Centenary of the synapse: from Sherrington to the molecular biology of the synapse and beyond.

Few concepts have meant more to neuroscience than the synapse, commonly understood to mean the junction between two excitable cells. The term was introduced by Charles Sherrington in 1897. The centenary of this event is an appropriate time to review the term's origins and utility. There are some surprises. The term didn't actually come from him. His concept was more functional than structural. The pioneering physiological and structural studies in the 1950s in fact did not lead to a rigorous definition. There is still confusion on how to define neurotransmitters. As molecular biological approaches are increasingly refining the concept of a fundamental synaptic unit, many types of neuronal interactions are appearing that do not fit with the synaptic concept. Are the neural circuits underlying behaviour strictly synaptic? In dealing with these questions, a longer perspective is useful for understanding how the term arose, how it has evolved to the present, and what kinds of challenges may be coming in the future.

The critical period for repair of CNS of neonatal opossum (Monodelphis domestica) in culture: correlation with development of glial cells, myelin and growth-inhibitory molecules.

A comparison was made of neurite growth across spinal cord lesions in the isolated central nervous system (CNS) of newborn opossums (Monodelphis domestica) at various stages of development. The aim was to define the critical period at which growth after injury ceases to occur, with emphasis on growth-inhibitory proteins, myelin and glial cells. In postnatal opossums 3-6 days old (P3-6), repair was observed 5 days after lesions were made in culture at the cervical level (C7) by crushing with forceps. Through-conduction of action potentials was re-established and axons stained by Dil grew into and beyond the crush. In a series of 66 animals 29 showed repair. In 28 animals at P11-12 with comparable lesions repair was observed in five preparations. At P13-14, the CNS was still viable in culture, but none of the 25 preparations examined showed any axonal growth into the crush or conduction through it. The rostro-caudal gradient of development permitted lesions to be made in mature cervical and immature lumbar regions of P11-12 spinal cord. Growth across crushes occurred in lumbar but not in cervical segments of the same preparation. The development of glial cells and myelin was assessed by electron microscopy and by staining with specific antibodies (Rip-1 and myelin-associated glycoprotein) in cervical segments of neonatal P6-14 opossums. At P8, oligodendrocytes and thin myelin sheaths started to appear followed at P9 by astrocytes stained with antibody against glial fibrillary acidic protein. By P14, astrocytes, oligodendrocytes and well-developed myelin sheaths were abundant. The cervical crush sites of P12 cords contained occasional astrocytes but no oligodendrocytes. Specific antibodies (IN-1) to neurite growth-inhibiting proteins (NI-35/250) associated with oligodendrocytes and myelin in the rat CNS cross-reacted with opossum proteins. Assays using the spreading of 3T3 fibroblasts and IN-1 showed that by P7 inhibitory proteins became apparent, particularly in the hindbrain and cervical spinal cord. The concentrations of NI-35/250 thereafter increased and became abundant in the adult opossum. Our finding of a well-defined critical period, encompassing only 5 days, in CNS preparations that can be maintained in culture offers advantages for analysing mechanisms that promote or prevent CNS repair.

The influence of 17 beta-oestradiol on K+ currents in smooth muscle cells isolated from immature rat uterus.

Potassium currents in cells isolated from myometrium of immature (23-26 days after birth) rats were measured by whole-cell patch clamp to analyse the effects of gonadal steroids. Predominantly outward K+ currents with an early transient component were recorded in response to depolarizing pulses from a holding potential of -90 mV. The transient current, which was rarely present in adult myometrial cells, was inactivated by holding the membrane potential at -40 mV and attenuated by 1 mM 4-aminopyridine. Under these conditions a second sustained outward current was apparent. Administration of 17 beta-oestradiol to the immature rat before isolation of the myometrial cells reduced the probability of occurrence of the transient currents from 78.6% of cells (n = 33) to 29.8% (n = 14). Progesterone had only a slight effect. The kinetics of the transient currents were differently influenced by the two steroids. Cells isolated from animals previously given 17 beta-oestradiol showed transient outward currents with significantly shorter time constants of decay than those for control cells. Conversely, progesterone caused the time constants to be increased. We conclude that the gonadal steroids exert an influence on the expression of different populations of ionic channels in isolated cells of the immature rat uterus. These channels may regulate the excitability and contractility of the uterus in vivo.

Repair of connections in injured neonatal and embryonic spinal cord in vitro.

A remarkable preparation for studying development and repair is the CNS of the newborn opossum which, removed in its entirety, survives in culture for more than 1 week. In suitable medium, cells continue to divide, mature and reflex activity is maintained. Moreover, nerve fibers grow rapidly, reliably and extensively across lesions made in the spinal cord. Restoration of conduction has been demonstrated by recording electrically; labeled fibres have been observed directly by light and electron microscopy as they traverse the lesion. Similar experiments have also been made in embryonic (E15) rat CNS in culture. Open questions concern the identity of the fibers that traverse the lesion and the specificity of connections that they make with targets. We are now also analysing mechanisms that favor repair in younger opossums and that prevent it in their older siblings. Of particular interest are oligodendrocytes and myelin that start to appear at about 8-9 days after birth.

Hormonal influence on ionic channels in myometrium.

Smooth muscle cells in culture isolated from myometrium were characterized by scanning microscope and immunohistochemistry. Using the whole-cell patch-clamp configuration, and the single channel bilayer technique, the properties of ionic channels expressed in both non-pregnant and pregnant myometrium have been described. The predominantly expressed potassium channel changes from a transient inactivating outward current seen before puberty, to a calcium sensitive delayed outward current present in the adult stage. A change in the calcium channel population occurs from the nonpregnant to the pregnant state. Finally, sodium channels are expressed with greater frequency towards the end of gestation suggesting that these channels may play a role in labor.

Expression of different potassium channels in cells isolated from human myometrium and leiomyomas.

OBJECTIVE: Electrophysiologic characteristics of human myometrial and leiomyomatous cells isolated in culture were investigated. Both types of cell were shown to be smooth muscle cells by immunofluorescence. STUDY DESIGN: Voltage-activated potassium currents were recorded by whole-cell patch-clamp techniques and analyzed for differences in expression, voltage-dependence, kinetics, and inactivation. RESULTS: Depolarizing-voltage steps from -90 mV to +30 mV elicited two types of noninactivating outward currents that differed in their kinetics in 83% (n = 36) of normal cells in culture for 3 to 5 days; 6% responded with fast (3.5 milliseconds) outward inactivating currents; 11% in culture for 1 day responded only with long-lasting inactivating currents (33.2 +/- 7.2 milliseconds). Cells isolated from leiomyomas responded preferentially (65%, n = 31) with fast (3.3 +/- 0.1 milliseconds) outward inactivating currents; 35% responded with noninactivating outward currents similar to those from normal cells. CONCLUSION: Different potassium channel currents, noninactivating and inactivating, are predominantly expressed in cells isolated from human myometrium and leiomyomas, respectively.

Characterization of large-conductance, calcium-activated potassium channels from human myometrium.

OBJECTIVES: The purpose of our study was to detect and characterize potassium channels in the plasma membrane of smooth muscle cells from human myometrium. STUDY DESIGN: Plasma membrane vesicles were incorporated into lipid bilayers to record single potassium channel activity. RESULTS: We predominantly found a "maxi" calcium-activated potassium channel (261 picosiemens). This channel was calcium (micromoles per liter range) and voltage sensitive, highly selective for K+ over Na+ and Cs+, and was sensitive to external tetraethylammonium (dissociation constant approximately 220 mumol/L) and charybdotoxin (dissociation constant approximately 23 nmol/L). External apamin and 4-aminopyridine had no effect on this channel. Another type of potassium channel that was less frequently observed was also identified. It had a smaller conductance (142 picosiemens) and it seemed to be calcium independent (up to 50 nmol/L). CONCLUSION: Human myometrium possesses abundant "maxi" calcium-activated potassium channels. This channel shares common characteristics with other "maxi" calcium-activated potassium channels, including calcium and voltage gating, high conductance and selectivity, and channel pharmacologic profile.

Progesterone increases Ca2+ currents in myometrial cells from immature and nonpregnant adult rats.

We examined the role of ovarian steroids in regulating Ca2+ channels in rat uterine smooth muscle. Ca2+ currents (ICa) in myometrial cells from nonpregnant adult rats and immature rats injected with either estrogen or progesterone or estrogen plus progesterone were measured with the whole cell patch-clamp method. ICa was more prominent in cells from diestrous rats than in cells from estrous rats. In cells from immature rats the ICa density was significantly greater in cells from progesterone-injected rats than in cells from estrogen-injected or noninjected rats. ICa in cells from rats injected simultaneously with progesterone and the progesterone antagonist RU-486 was not significantly greater than those from noninjected rats. These increases in ICa density are not the result of changes in ICa activation kinetics or voltage dependence, since both are unaffected by steroid injection. The kinetics and voltage dependence of the ICa current in cells from immature and nonpregnant adult rats are similar, suggesting that they represent a single population of Ca2+ channels.

Macrophages direct process elongation from adult frog motorneurons in culture.

Motorneurons and macrophages have been isolated and identified in primary cultures from adult frog (Rana pipiens) spinal cord. Time-lapse video microscopy revealed that during the first two weeks migrating macrophages contact the growth cones of motorneurons. As they continue to migrate, the motorneuron processes elongate in close association with the moving macrophages. Elongating motorneuron processes are thereby brought into contact with other motorneurons and networks are formed. At later stages, the macrophages die but the motorneurons and the networks survive for at least another two weeks. These experiments show that macrophages can promote a directed elongation of motorneuron processes and suggest that they play a similar role during regeneration in vivo.

Reflexes, fictive respiration and cell division in the brain and spinal cord of the newborn opossum, Monodelphis domestica, isolated and maintained in vitro.

1. The entire central nervous system (CNS) was isolated from 1- to 4-day-old newborn South American opossums (Monodelphis domestica). At this stage the CNS has only an embryonic forebrain (two-layered) and no cerebellum and corresponds to a 14-day rat embryo. Its eyes, ears and hind-limbs are only at an early stage of formation. The isolated CNS preparations continue to develop and to produce electrical signals for up to 4 days in oxygenated Krebs' fluid at 23 degrees C. 2. The longitudinal axis of the CNS showed markedly different stages of development. More neuroblast cells were present in the proliferative zone in lumbosacral than in cervical or thoracic regions of the cord. 3. The progeny of dividing cells were labelled in isolated preparations by applying bromodeoxyuridine (BrdU) to the bathing solution for 2 h. Stained precursor cells were observed in CNS that had been left in Krebs' fluid for 4 days before applying BrdU and also in CNS that had been exposed to BrdU shortly after dissection and then left for 4 days. 4. Compound action potentials were evoked from the isolated CNS by stimulation with extracellular electrodes. Compound action potentials increased in amplitude with stronger stimulation and showed discrete peaks of conduction velocity. All electrical activity was eliminated reversibly by 0.1 mumol l-1 tetrodotoxin applied to the bathing solution. Block and recovery occurred with a half-time of approximately 5 min. High concentrations of magnesium (20 mmol l-1) reversibly blocked slower components of the volley. 5. Reflexes in cervical and thoracic segments of the spinal cord continued to function in isolated preparations. Stimulation of a dorsal root evoked bursts of impulses in the appropriate ventral root. Spontaneous and evoked activity in ventral roots was eliminated reversibly by 20 mmol l-1 magnesium. 6. In thoracic segments, spontaneous rhythmical bursts of action potentials were recorded. Burst activity was correlated with respiratory movements of the ribs in semi-intact preparations in which a few ribs and muscles were left attached to the isolated CNS. 7. At raised temperatures of 28 degrees C compared to 23 degrees C both spontaneous and evoked electrical activity were reversibly reduced. 8. Together these results show that the isolated CNS of the newborn opossum survives well in culture. The preparation offers advantages for pharmacological and physiological studies of spinal reflexes, for analysis of the mechanisms underlying rhythmical respiratory activity and for following the time course of CNS development in vitro.

Hormonal regulation of potassium currents in single myometrial cells.

Three potassium currents (IK) were recorded from myometrial cells isolated from the uterus of rats at estrus and diestrus and kept in culture for 1-6 days. IK were differentiated by their modulation with norepinephrine and/or by their onset kinetics. At +50 mV the activation time constants were about 0.7 ms, 6 ms, and 15 ms for the fast, the intermediate, and the slow IK, respectively. Norepinephrine (1 microM) potentiated the fast IK and reduced the intermediate IK. In addition, differences were found with respect to cells from animals at estrus and diestrus. The fast IK was preferentially expressed in cultures from animals at estrus, whereas the intermediate IK was more frequent in cells from rats at diestrus. These results indicate that K+ channels from myometrial cells are multiregulated. Regulation may occur by short-term signals (neurotransmitters) and/or by preferentially expressing distinct types of channels depending on the hormonal status of the animal.

A hypothesis for the superior sensitivity of wheat germ agglutinin as a neuroanatomical probe.

We have shown that a conjugate of wheat germ agglutinin (WGA), with horseradish peroxidase (HRP), is more sensitive than native HRP as a probe of neuroanatomic connections involving the retrograde transport of the lectin. It has also been shown in our laboratory that WGA-HRP remains at the site of injection twice as long as HRP. The purpose of the present morphometric study was to investigate the basis for the higher sensitivity of WGA-HRP over HRP as a retrogradely transported tracer molecule. To do this, we modified the experiment of Heuser and Reese which utilized the tracing of HRP in the frog neuromuscular junction (Heuser, J.E. and Reese, T.S., J. Cell Biol., 57 (1973) 315-344). Instead of using HRP alone, we examined, in double labeling experiments, fluid and adsorptive endocytosis with free HRP and WGA coupled to ferritin (WGA-ferritin) respectively. Immediately after nerve stimulation, both markers are taken up simultaneously into cisternae, and in tubular structures strikingly similar to the described compartment of uncoupling of receptor from ligand (CURL). Frequently, cisternae were connected with putative CURL. This early double labeling of cisternae and putative CURL was followed by the appearance of synaptic vesicles labeled with WGA-ferritin only (72-79%), HRP only (6-11%), and both labels (13-16%). In contrast to the labeling pattern of synaptic vesicles, the majority of cisternae and putative CURL had both labels throughout the duration of the experiments (77-80%). The results of this study indicate that most of WGA-ferritin and HRP are co-localized in cisternae and putative CURL, compartments involved in endocytosis and surface receptor recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term cultures of neurons from adult frog brain express GABA and glutamate-activated channels.

Cells from adult Xenopus laevis brainstem and spinal cord were dissociated with mild enzymatic treatment and grown in long-term cell culture. These cells had specific attachment/substrate and medium/serum requirements. Cells with bipolar and multipolar morphology were positively identified as neurons using immunohistochemistry with monoclonal antibodies to rat and bovine neurofilament proteins which we show here cross-react with similar amphibian proteins. Patch clamp recordings demonstrated that these neurons have populations of ionic channels which are activated by L-glutamate or gamma-aminobutyric acid (GABA). The characteristics of these channels were similar to those previously described for GABA- and glutamate-activated channels in embryonic mammalian neurons isolated in culture. Cell cultures of neurons isolated from adult Xenopus laevis brain may be a useful and simple preparation with which to examine the modulation of neuronal properties by various agents over longer time intervals then has been previously possible.

5 alpha-dihydrotestosterone has nonspecific effects on membrane channels and possible genomic effects on ACh-activated channels.

1. Myotubes cultured from adult male Xenopus laevis laryngeal muscle have been found to express androgen receptors. 2. Using patch-clamp techniques, it was found that 5 alpha-dihydrotestosterone (5 alpha-DHT) can act directly at membranes of these myotubes to alter the kinetic properties of acetylcholine- (ACh) activated single channels. 3. When the culture medium contains 5 alpha-DHT for greater than 6 days, the androgen acts through its receptors to markedly increase ACh-activated, single-channel conductances and alter single-channel kinetics. These effects do not occur if the antiandrogen, flutamide, which prevents the androgen from combining with its receptor, is added to the medium before the addition of the 5 alpha-DHT. These effects also do not occur in myotubes cultured from quadriceps femoris muscles that have much lower levels of specific androgen receptor. 4. When alpha-bungarotoxin (alpha-BTX) is added to the medium for 3 h before recording, no ACh channels can be recorded from myotubes in control medium within 5 h after washout of the alpha-BTX. However, when the culture medium contains 5 alpha-DHT for greater than or equal to 6 days, ACh channels can be recorded within 8 min of the washout of the alpha-BTX. 5. The results suggest that 5 alpha-DHT may act to alter the properties of ACh-activated ion channels at multiple sites in excitable cells.

Presumptive neurons derived by differentiation of a human embryonal carcinoma cell line exhibit tetrodotoxin-sensitive sodium currents and the capacity for regenerative responses.

Cloned human embryonal carcinoma cells (NTERA-2 cl.D1) differentiate into neuron-like cells upon exposure to retinoic acid. Using whole-cell patch-clamp techniques, these putative neurons exhibited rapidly activating and inactivating inward currents upon depolarization as well as outward currents. The electrical characteristics and tetrodotoxin (TTX) sensitivity of the inward currents suggest that they were sodium currents. By contrast, only outward potassium currents were seen in the undifferentiated stem cells. Under current clamp conditions, the neuron-like cells showed regenerative responses. The peaks of these responses never exceeded the O-mV level, perhaps due to the low mean inward current density of 93.8 +/- 17.8 (SEM) microA/cm2:n = 9. The electrophysiological characteristics of these human teratocarcinoma-derived neuron-like cells were consistent with our previous identification of these cells as neurons, but suggest that they may resemble immature embryonic, rather than adult, neurons.

Sexual dimorphism in the fibers of a "clasp" muscle of Xenopus laevis.

When antibodies specific to fast-twitch, slow-twitch, and slow-tonic myosins were used to stain the clasp muscle, m. sternoradialis, of Xenopus laevis, three predominant fiber types were identified in both males and females. Three fiber types can also be distinguished by the diameters and conduction velocities of their motor nerves. Significant differences in the numbers of slow-tonic fibers were identified between the genders and between control and castrated male animals. This finding suggests that these slow-tonic fibers, which probably dominate the clasp reflex during mating, may be under hormonal control.

Dye and electrical coupling between frog motoneurons.

Coupling between lumbar motoneurons in the isolated frog spinal cord was studied by using intracellular recordings and intracellular injections of Lucifer Yellow CH. Physiological studies revealed intra- and intersegmental, short latency electrical interactions between many motoneurons. Injections of Lucifer Yellow into motoneurons that were found to be coupled electrically revealed intra- and intersegmental dye-coupling.