Why does the central nervous system not regenerate after injury?

Spinal cord injuries in humans and in other mammals are never followed by regrowth. In recent years, considerable progress has been made in analyzing mechanisms that promote and inhibit regeneration. The focus of this review is changes that occur in the transition period in development when the central nervous system (CNS) changes from being able to regenerate to the adult state of failure. In our experiments we have used the neonatal opossum (Monodelphis domestica), which corresponds to a 14-day embryonic rat or mouse. The CNS isolated from an opossum pup and maintained in culture shows dramatic regeneration. Fibers grow through and beyond lesions and reform synaptic connections with their targets. Similarly, anesthetized neonatal pups attached to the mother recover the ability to walk after complete spinal cord transection. Although the CNS isolated from a 9-day-old animal will regenerate in vitro, CNS from a 12-day-old will not. This is the stage at which glial cells in the CNS develop. Present research is devoted toward molecular screening to determine which growth-promoting molecules decrease during development, which inhibitory molecules increase, and which receptors on growing axons become altered. Despite progress in many laboratories, major hurdles must be overcome before patients can hope to be treated. Nevertheless, the picture today is not as discouraging as it was: one can think of strategies for research on spinal cord injury so as to promote regeneration and restore function.

Procedures for whole-mount immunohistochemistry and in situ hybridization of immature mammalian CNS.

Whole-mount labeling techniques for staining in invertebrates or lower vertebrates cannot simply be applied to the mammalian central nervous system (CNS) because of its large size. Such techniques if possible would offer advantages over conventional methods based on sections since an immediate and 3-dimensional view of the stained components in a transparent CNS is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The aim of our experiments is to follow developing and regenerating expression of proteins and mRNAs in the CNS of mouse embryos and newborn opossums (Monodelphis domestica). Accordingly, we have devised three techniques applicable to whole-mounts: (i) An effective immunohistochemical procedure. This comprises a peroxidase-antiperoxidase method (PAP-WM) based on protocols initially developed for Xenopus embryos and oocytes, including a variation to detect exogenously applied nucleotide analogs such as 5-bromo-2'-deoxyuridine (PAP[BrdU]-WM). For greater resolution we have introduced a novel gold-silver method (IGSS-WM). (ii) An in situ hybridization procedure (ISH[PAP]-WM) which combines PAP-WM with protocols described for Xenopus. (iii) A deconvolution (optical sectioning) procedure which improves resolution for bright-field microscopy. We show that reliable whole-mount staining can be obtained using isolated CNS aged up to mouse embryonic day 17 and newborn opossum up to 15 days. Examples are shown of preparations in which one can directly localize nerve cells containing neurotransmitters, cytoskeletal proteins, nucleotide analogs and growth factor messages.

Neurite outgrowth through lesions of neonatal opossum spinal cord in culture.

The aim of these experiments was to analyze neurite outgrowth during regeneration of opossum spinal cord isolated from Monodelfis domestica and maintained in culture for 3-5 days. Lesions were made by crushing with forceps. In isolated spinal cords of animals aged 3 days, neurites entered the crush and grew along the basal lamina of the pia mater. Growth cones with pleiomorphic appearance containing vesicles, mitochondria and microtubules were abundant in the marginal zone, as were synaptoid contacts with active zones facing basal lamina. In preparations from animals aged 11-12 days, the lesion site was disrupted and contained only degenerating axons, debris and vesicles. Axons and growth cones entered the edge of the lesion but did not extend into it. Lesions in young animals extended over distances of more than 1 mm and contained no radial glia. The damaged area in older preparations was restricted to the crush site with normal astrocytes, oligodendrocytes and neurons immediately adjacent to the lesion. Thus, similar crushes produced more extensive damage in younger spinal cords that were capable of regeneration than in older cords that were not. Dorsal root ganglion fibers labeled with carbocyanine dye (DiI) were observed by video imaging as they grew through lesions. Individual growth cones examined subsequently by electron microscopy had grown again along pial basal lamina. After 5 days in culture dorsal root stimulation gave rise to discharges in ventral roots beyond the lesion indicating that synaptic connections were formed by growing fibers.

Torque/velocity properties of human knee muscles: peak and angle-specific estimates.

Angle-specific (AS) torque/velocity data have been used to avoid angle related variation in peak torque capacity. However, series elastic structures cause the contractile velocity of active force-producing tissue to differ from external joint velocity except at peak torque. Alternatively, angle related variation may be removed by normalizing peak torque to the isometric maximum at that angular position. The AS, peak (P), and normalized peak (NP) methods were compared in isovelocity knee flexion and extension at velocities between 50 and 250 degrees s-1 for 8 male subjects. The P and NP methods gave more similar torque/velocity relations than the AS method. Further, very little variation in peak torque was attributed to differences in joint angle. Both the P and AS methods illustrate that relative quadriceps/hamstrings torque capability (flexor/extensor ratio) increases slightly with velocity. It is proposed that antagonist muscle torque capabilities should be compared at different angular positions to assess muscular imbalance.

Anterograde and retrograde effects of synapse formation on calcium currents and neurite outgrowth in cultured leech neurons.

The aim of our experiments has been to analyse how formation of chemical synapses affects the distribution of calcium (Ca2+) currents and neurite outgrowth of leech Retzius cells. Previous results showed that Ca2+ currents measured in the initial process or 'stump' of postsynaptic cells were significantly smaller than those in corresponding sites on presynaptic neurons. In the present experiments, neurons were plated together in close apposition as pairs or as triads, with the tip of one Retzius cell touching the soma of another. Ca2+ currents from selected areas of the neuronal surfaces were measured by loose-patch recording before and after the formation of chemically mediated synaptic connections, which developed in about 8 h. With three cells arranged in a row, the last of the series, which was purely postsynaptic (i.e. with no target), also showed a dramatic reduction in Ca2+ currents in its initial segment, compared with the currents seen in either the first cell (purely presynaptic) or the second cell of the chain (which was both postsynaptic to the first cell and presynaptic to the third). This suggests that retrograde as well as anterograde effects on Ca2+ currents occurred as a result of synapse formation: the Ca2+ currents in the middle cell did not decrease although a synapse had been formed on it. To test for additional consequences of synapse formation, neurite outgrowth was measured in postsynaptic cells and in single cells plated on an extract of extracellular matrix containing laminin (ECM-laminin). After 48 h, the total length of neuritic outgrowth in postsynaptic cells was only about one third of that in single cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal serum human chorionic gonadotropin levels in twin pregnancies.

Introduction of combined screening with maternal serum alpha-fetoprotein and human chorionic gonadotropin (MShCG) assays for fetal chromosome defects requires establishment of the normal range for twins. This report documents that the normal range for MShCG between 15 and 19 weeks in twin gestations was 1.84-2.41 multiples of the singleton median. Of the 192 twin pregnancies studied, 31.7 and 47.9 per cent had MShCG values greater than or equal to 2.5 and greater than or equal to 2.0 multiples of the singleton median, respectively.

Presynaptic calcium currents and facilitation of serotonin release at synapses between cultured leech neurones.

1. The role of presynaptic Ca2+ entry in facilitation of transmitter release has been analysed by voltage-clamp measurements at synapses formed in culture by Retzius and P neurones isolated from the central nervous system (CNS) of the leech. The transmitter released by Retzius cells is serotonin. 2. Synaptic transmission persisted in solutions containing raised concentrations of divalent cations, reduced concentrations of Na+, and tetraethylammonium (TEA+) and 4-AP (to block K+ currents). Ca2+ and Sr2+ were more effective in promoting transmitter release than Ba2+, as assessed by the postsynaptic potentials in P cells. The degree and time course of facilitation in Ca2+- and Sr2+-containing solutions were similar to those observed for synapses bathed in normal L-15 medium. 3. Transmitter release depended upon the amplitude and the duration of presynaptic depolarization and inward Ca2+ current. Peak Ca2+ currents and postsynaptic potentials occurred with depolarizing steps to +15 mV. Frequent or prolonged pulses depressed the postsynaptic potentials. 4. Pairs of depolarizing pulses that caused facilitation were accompanied by identical inward Ca2+ currents. These results indicate that the mechanism responsible for facilitated serotonin release must occur following Ca2+ entry and that residual Ca2+ plays a role.

Na+, K+ and Ca2+ currents in identified leech neurones in culture.

1. Na+, K+ and Ca2+ currents have been measured by voltage-clamp in Retzius (R), anterior pagoda (AP) and sensory (pressure, touch and nociceptive) cells dissected from the central nervous system (CNS) of the leech. These cells maintain their distinctive membrane properties and action potential configurations in culture. Currents carried by the individual ions were analysed by the use of channel blockers and by their kinetics. Since the cells are isopotential they can be voltage-clamped effectively. 2. Depolarization, as expected, gave rise to an early inward Na+ current followed by a delayed outward K+ current. In Na+-free medium containing tetraethylammonium (TEA+), and in the presence of 4-aminopyridine (4-AP), inward Ca2+ currents were revealed that inactivated slowly and were blocked by Cd2+ and Mn2+. 3. Na+ and Ca2+ currents were similar in their characteristics in R. AP and sensory neurones. In contrast, K+ currents showed marked differences. Three principal K+ currents were identified. These differed in their time courses of activation and inactivation and in their responses to Ca2+ channel blockers. 4. K+ currents of the A-type (IA) activated and inactivated rapidly, were not affected by Ca2+ channel blockers and were eliminated by steady-state inactivation at holding potentials of -30 mV. A-type K+ currents were found in AP cells and as a minor component of the outward current in R cells. A Ca2+-activated K+ current (IC), that inactivated more slowly and was reduced by Ca2+ channel blockers, constituted the major outward current in R cells. The third K+ current resembled the delayed rectifier currents (IK1 and IK2) of squid axons with slow activation and inactivation kinetics. Such currents were found in R cells and in the sensory neurones (T, P and N). 5. The principal differences in membrane properties of identified leech neurones can be explained in terms of the numbers of Na+ channels and the distinctive kinetics of K+ channels in each type of cell.

Slow depolarizing and hyperpolarizing currents which mediate bursting in Aplysia neurone R15.

Interruption of normal bursting activity by application of a voltage clamp reveals that action potentials in Aplysia neurone R15 are followed by two slow currents that long outlast the currents produced during the action potentials. Similar currents are seen following simulation of an action potential with a brief depolarizing pulse delivered under continuous voltage clamp. One of these currents, herein called ID, is an inward, or depolarizing current 0.5-5 nA in amplitude that reaches a peak 300-500 ms after the action potential. It produces the depolarizing after-potential that follows action potentials in this cell and is responsible also for the grouping together of action potentials into bursts. The second current, herein called IH, is an outward, or hyperpolarizing current 0.1-2 nA in amplitude that reaches a peak in 2-10 s and is still present for many tens of seconds following the action potential. IH mediates the interburst hyperpolarization. Both currents summate temporally during the burst. Despite changes in the amplitude and duration of action potentials during the burst, each action potential adds nearly constant increments to the summated amplitudes of ID and IH. The summated amplitude of ID grows during the first few action potentials and gives rise to the increased rate of depolarization and the increased firing rate seen during the first half of the burst. Due to its slower kinetics, IH summates throughout the burst until its summated amplitude is large enough to cause the cell to hyperpolarize, thereby bringing the burst to an end. When the normal burst is interrupted by application of the voltage clamp, the ID and IH current peaks are followed by a current which approaches a more negative steady-state level with a time course that consists of at least two phases. The first phase is exponential with a time constant of 15-30 s. Under continuous voltage clamp, the current following a train of depolarizing pulses returns to the holding current with a similar time course. These observations, together with time constants for IH that are longer than the interburst interval, suggest that IH is always partially activated during normal bursting. A computer simulation demonstrates that opposing inward and outward currents with different kinetics, i.e. ID and IH, are sufficient to give rise to bursting activity, in the absence of non-linear voltage-dependent conductances. Such voltage-dependent conductances, which are present in the normal cell, contribute to but are not necessary for bursting activity.

Voltage and ion dependences of the slow currents which mediate bursting in Aplysia neurone R15.

The previous paper described a slow depolarizing tail current, ID, and a slow hyperpolarizing tail current, IH, that are activated by action potentials and by brief depolarizing pulses in Aplysia neurone R15. ID and IH are necessary for the generation of bursting pace-maker activity in this cell. In this paper, the voltage and ion dependence of ID and IH are studied in an effort to determine the charge carriers for the two currents. When the slow currents are activated by brief depolarizing pulses delivered under voltage clamp in normal medium, an increase in the size of the pulse of 5-10 mV is usually sufficient to bring about full activation of ID. The apparent threshold in normal medium is approximately -20 mV. In medium in which K+ channels are blocked, full activation of an inward tail current that resembles ID requires increasing the pulse amplitude by only 1-2 mV. In contrast, IH is activated in a graded fashion over a 40 mV range of pulse amplitudes. After activating the currents with action potentials or with supramaximal pulses, ID remains an inward current and IH an outward current over a range of membrane potentials spanning -20 to -120 mV. In normal medium, ID is dependent on both extracellular Na+ concentration ( [Na+]o) and extracellular Ca2+ concentration ( [Ca2+]o). When K+ channels are blocked, ID can be supported by either [Na+]o or [Ca2+]o. IH depends only on [Ca2+]o as long as [Na+]o is at least 50 mM. Neither ID nor IH is decreased by decreasing the K+ gradient or by application of K+ channel blockers. These treatments increase somewhat the apparent amplitude of ID, probably by unmasking it from the large K+ tail current that follows the depolarizing pulse. A direct comparison in the same cell of the tetraethylammonium sensitivity of IH and of the Ca2+-activated K+ current demonstrates that these two currents flow through separate and distinct populations of channels. We conclude that in R15, ID arises in response to the triggering of an axonal action potential which in turn, through an as yet unknown mechanism, causes an increased influx of Na+ and/or Ca2+. We conclude that the apparent outward current IH, which is responsible for the interburst hyperpolarization in a normally bursting R15, in fact arises from a decrease in a resting inward Ca2+ current, possibly as the result of Ca2+-induced inactivation of Ca2+ channels.

Intracellular injection of protein kinase inhibitor blocks the serotonin-induced increase in K+ conductance in Aplysia neuron R15.

Previous work has shown that serotonin induces an increase in membrane K+ conductance in Aplysia neuron R15 and that this response is mediated by cAMP. The present study examines the role of protein phosphorylation in the response to serotonin. A specific inhibitor of cAMP-dependent protein kinase was injected intracellularly into neuron R15. The injection blocked the serotonin-induced increase in K+ conductance completely for at least 4 hours. The blockage was selective because the cell's response to dopamine was not inhibited. Furthermore, the blockage was specifically produced by protein kinase inhibitor because injection of other proteins (alpha-bungarotoxin and bovine serum albumin) did not affect the serotonin response. The serotonin response recovered fully 5-13 hours after the injection, presumably as a result of intracellular proteolysis of the protein kinase inhibitor. The results indicate that protein phosphorylation is a necessary step in the process that leads to activation of K+ channels by serotonin in neuron R15.

Triplicated bursting neuron R15 in an Aplysia abdominal ganglion: non-symmetrical coupling, common synaptic inputs and response to dopamine.

An abdominal ganglion of the mollusc Aplysia californica was found to contain 3 neurons in the place normally occupied by a single R15 cell. The 3 neurons exhibited properties characteristic of R15 neurons including spontaneous bursts. The bursts appeared asynchronously in spite of electrotonic coupling between them. The coupling function approximated a low pass filter with a cut-off frequency between 0.02 and 0.05 Hz in accordance with a measured coupling time-constant of 5--10 sec. Coupling measured in the cell body was found to be stronger for hyperpolarizing currents than for depolarizing currents injected into any of the 3 cells. This 'symmetrical rectification' can be explained by a rectifying axonal membrane interposed between the site of coupling and the site of recording. All 3 cells were found to have dopamine receptors and to receive common synaptic inputs. Since the coupling efficiency was found to vary depending on the direction of current flow, depolarizing synaptic inputs and spike burst generation remain autonomous.

Cyclic AMP modulation of a specific ion channel in an identified nerve cell: possible role for protein phosphorylation.

Multidisciplinary studies of the role of cAMP in synaptic transmission have been made possible by the favorable properties of the molluscan nervous system, and there is now evidence from several laboratories implicating cAMP in physiological responses in various Aplysia nerve and muscle cells (9,10,15,18,21). The results we have obtained satisfy all the criteria (8) necessary to establish that cAMP mediates the response to a neurotransmitter: a) the response is mimicked by intra- or extracellular application of cAMP derivatives, and by activation of adenylate cyclase within R15; b) a phosphodiesterase inhibitor enhances the response to low concentrations of serotonin; c) serotonin causes cAMP to accumulate within R15, and stimulates adenylate cyclase activity in membranes prepared from R15 cell bodies; and d) the serotonin receptors mediating adenylate cyclase stimulation and R15 hyperpolarization are pharmacologically very similar. This is the first time all these criteria have been satisfied in a neuronal system, and thus we conclude that the serotonin-induced increase in potassium conductance in neuron R15 is mediated by cAMP.

Mechanism of long-lasting synaptic inhibition in Aplysia neuron R15.

1. Long-lasting inhibition is a synaptically mediated response found in certain molluscan nerve cells that fire action potentials in bursts. It is elicited by repetitive stimulation of a presynaptic nerve and may last for minutes or hours after stimulation. 2. Voltage-clamp techniques were employed to measure the voltage dependence of the synaptically elicited current. Current-voltage curves were obtained by stepping or sweeping the voltage over the range -40 to -120 mV. 3. Long-lasting inhibition was found to be mediated by two separate conductance mechanisms. A component that reverses near -80 mV is most prominent at times up to 5 min following stimulation. A component with no reversal potential between -40 and -120 mV predominates at later times. 4. The reversible component is attenuated by reducing the intensity of stimulation of the presynaptic nerve, by injection of TEA into the postsynaptic cell, or by activation of a potassium conductance with serotonin prior to stimulation of the nerve. Thus, the reversible component appears to be mediated by an increase in potassium conductance. 5. The effects of the nonreversible component measured in the soma appear to be too large to attribute it to a conductance change that is electrically "distant" from the soma. It is attenuated by turning off a resting inward ion conductance with dopamine prior to stimulation of the nerve. It is not affected by short exposure to ouabain, but is attenuated by longer exposures that reduce the sodium and calcium gradients. Thus, the nonreversible component may be mediated by a decrease in voltage-dependent inward current flow carried by sodium or calcium.

Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

An inherited, structurally abnormal and superactive form of the enzyme 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) synthetase (EC 2.7.6.1) has been characterized in fibroblasts cultured from a 14-yr-old male (S.M.) with clinical manifestations of uric acid overproduction present since infancy. PP-ribose-P synthetase from the cells of this child showed four- to fivefold greater than normal resistance to purine nucleotide (ADP and GDP) feedback inhibition of enzyme activity and hyperbolic rather than sigmoidal inorganic phosphate (Pi) activation in incompletely dialyzed extracts. Excessive maximal velocity of the enzyme reaction catalyzed by the mutant enzyme was indicated by: enzyme activities twice those of normal at all concentrations of Pi in chromatographed fibroblast extracts; normal affinity constants for substrates and for the activator, Mg2+; and twofold greater than normal activity per immunoreactive enzyme molecule. The mutant enzyme thus possessed deficient regulatory and superactive catalytic properties, two mechanisms previously demonstrated individually to underlie the excessive PPRribose-P and uric acid synthesis of affected members of families with superactive PP-ribose-P synthetases. Increased PP-ribose-P concentration (4-fold) and generation (2.7-fold) and enhanced rates of PP-ribose-P dependent purine synthetic reactions, including purine synthesis de novo, in S.M. fibroblasts confirmed the functional significance of this patient's mutant enzyme. Diminished stability of the variant PP-ribose-P synthetase was manifested in vitro by increased thermal lability and in vivo by deficiency of enzyme activity at Pi concentrations greater than 0.3 mM in hemolysates and by an accelerated, age-related decrement in enzyme activity in lysates of erythrocytes separated by specific density. Despite the diminished amount of PP-ribose-P synthetase in the S.M. erythrocyte population, S.M. erythrocytes had increased PP-ribose-P concentration and increased rates of incorporation of [14C]adenine and hypoxanthine into acid-soluble nucleotides during incubation at 1 mM Pi. These findings provided further confirmation of the extent to which PP-ribose-P synthesis is modulated in the normal cell at physiological Pi concentration by purine nucleotide inhibition of PP-ribose-P synthetase. The activity and kinetic characteristics of PP-ribose-P synthetase from fibroblasts of the mother of patient S.M. indicated that this woman was a heterozygous carrier of the enzyme defect expressed in hemizygous manner by her son.

Synaptic and hormonal modulation of a neuronal oscillator: a search for molecular mechanisms.

1. The central ganglia of a number of gastropod molluscs (including the marine snail Aplysia californica and the terrestrial snail Helix pomatia) contain neurones which exhibit endogenous patterns of oscillatory activity. 2. This oscillatory activity can be modulated for long periods of time by synaptic and hormonal stimulation. 3. Stimulation of appropriate pre-synaptic nerves causes long-lasting hyperpolarization in these neurones, with complete abolition of oscillatory activity. This synaptic response is mediated by an increase in K+ conductance, together with a decrease in inward (Na+/Ca2+) conductance. The ionic conductances affected by synaptic stimulation are those responsible for producing the rhythmic oscillations. 4. The oscillatory activity can also be modulated by the vertebrate neurohyophyseal peptides, vasopressin and oxytocin, and by an endogenous peptide-containing extract of molluscan ganglia. In contrast to synaptic stimulation, these agents cause an increase in oscillatory activity. 5. The endogenous molluscan factor which produces an increase in oscillatory activity can be purified by affinity chromatography on bovine neurophysin linked to Sepharose. This indicates that the molluscan nervous system may contain a neurohypophyseal-like peptide. 6. Oscillatory activity can be modulated by manipulation of cyclic nucleotide metabolism in these neurones. Increases in cAMP alone are associated with abolition of oscillatory activity; this mimics long-lasting synaptic hyperpolarization. Increases in cAMP and cGMP together are associated with an increase in oscillatory activity and mimic the effects of the vertebrate and molluscan peptides. Thus, it is possible that cyclic nucleotides play a role in these physiological responses.

Occurrence of glutathione in bacteria.

Glutathione and soluble thiol content were examined in a broad spectrum of bacteria. Significant soluble thiol was present in all cases. The thiol compound was glutathione in most of the gram-negative bacteria but not in most of the gram-positive bacteria studied. Glutathione was absent in four anerobes and one microaerophile but was present in a blue-green bacterium. The glutathione content of Escherichia coli increased significantly during transition from exponential to stationary phase.