent fibres have shown spontaneous primary afferent depolarizations (PAD) which underlie the generation of the spontaneous dorsal root activity. 5. The evoked dorsal root reflex has been shown to spread up to 16 spinal segments both rostrally and caudally from the stimulated dorsal root, and to the contralateral side of the cord. 6. The spontaneous dorsal root activity in widely separated segments has been shown by cross-correlation analysis to be linked both ipsi- and contra-laterally. 7. The significance of such a widespread system for the generation of PAD is discussed.     

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.     

Clonal analysis of astrocyte diversity in neonatal rat spinal cord cultures.

Within the mammalian CNS, astrocytes appear to be a heterogeneous class of cells. To assay the number of distinct types of astrocytes in the rat spinal cord, cell lineage and phenotypic analyses were carried out on cultures from newborn rat spinal cord and five distinct types of astrocytes were observed. Proliferating precursors for each class of astrocyte were isolated by low density culture and shown to give rise to 5 distinct and morphologically homogeneous clusters of GFAP + astrocytes. Immunocytochemical analysis with antibodies A2B5 and Ran-2, which identify different glial lineages in optic nerve cultures, demonstrated that many clusters included both A2B5+ and A2B5- cells. Similarly, many clusters also possessed a mixture of Ran-2+ and Ran-2-cells, suggesting that in spinal cord cultures, in contrast to optic nerve cultures, expression of these antigens is regulated by individual cells rather than by cell lineage. Single-cell cloning studies, revealed that the abundance and proliferative capacity of individual astrocyte precursors differed depending on the type of astrocyte. To assay the effects of a complex cellular environment on the composition of astrocyte clones, lineage analysis was performed in complete spinal cord cultures using a replication deficient retrovirus. Although similar morphologically homogeneous clones of cells to those seen with single-cell clones were observed, the proliferative capacity and relative abundance of the distinct astrocyte precursors differed from that seen in single-cell cloning studies. Together these observations suggest that in spinal cord, gliogenesis is considerably more complex than in the optic nerve and that cultures of newborn rat spinal cord contain multiple, distinct populations of astrocytes.     

Formation of functional synaptic connections between heterogeneous brain formations in organotypic nerve tissue culture

The principles of oriented growth of nerve fibers and the formation of functional synaptic connections during combined culture of brain structures with no direct anatomical or functional connections (the spinal cord and olfactory bulbs of mouse embryos) were investigated by neuromorphological and electrophysiological methods. During the first week of culture connections, mainly glio-neuronal bridges, formed between the explants of spinal cord and olfactory bulb. Glial cells forming an oriented substrate, facilitating growth and fasciculation (the formation of bundles) of axons that developed subsequently, play an active role in the formation of such connections. In preparations impregnated with silver, connections formed by bundles of axons or by single nerve fibers were seen between the explants. The results of electrophysiological investigations of combined cultures of heterogeneous brain structures showed that by the second week of culture functional synaptic connections have formed between explants of spinal cord and olfactory bulbs. Electrical stimulation of spinal cord explants led to the appearance of short- and long-latency unit responses in explants of the olfactory bulbs. The formation of nonspecific functional synaptic connections for these brain structures during combined culture, revealed by this investigation, is evidence of the high level of morphogenetic plasticity of growing or regenerating axons and of the active role of neuroglial cells in preparation and provision for oriented growth of nerve fibers.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 490–497, September–October, 1980.     

Recovery of CNS Pathway Innervating the Sciatic Nerve Following Transplantation of Human Neural Stem Cells in Rat Spinal Cord Injury

Stem cell research has been attained a greater attention in most fields of medicine due to its potential for many incurable diseases through replacing or helping the regeneration of damaged cells or tissues. Here, we demonstrated the functional recovery and structural connection of the central nervous system pathway innervating the sciatic nerve after total transection of the spinal cord followed by the transplantation of human neural stem cells (hNSC) in the injured rat spinal cord site. The limb function of hNSC-treated group recovered dramatically compared with that in the sham group by Basso–Beattie–Bresnahan (BBB) scores. Transplanted hNSC differentiated into astrocytes and neurons in the injured site. In addition, immunohistochemistry for growth-associated protein 43 showed axonal regeneration in the injured spinal cord site. The pseudorabies viral-Ba (PRV-Ba) tracing method revealed that transplanted hNSC and their differentiated neurons showed positive labeling after sciatic nerve injection. In addition, the PRV-Ba labeling was also observed in several nuclei in the brain innervating the sciatic nerve. This result implies that the rat CNS motor pathway could be reconstructed by hNSC transplantation, and it may contribute to the functional recovery of the limb.     

Synaptic modulation of neuronal coupling

Electrotonic coupling among neurons in the vertebrate, and more specifically the mammalian, brain has now been demonstrated to exist in all major brain subdivisions and in the spinal cord. For many of these brain areas, recent studies have investigated the possibilities of modulation of that coupling by synaptically released transmitters and/or neuromodulators. Reviewed here is the evidence for coupling, the synaptically related factors that play roles in up- or downregulation of this type of intercellular interaction and, to the extent that they have been investigated, the intracellular mechanisms operative in changing the extent of coupled networks in the brain. The functional significance of coupling and its modulation is discussed for some of these areas.     

The pharmacological characteristics, immunochemical identification and study of the location of quisqualate receptors in the rat and human brains]

The kinetics of [3H]-L-glutamate binding to brain synaptic membranes (SM) and to glutamate-binding proteins (GBP) was determined with agonist and monoclonal antibodies (MAbs). It was revealed, that rat and human brain GBP have individual protein components with M(r) from 14 to 92 kDa. Quisqualate inhibited [3H]-L-glutamate binding to solubilized and to purified 68 kDa protein component. MAbs have the most activity, and NMDA was failure. It has been shown that 68 kDa component antigen determinants are similar to those of bovine, frog and rat brain synaptic membranes. Anti-GBP monoclonal antibodies blocked functional non-NMDA receptors in isolated frog spinal cord. Immunocytochemistry was done on rat and human brain sections. Distribution of quisqualate receptors was determined with light and electron microscopy. Some properties of vertebrate CNS non-NMDA receptors are discussed.     

Chordate origins of the vertebrate central nervous system.

Fine structural, computerized three-dimensional (3D) mapping of cell connectivity in the amphioxus nervous system and comparative molecular genetic studies of amphioxus and tunicates have provided recent insights into the phylogenetic origin of the vertebrate nervous system. The results suggest that several of the genetic mechanisms for establishing and patterning the vertebrate nervous system already operated in the ancestral chordate and that the nerve cord of the proximate invertebrate ancestor of the vertebrates included a diencephalon, midbrain, hindbrain, and spinal cord. In contrast, the telencephalon, a midbrain-hindbrain boundary region with organizer properties, and the definitive neural crest appear to be vertebrate innovations.     

Monoaminergic Modulation of Spinal Viscero-Sympathetic Function in the Neonatal Mouse Thoracic Spinal Cord

Descending serotonergic, noradrenergic, and dopaminergic systems project diffusely to sensory, motor and autonomic spinal cord regions. Using neonatal mice, this study examined monoaminergic modulation of visceral sensory input and sympathetic preganglionic output. Whole-cell recordings from sympathetic preganglionic neurons (SPNs) in spinal cord slice demonstrated that serotonin, noradrenaline, and dopamine modulated SPN excitability. Serotonin depolarized all, while noradrenaline and dopamine depolarized most SPNs. Serotonin and noradrenaline also increased SPN current-evoked firing frequency, while both increases and decreases were seen with dopamine. In an in vitro thoracolumbar spinal cord/sympathetic chain preparation, stimulation of splanchnic nerve visceral afferents evoked reflexes and subthreshold population synaptic potentials in thoracic ventral roots that were dose-dependently depressed by the monoamines. Visceral afferent stimulation also evoked bicuculline-sensitive dorsal root potentials thought to reflect presynaptic inhibition via primary afferent depolarization. These dorsal root potentials were likewise dose-dependently depressed by the monoamines. Concomitant monoaminergic depression of population afferent synaptic transmission recorded as dorsal horn field potentials was also seen. Collectively, serotonin, norepinephrine and dopamine were shown to exert broad and comparable modulatory regulation of viscero-sympathetic function. The general facilitation of SPN efferent excitability with simultaneous depression of visceral afferent-evoked motor output suggests that descending monoaminergic systems reconfigure spinal cord autonomic function away from visceral sensory influence. Coincident monoaminergic reductions in dorsal horn responses support a multifaceted modulatory shift in the encoding of spinal visceral afferent activity. Similar monoamine-induced changes have been observed for somatic sensorimotor function, suggesting an integrative modulatory response on spinal autonomic and somatic function.     

Developmental genetic evidence for a monophyletic origin of the bilaterian brain

The widely held notion of an independent evolutionary origin of invertebrate and vertebrate brains is based on classical phylogenetic, neuroanatomical and embryological data. The interpretation of these data in favour of a polyphyletic origin of animals brains is currently being challenged by three fundamental findings that derive from comparative molecular, genetic and developmental analyses. First, modern molecular systematics indicates that none of the extant animals correspond to evolutionary intermediates between the protostomes and the deuterostomes, thus making it impossible to deduce the morphological organization of the ancestral bilaterian or its brain from living species. Second, recent molecular genetic evidence for the body axis inversion hypothesis now supports the idea that the basic body plan of vertebrates and invertebrates is similar but inverted, suggesting that the ventral nerve chord of protostome invertebrates is homologous to the dorsal nerve cord of deuterostome chordates. Third, a developmental genetic analysis of the molecular control elements involved in early embryonic brain patterning is uncovering the existence of structurally and functionally homologous genes that have comparable and interchangeable functions in key aspects of brain development in invertebrate and vertebrate model systems. All three of these findings are compatible with the hypothesis of a monophyletic origin of the bilaterian brain. Here we review these findings and consider their significance and implications for current thinking on the evolutionary origin of bilaterian brains. We also preview the impact of comparative functional genomic analyses on our understanding of brain evolution.     

Species-specific effects on the optical signals of voltage-sensitive dyes

Summary The absorption changes of two merocyanine dyes in response to membrane potential changes were measured on several nueronal preparations to see whether the dyes would be useful in recording from these cells.We were able to record large signals without averaging from barnacle and leech neurons. The greatest signal with WW375 was seen at 750 nm. Much smaller increases in transmitted light intensity were seen at all other wavelengths between 500 and 780 nm. In contrast, vertebrate neuronal preparations produced much smaller signals with an entirely different action spectrum. Essentially the same spectrum was seen in cells of the sympathetic ganglion of the bullfrog,Rana catesbiana, dissociated chick spinal cord neurons, or dissociated rat superior cervical ganglion neurons. In each case an action potential was accompanied by increases in transmitted light intensity between 500 and 600 nm and 730 and 780 nm, and decreases in intensity between 600 and 730 nm with the dye WW375, the best dye tested. Similar results were obtained with dye NK2367 on both vertebrate and invertebrate preparations, except that the spectral properties were shifted 30 nm towards the blue. Both dyes caused some photodynamic damage to the cultured neurons after a few minute's exposure to the illuminating light. Several analogues of these dyes were also tried, but did not produce larger signals.