Showing papers in "Cellular and Molecular Neurobiology in 1985"

Activity-dependent presynaptic facilitation: an associative mechanism in Aplysia.

Abstract: In studying the classical conditioning of the siphon withdrawal reflex in Aplysia, we have identified a neuronal mechanism that plays an important role in this conditioning: activity-dependent presynaptic facilitation. This review describes our analysis of the cellular basis of this associative mechanism. During the conditioning of the withdrawal reflex, the unconditioned stimulus, a tail shock, produces presynaptic facilitation of synaptic transmission from the siphon sensory neurons in the conditioned stimulus pathway. The facilitation is enhanced if a sensory neuron has fired action potentials just prior to receiving facilitatory input, as occurs during training when the conditioned stimulus precedes the unconditioned stimulus. This activity-dependent enhancement of presynaptic facilitation provides a mechanism for the temporal specificity in conditioning of the reflex. Activity-dependent facilitation appears to involve the same cyclic AMP (cAMP)-dependent cascade that underlies presynaptic facilitation in these neurons in the absence of paired spike activity. Our evidence suggests that it is the transient elevation of intracellular Ca2+ that is responsible for the enhancement of the facilitation response by paired spike activity. Moreover, our preliminary results indicate that Ca2+/calmodulin is able to potentiate the activation of adenylate cyclase in Aplysia neurons by facilitatory transmitter. Thus, the dual activation of the calmodulin-dependent cyclase by Ca2+ and transmitter may give this enzyme an important associative role in learning. In the conclusion, the possible phylogenetic generality of this associative mechanism is discussed as well as its possible role in activity-dependent processes in neuronal development.

Activity and synapse elimination at the neuromuscular junction

Abstract: 1. The neuromuscular junction undergoes a loss of synaptic connections during early development. This loss converts the innervation of each muscle fiber from polyneuronal to single. During this change the number of motor neurons remains constant but the number of muscle fibers innervated by each motor neuron is reduced. Evidence indicates that a local competition among the inputs on each muscle fiber determines which inputs are eliminated. 2. The role of synapse elimination in the development of neuromuscular circuits, other than ensuring a single innervation of each fiber, is unclear. Most evidence suggests that the elimination plays little or no role in correcting for errant connections. Rather, it seems that connections are initially highly specific, in terms of both which motor neurons connect to which muscles and which neurons connect to which particular fibers within these muscles. 3. A number of attempts have been made to determine the importance of neuromuscular activity during early development for this rearrangement of synaptic connections. Experiments reducing neuromuscular activity by muscle tenotomy, deafferentation and spinal cord section, block of nerve impulse conduction with tetrodotoxin, and the use of postsynaptic and presynaptic blocking agents have all shown that normal activity is required for normal synapse elimination. Most experiments in which complete muscle paralysis has been achieved show that activity may be essential for the occurrence of synapse elimination. Furthermore, experiments in which neuromuscular activity has been augmented by external stimulation show that synapse elimination is accelerated. 4. A plausible hypothesis to explain the activity dependence of neuromuscular synapse elimination is that a neuromuscular trophic agent is produced by the muscle fibers and that this production is controlled by muscle-fiber activity. The terminals on each fiber compete for the substance produced by that fiber. Inactive fibers produce large quantities of this substance; on the other hand, muscle activity suppresses the level of synthesis of this agent to the point where only a single synaptic terminal can be maintained. Inactive muscle fibers would be expected to be able to maintain more nerve terminals. The attractiveness of this scheme is that it provides a simple feedback mechanism to ensure that each fiber retains a single effective input.

Immunocytochemical localization of actin in dendritic spines of the cerebral cortex using colloidal gold as a probe

Abstract: 1. Immunocytochemical localization of actin in rat cerebral cortex embedded in the resin LR White was performed using 5 nm colloidal gold as a probe. Antigenicity is maintained throughout the embedding procedure and the low electron opacity of LR White permits fine filamentous structures to be visualized. Control experiments included incubating the sections with normal goat serum or mouse IgG instead of the primary antibody, preadsorbing the antibody with actin from bovine muscle or liver acetone powder, and heat treating the primary antibody. 2. Immunoreactive actin was identified primarily in dendritic spines, particularly in the postsynaptic density (PSD), the subsynaptic web, and the spine apparatus and endothelial and smooth muscle cells of blood vessels. 3. Within dendritic spines, actin which is labeled in the PSD is in continuity with the filaments of the subsynaptic web. These filaments, in turn, are in continuity with the spine apparatus and/or the spine membranes adjacent to the PSD. The PSD may therefore function like other submembranous filamentous arrays which communicate events occurring at the membrane, in this case, the postsynaptic membrane, to the underlying cytoskeletal network, i.e., the subsynaptic web of the spine. It is also suggested that the actin present in the spine may play a role in changes in spine shape and synaptic curvature. Some actin was also seen in the presynaptic process in association with synaptic vesicles, the filamentous network that is contiguous with the synaptic vesicle membrane, and the presynaptic dense projections. Actin may be involved in dynamic processes in the presynaptic ending which include vesicle translocation.

A possible mechanism of morphometric changes in dendritic spines induced by stimulation

Abstract: 1. A number of experimental procedures which induce increased electrical activity (including long-term potentiation) were shown to be accompanied by morphometric changes in dendritic spines. These changes include an enlargement of the spine head, shortening and widening of the spine stalk, and an increase in the length of synaptic apposition. 2. A possible mechanism is suggested which takes into account specific cytological features of the spine and the existence of contractile proteins in neurons. Dendritic spines are defined as special domains of the neuron which have a unique organization of the cytoplasm. Actin filaments form a very dense network in the spine head, and they are longitudinally organized within the spine stalk. Spines were also shown to contain myosin and other actin-regulatory proteins. The high density of the actin network could explain the characteristic absence of the cytoplasmic organelles from dendritic spines. 3. In analogy with other cells, such an actin organization indicates low levels of free cytosolic calcium. Even in the resting state, calcium levels may be unevenly distributed through the neuron, being lowest within the subplasmalemmal region. Due to the high surface-to-volume ratio in spines, the cytoplasm is formed mostly by the subplasmalemmal region. The spine apparatus or the smooth endoplasmic reticulum, which is recognized as a calcium-sequestering site in spines, may also contribute to the low calcium levels there. 4. However, when in the stimulated spine the voltage-dependent calcium channels open, then, given the spine's high surface-to-volume ratio, the concentration of calcium may very quickly attain levels that will activate the actin-regulatory proteins and myosin and thus trigger the chain of events leading to the enlargement of the spine head and to the contraction (i.e., widening and shortening) of the spine stalk. The increased free cytosolic calcium may also activate the protein-producing system localized at the base of the spine, which, under certain conditions, could stabilize the morphometric changes of the spine.

The role of brain extracellular proteins in neuroplasticity and learning

Abstract: 1. Double labeling studies of the pattern of protein synthesis in goldfish and mouse brain identified a class of glycoproteins (the ependymins) whose turnover rate was enhanced after training. A variety of control experiments indicated that these macromolecules have an important role in the molecular and cell biology of learning. Antisera to the ependymins when injected into the brains of trained goldfish cause amnesia of a newly acquired behavior. 2. Isolation and localization studies by immunocytochemical methods indicate that the ependymins are released into the brain extracellular fluid by a class of neurosecretory cells. In mammalian brain ependymin-containing cells are highly concentrated in the limibic system. 3. The ependymins are constituted from two disulfide-linked acidic polypeptide chains (M.W. 37K and 31K). They contain at least 5% covalently bound carbohydrate per chain with mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid as the predominant components. 4. The highly soluble ependymins can rapidly polymerize to form an insoluble fibrous matrix if calcium is removed from solution by the addition of a Ca2+ -chelating agent or dialysis. 5. The self-aggregation property of the ependymins can be triggered by the depletion of Ca2+ from the extracellular space. Studies of the kinetics of the aggregation phenomenon by measurements of turbidity changes indicate that the process can be terminated but not reversed by restoring Ca2+ to its normal CSF level. 6. Immunohistochemical studies of the brains of trained goldfish show the presence of punctate statining sites in the perimeter of certain cells located in specific brain regions. This suggests that ependymin aggregation might occurin vivo during learning. 7. A molecular hypothesis relating the aggregation properties of the ependymins to neuroplasticity and learning is proposed.

Quantitative autoradiographic characterization of receptors for angiotensin II and other neuropeptides in individual brain nuclei and peripheral tissues from single rats

Abstract: 1. Autoradiographic techniques coupled with computerized microdensitometry and comparison with125I standards were used to characterize and quantitate receptors for neuropeptides in rat brain and adrenal and pituitary glands. 2. These techniques are rapidly performed, anatomically precise, and more sensitive than membrane binding techniques. They permit the determination of complete saturation curves and Scatchard analysis in discrete nuclei of the rat brain and in single rat pituitary and adrenal glands. 3. Angiotensin II (AII) receptors were quantitated after incubation of 16-µm tissue sections with the AII agonist125I-[Sar1]-AII. 4. High-affinity, high-density AII receptors were present in the organon subfornicalis, organon vasculosum laminae terminalis and nuclei triangularis septalis, suprachiasmatis, and paraventricularis of the rat and in rat adrenal capsule-zona glomerulosa area, adrenal medulla, and anterior pituitary. 5. These techniques could be used for precise localization and quantitation of other neuropeptide receptors in single rat brain nuclei, after optimizing the assay conditions and provided that suitable125I ligands are available.

Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.

Abstract: 1. During regeneration of the optic nerve in goldfish, the ingrowing retinal fibers successfully seek out their correct places in the overall retinotopic projection on the tectum. Chemospecific cell-surface interactions appear to be sufficient to organize only a crude retinotopic map on the tectum during regeneration. 2. Precise retinotopic ordering appears to be achieved via an activity-dependent stabilization of appropriate synapses and is based upon the correlated activity of neighboring ganglion cells of the same receptive-field type in the retina. Four treatments have been found to block the sharpening process: (a) blocking the activity of the ganglion cells with intraocular tetrodotoxin (TTX), (b) rearing in total darkness, (c) correlating the activation of all ganglion cells via stroboscopic illumination and (d) blocking retinotectal synaptic transmission withα-bungarotoxin (αBTX). 3. These experiments support a role for correlated visually driven activity in sharpening the diffuse projection and suggest that this correlated activity interacts within the postsynaptic cells, probably through the summation of excitatory postsynaptic potentials (EPSPs). 4. Other experiments support the concept that effective synapses are stabilized: a local postsynaptic block of transmission causes a local disruption in the retinotectal map. The changes that occur during this disruption suggest that each arbor can move to maximize its synaptic efficacy. 5. In development, initial retinotectal projections are often diffuse and may undergo a similar activity-dependent sharpening. 6. Indirect retinotectal maps, as well as auditory maps, appear to be brought into register with the direct retinotopic projections by promoting the convergence of contacts with correlated activity. 7. A similar mechanism may drive both the formation of ocular dominance patches in fish tectum and kitten visual cortex and the segregation of different receptive-field types in the lateral geniculate nucleus. 8. Activity-dependent synaptic stabilization may therefore be a general mechanism whereby the diffuse projections of early development are brought to the precise, mature level of organization.

The effects of exposure to exogenous fatty acids and membrane fatty acid modification on the electrical properties of NG108-15 cells

Abstract: 1. The role of membrane lipid composition in determining the electrical properties of neuronal cells was investigated by altering the available fatty acids in the growth medium of cultured neuroblastoma × glioma hybrid cells, clone NG108-15. 2. Growth of the cells for several days in the presence of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic) caused a pronounced decrease in the Na+ action-potential rate of rise (dV/dt) and smaller decreases in the amplitude, measured by intracellular recording. Oleic acid had no effect on the action potentials generated by the cells. In contrast, a saturated fatty acid (palmitate) and atrans monounsaturated fatty acid (elaidate) caused increases in both the rate of rise and the amplitude. 3. No changes in the resting membrane potentials or Ca2+ action potentials of fatty acid-treated cells were observed. The membrane capacitance and time constant were not altered by exposure to arachidonate, oleate, or elaidate, whereas arachidonate caused a small increase in membrane resistance. 4. Examination of the membrane phospholipid fatty acid composition of cells grown with various fatty acids revealed no consistent alterations which could explain these results. 5. To examine the mechanism for arachidonate-induced decreases indV/dt, the binding of3H-saxitoxin (known to interact with voltage-sensitive Na+) channels was measured. Membranes from cells grown with arachidonate contained fewer saxitoxin binding sites, suggesting fewer Na+ channels in these cells. 6. We conclude that conditions which lead to major changes in the membrane fatty acid composition have no effect on the resting membrane potential, membrane capacitance, time constant, or Ca2+ action potentials in NG108-15 cells. Membrane resistance also does not appear to be very sensitive to membrane fatty acid composition. However, changes in the availability of fatty acids and/or changes in the subsequent membrane fatty acid composition lead to altered Na+ action potentials. The primary mechanism for this alteration appears to be through changes in the number of Na+ channels in the cells.

Eye-specific segregation of optic afferents in mammals, fish, and frogs: The role of activity

Abstract: 1. Eye-specific patches or stripes normally develop in the visual cortex and superior colliculus of many (but not all) mammals and are also formed, after surgically produced binocular innervation, in the optic tectum of fish and frogs. The segregation of ocular dominance patches or columns has been studied using a variety of anatomical pathway-tracing techniques, by electrophysiological recording of postsynaptic units or field potentials, and by the 2-deoxyglucose method following visual stimulation of only one eye. 2. In the tectum of both fish and frogs and in the cortex and colliculus of mammals, eye-specific patches develop from initially diffuse, overlapping projections. 3. Of the various mechanisms that might cause such segregation, the evidence favors an activity-dependent process that stabilizes synapses from the same eye because of their correlated activity. First, several environmental manipulations affect the segregation of afferents in visual cortex: strabismus and alternate monocular exposure apparently enhance segregation, whereas dark rearing slows the segregation process, and monocular deprivation causes the experienced eye to form larger patches at the expense of those of the deprived eye. Second, blocking activity in both eyes is effective in preventing the segregation both in the tectum of fish and frog and in the visual cortex of cat. With the eyes blocked, alternate stimulation of the optic nerves permits the segregation of ocular dominance, at least onto single cells in the cat visual cortex. 4. These findings are discussed in terms of an activity-dependent stabilization of those synapses having correlated activity (those from neighboring ganglion cells within one eye) but not of those lacking correlated activity (those from left and right eyes). We suggest that the eye-specific patches represent a compromise between total segregation of the projections from the two eyes and the formation of a single continuous retinotopic map across the surface of the cortex or tectum.

Further study of the correlation between Na-pump activity and membrane chemosensitivity.

Abstract: 1. Using internally dialyzed neurons ofHelix, we have examined the effects of sodium-pump activity and intracellular ATP concentration on transmembrane currents induced by acetylcholine (ACh) andγ-aminobutyric acid (GABA). We also report on the effects of pump activity and levels of intracellular ATP on binding byHelix ganglia of3H-α-bungarotoxin (3H-α-BT) and3H-GABA. 2. Both ouabain-containing and potassium-free solutions depressed the neurotransmitter-induced transmembrane current of one type of dialyzed neurons. 3. An increase in the intracellular ATP concentration led to a depression of ACh-induced currents and to the disappearance of the blocking effect of ouabain on these currents. Intracellular ADP had a similar but smaller effect on transmitter-induced currents, and intracellular AMP was ineffective. The depressing effect of internal ATP on ACh-induced currents was absent in the presence of an inhibitor of membrane phosphorylation (dinitrophenol). 4. The binding of tritium-labeledα-BT and GABA to the membranes was depressed by both ouabain-containing and K-free solutions and also by compounds (theophylline and NaF) which increase the levels of intracellular ATP. 5. The results suggest that the Na pump modulates the affinity of ACh and GABA membrane receptors by the regulation of the phosphorylated state of membrane receptors.

Adaptive plasticity in the spinal stretch reflex: an accessible substrate of memory?

Abstract: 1. The study of the substrates of memory in higher vertebrates is one of the major problems of neurobiology. A simple and technically accessible experimental model is needed. 2. Recent studies have demonstrated long-term adaptive plasticity, a form of memory, in the spinal stretch reflex (SSR). The SSR is due largely to a two-neuron monosynaptic arc, the simplest, best-defined, and most accessible pathway in the primate central nervous system (CNS). 3. Monkeys can slowly change SSR amplitude without a change in initial muscle length or alpha motoneuron tone, when reward is made contingent on amplitude. Change occurs over weeks and months and persists for long periods. It is relatively specific to the agonist muscle and affects movement. 4. The salient features of SSR adaptive plasticity, combined with clinical and laboratory evidence indicating spinal cord capacity for intrinsic change, suggest that SSR change eventually involves persistent segmental alteration. If this is the case, SSR plasticity should be a powerful model for studying the neuronal and synaptic substrates of memory in a primate.

The anatomy of geniculocortical connections in monocularly deprived cats.

Abstract: 1. In monocularly deprived (MD) cats, many cells in the lateral geniculate nucleus (LGN) but few cells in the visual cortex respond to input from the deprived eye, suggesting that the connections to visual cortex from the deprived geniculate laminae may have been disrupted. 2. It has been known for some time that the afferents representing the deprived eye terminate over a smaller percentage of layer IV than do those representing the experienced eye, but it is becoming increasingly clear that this alone cannot explain the inability of the deprived pathway to activate cortical cells. 3. 2-Deoxyglucose studies of ocular dominance columns in MD cats have shown that the columns are often (a) restricted to layer IV, suggesting that intracortical connections may be disrupted, and (b) very faint, suggesting that MD alters the efficacy of the deprived pathway in addition to restricting its territory. 4. Electron microscopy has shown that both deprived and experienced afferents end in terminals that contain mitochondria and round synaptic vesicles and that make asymmetric contacts with dendritic profiles. However, the terminals of deprived afferents differ from those of experienced afferents: they are 25% smaller, contain 33% fewer mitochondria, are more likely to make synapses that are presynaptically convex (and thus, perhaps, immature), make fewer perforated synapses, and synapse onto smaller spines. Further, the geniculocortical axons from deprived laminae appear to end in fewer synaptic terminals, than do those from the experienced laminae. 5. The finding that the synaptic terminals of deprived afferents are both abnormal morphologically and fewer in number can help to explain the reduced effectiveness of the deprived eye in driving cortical cells but does not rule out additional effects such as suppression and loss of intracortical connectivity.

The role of visual experience in the development of cat striate cortex.

Abstract: 1. By the third postnatal week, intrinsic developmental programs have established a framework within the cat visual system; this will be used to guide the course of subsequent experience-dependent development. Key elements in this framework are precociously mature cells in visual cortex area 17. These orientation-selective cells are predominantly first-order neurons, they are concentrated in layers IV and VI of area 17, most of them are activated monocularly, many may receive their direct excitatory input from lateral geniculate nucleus X cells, and the distribution of their preferred orientations is biased toward horizontal and vertical. 2. Between the third and the sixth postnatal week, most of the remaining cells in area 17 develop orientation selectivity; this extension of orientation selectivity is blocked or delayed if kittens are deprived of normal patterned visual stimulation. Furthermore, exposure to a limited range of stimulus orientations can lead to an increase in the proportion of orientation-selective cells, and the range of orientation preferences that the cells acquire is restricted by the range of orientations to which the animal is exposed. This occurs with no apparent change in the physiology or morphology of intrinsically selective area 17 cells. Thus selective exposure may have its effect by influencing the connections between the intrinsically selective cells and higher-order neurons in area 17. 3. Experience-dependent changes in the visual system may function to “fine-tune” sensory processing and thus optimize the system's response to the dominant features of the environment. This experience-dependent process could help the young animal to focus its “attention” on those features of its environment that are critical to its survival.

Role of uptake in gamma-aminobutyric acid (GABA)-mediated responses in guinea pig hippocampal neurons.

Abstract: 1. Intracellular recordings were obtained from hippocampal pyramidal neurons maintainedin vitro. Measurements were made of the conductance change induced by iontophoretically appliedγ-aminobutyric acid (GABA) and, using voltage-clamp techniques, of inhibitory postsynaptic currents resulting from activation of inhibitory pathways. 2. Analysis of GABA iontophoretic charge-response curves indicated that there was considerable variation among neurons with respect to the slope of this relation. 3. The placement of the GABA-containing pipette did not appear to be responsible for the observed variation, since vertical repositioning of the pipette did not alter the slope of the charge-response relationship. 4. Steady iontophoresis of GABA from one barrel of a double-barreled pipette markedly affected the charge-response relation obtained when short pulses were applied to the other barrel. The curve was shifted to the left, and the slope was decreased. Concomitantly, the enhanced GABA-induced responses were prolonged. 5. Similar alterations in GABA responsiveness were observed when the uptake blocker, nipecotic acid, was iontophoretically applied. Furthermore, bath application of saline containing a reduced sodium concentration (25% of control) also produced a prolongation of GABA-mediated responses. 6. Under voltage clamp, inhibitory postsynaptic currents were observed to have biphasic decays. The initial, fast decay was prolonged by an average of 18% by nipecotic acid, whereas the later, slow phase was prolonged by 23%. 7. The results of these studies support the hypothesis that a saturable GABA uptake system is responsible for the observed variation in the charge-response curves and, in turn, underlies the apparent sensitizing effect of excess GABA application. The results also suggest that a reduction of transmitter uptake affects the time course of inhibitory postsynaptic currents in the hippocampus.

The role of visual experience in the formation of binocular projections in frogs.

Abstract: Many parts of the visual system contain topographic maps of the visual field. In such structures, the binocular portion of the visual field is generally represented by overlapping, matching projections relayed from the two eyes. One of the developmental factors which helps to bring the maps from the two eyes into register is visual input. The role of visual input is especially dramatic in the frog, Xenopus laevis. In tadpoles of this species, the eyes initially face laterally and have essentially no binocular overlap. At metamorphosis, the eyes begin to move rostrodorsally; eventually, their visual fields have a 170 degree region of binocular overlap. Despite this major change in binocular overlap, the maps from the ipsilateral and contralateral eyes to the optic tectum normally remain in register throughout development. This coordination of the two projections is disrupted by visual deprivation. In dark-reared Xenopus, the contralateral projection is nearly normal but the ipsilateral map is highly disorganized. The impact of visual input on the ipsilateral map also is shown by the effect of early rotation of one eye. Examination of the tectal lobe contralateral to the rotated eye reveals that both the contralateral and the ipsilateral maps to that tectum are rotated, even though the ipsilateral map originates from the normal eye. Thus, the ipsilateral map has changed orientation to remain in register with the contralateral map. Similarly, the two maps on the other tectal lobe are in register; in this case, both projections are normally oriented even though the ipsilateral map is from the rotated eye. The discovery that the ipsilateral eye's map reaches the tectum indirectly, via a relay in the nucleus isthmi, has made it possible to study the anatomical changes underlying visually dependent plasticity. Retrograde and anterograde tracing with horseradish peroxidase have shown that eye rotation causes isthmotectal axons to follow abnormal trajectories. An axon's route first goes toward the tectal site where it normally would arborize but then changes direction to reach a new tectal site. Such rearrangements bring the isthmotectal axons into proximity with retinotectal axons which have the same receptive fields. Anterograde horseradish peroxidase filling has also been used to study the trajectories and arborizations of developing isthmotectal axons. The results show that the axons enter the tectum before the onset of eye migration but do not begin to branch profusely until eye movement begins to create a zone of binocular space.(ABSTRACT TRUNCATED AT 400 WORDS)

Action of excitatory amino acids and their antagonists on hippocampal neurons.

Abstract: 1. Intracellular recordings were obtained from guinea pig hippocampal neurons maintainedin vitro. Current- and voltage-clamp techniques were used to study the effect of microiontophoresis of excitatory amino acid agonists. Modification of agonist responses by bath application of known concentrations of antagonist agents was also examined. 2. All agonists used, glutamate, aspartate,N-methyl-d-aspartic acid (NMDA), and quisqualate, depolarized hippocampal neurons and caused repetitive firing. NMDA was also noted to induce burst-firing in some neurons. Quisqualate and NMDA were more potent than glutamate or aspartate. 3. In slices perfused with a nominally calcium-free saline containing tetrodotoxin and manganese, quisqualate application produced a depolarization associated with a conductance increase. Under those conditions, NMDA-induced depolarizations caused apparent decreases as well as increases in conductance. The apparent decreases in conductance were observed in the voltage range of -40 to -70 mV, whereas increases in conductance were observed at membrane potentials more positive than-35 mV. 4. Under voltage-clamp conditions, quisqualate produced an inward current whose amplitude increased with hyperpolarization and decreased upon depolarization, reversing near 0 mV. The conductance change induced by quisqualate was independent of voltage. NMDA application resulted in an inward current that was maximal around the resting potential and decreased with both hyperpolarization and depolarization. Response reversal was not observed with hyperpolarization to-100 mV but was apparent with depolarization beyond 0 mV. Conductance changes induced by NMDA were voltage dependent, and the application of this agent was associated with the appearance of a region of negative slope conductance in the current-voltage relationship. 5. Apparent decreases in conductance in response to NMDA were reduced when the extracellular magnesium concentration was lowered. Response amplitudes were not affected. 6. The NMDA receptor antagonistdl-2-amino-5-phosphonovalerate (dl-APV) was a potent and selective blocker of NMDA responses, whereas the antagonistdl-2-amino-4-phosphonobutyric acid (dl-APB) was less potent and did not select between NMDA and quisqualate responses. Analysis of iontophoretic dose-response curves indicated thatdl-APV was a competitive antagonist. 7. The results of these experiments indicate that hippocampal CA1 pyramidal neurons possess separate receptors for quisqualate and NMDA, with different pharmacological and electrophysiological profiles.

Choline fluxes in synaptosomal membrane vesicles

Abstract: 1. Synaptic plasma membrane vesicles isolated from the highly cholinergic nervous tissue of insects were used to study the translocation of choline across the membrane via a high-affinity carrier-mediated mechanism energized by ion gradients as the sole driving force. 2. The uphill movement of choline, energized mainly by the Na+ gradient, attained levels of choline severalfold the final equilibrium value at the peak of the overshoot. 3. Efflux of choline required the presence of internal sodium ions and was promoted by external choline if Na+ was present. External choline inhibited choline efflux in the absence of sodium. 4. It is concluded that the efflux of choline is in many aspects symmetrical with its uptake.

Quantitative autoradiography of the development of mu opiate binding sites in rat brain

Abstract: 1. The regional developmental appearance of mu binding sites in rat brain was examined by quantitative autoradiography of3H-dihydromorphine binding in rats 2, 14, 21, and 28 days old. 2. Labeling with3H-dihydromorphine was heterogeneous in adult rat brains, as previously reported by other laboratories. Levels of3H-dihydromorphine binding ranged from approximately 250 nCi/g tissue in the interpeduncular nucleus and 100 nCi/g tissue in the habenula to 40 nCi/g tissue in the hypothalamus and periaqueductal gray. Some areas, particularly white matter regions, had no detectable specific binding. 3. The density of3H-dihydromorphine binding increased in all regions between 2 and 28 days of age. 4. The increases in3H-dihydromorphine binding in various regions of rat brain developed at different rates. Maximal densities were seen by 14 days of age in most regions examined, including the caudate, hippocampus, amygdala, and hypothalamus. Binding in the medial thalamus and quadrigeminal plate, however, did not reach maximal levels until 21 days. 5. Although quantitative autoradiography offers major advantages in the examination of the regional distribution of opiate binding sites, variability both between sections from the same brain and between sections from different brains demonstrate some of the difficulties associated with this type of experimental approach.

Serotonin decreases a background current and increases calcium and calcium-activated current in pedal neurons of Hermissenda.

Abstract: 1. The effects of serotonin (5-HT) on membrane potential, membrane resistance, and select ionic currents were examined in large pedal neurons (LP1, LP3) of the molluskHermissenda. 2. Calcium (Ca) action potentials were evoked in sodium-free artificial seawater containing tetramethylammonium, tetraethylammonium, and 4-aminopyridine (0-Na, 4-AP, TEA ASW). They failed at stimulation rates greater than 0.5/sec and were blocked by cadmium (Cd). Under voltage clamp the calcium current (ICa) responsible for them also failed with repeated stimulation. Thus,ICa inactivation accounts for refractoriness of the Ca action potential. 3. The addition of 10µM 5-HT to 0-Na, 4-AP, TEA ASW produced a slight depolarization and increased excitability and input resistance. Under voltage clamp the background current decreased. The voltage-dependent inward, late outward, and outward tail currents, sensitive to Cd, increased.ICa inactivation persisted. 4. Under voltage clamp with Ca influx blocked by Cd, the addition of 10µM 5-HT decreased the remaining current uniformly over membrane potentials of — 10 to — 100 mV. Thus, 5-HT reduces a background current that is active within the physiological range of the membrane potential, voltage insensitive, independent of Ca influx, noninactivating, and not blocked by 4-AP or TEA.

The effect of nerve activity on the distribution of synaptic vesicles.

Abstract: 1. Certain gymnotid fish (apteronotids) continuously emit a high-frequency electric-organ discharge and thus continuously drive their electroreceptor afferents at high rates. Electroreceptor afferents terminate in one lamina of the electrosensory lateral line lobe (ELL) and can be readily sampled. Normally these terminals have many small vesicles clustered adjacent to the presynaptic membrane. 2. When afferent activity is blocked for 24 hr by an injection of tetrodotoxin (TTX) into the electroreceptor nerve, the density of vesicles adjacent to the synaptic membrane declines; the volume of the remaining vesicles increases. If the nerve of a TTX-treated fish is stimulated proximal to the injection site, these changes can be reversed. 3. These results imply that the migration of vesicles toward the presynaptic membrane is influenced by the level of activity in the nerve.

Interaction of heparin with multimolecular aggregates of acetylcholinesterase.

Abstract: 1. It has been reported previously that heparin, a sulfated glycosaminoglycan, releases the asymmetric 16 S form of acetylcholinesterase (AChE) from cholinergic synapses. Here it is shown that heparin releases the synaptic AChE not as individual 16 S species but as multimolecular aggregates (30 S) of such molecules. 2. Heparin is able to convert low-ionic strength AChE aggregates into a heparin type of AChE aggregates. 3. Our results suggest that the AChE aggregates detached by heparin are likely to be the physiologically important state of aggregation of the 16 S AChE form in the synaptic basal lamina.

Long-term potentiation and 4-aminopyridine.

Abstract: 1. Long-term potentiation (LTP) of excitatory postsynaptic potentials (epsp's) was investigated with extracellular field potential recording in hippocampal slices from rats. 2. In the presence of 100µM 4-aminopyridine (4-AP) the probability of eliciting LTP was unchanged or increased; the extent of potentiation was not significantly different from normal. 3. During LTP saturation, 4-AP further enhanced the epsp. 4. These data are inconsistent with an involvement of A-current reduction in LTP.

A comparative study of the effects of tetrodotoxin and the removal of external Na+ on the resting potential: evidence of separate pathways for the resting and excitable Na currents in squid axon.

Abstract: 1. To investigate whether the Na permeability of the resting membrane is determined predominantly by the excitable Na channel, we examined the effects of tetrodotoxin (TTX) and the complete removal of external Na+ on the resting potential. 2. In the intact squid axon bathed in K-free artificial seawater, both TTX and the removal of Na+ produced small hyperpolarizations. The effect of Na removal, however, was larger than that of TTX. 3. In the perfused squid axon, the hyperpolarization produced by the removal of external Na+ was greatly enhanced when the internal K concentration ([K+]i) was reduced. The effect of TTX, on the other hand, was not sensitive to the [K+]i or to the membrane potential. For [K+]i = 50 mM and [K+]o = 0, the average hyperpolarization produced by TTX was 1.2 mV, while the hyperpolarization produced by Na removal was approximately 21 mV. 4. The difference between these two effects suggests that the majority of the resting Na current passes through pathways other than the excitable Na channel.

Interactions of gamma-aminobutyric acid (GABA), pentobarbital, and homopantothenic acid (HOPA) on internally perfused frog sensory neurons.

Abstract: Augmentatory actions among Cl- currents (ICl) induced by gamma-aminobutyric acid (GABA), pentobarbital (PB), and homopantothenic acid (HOPA) were investigated in isolated frog sensory neurons after suppression of Na+, K+, and Ca2+ currents using a suction pipette technique which combines internal perfusion with voltage clamp. GABA-sensitive neurons responded to both PB and HOPA, and the responses behaved as a simple Cl- electrode and reversed at the Cl- equilibrium potential (ECl). The dose-response curve for GABA-induced Cl- conductance was sigmoidal with the GABA concentration producing a half-maximum response (4.2 X 10(-5) M). Both GABA and HOPA dose-response curves shifted to the left in the presence of PB, though the facilitatory action of PB on GABA- and HOPA-induced ICl was more effective in the former. There was a significant facilitatory interaction between GABA- and HOPA-induced ICl. It is concluded that HOPA affects the GABA-GABA or PB-PB receptor interactions.

Activity-dependent synaptic stabilization in development and learning: how similar the mechanisms?

Abstract: Correlated activation of inputs resulting in a selective increase in their synaptic efficacy has long been associated with models of learning (Hebb, 1949). More recently, however, it has become associated with models of development and has been proposed as a mechanism for the refinement of neuronal connections. In April of 1984, a conference held at the State University of New York at Albany compared recent findings in the two fields to ascertain the degree to which they are in fact dealing with the same phenomena. In this issue, the proceedings of that conference are presented as a set of review articles. In general, they support an emerging view that, during development of the nervous system, activity has a marked ability to modify synaptic connections and that, as the animal matures, this ability is retained in some areas more than in others. Activity-Dependent Stabilization in Development. Early projections tend to be diffuse, and in many cases, activity is involved in their sharpening to the precise mature pattern. Several examples come from the visual system of vertebrates: (1) the formation of eye-specific stripes that occurs normally in visual cortex but also in dually innervated tectum of goldfish and frog (Tieman, 1985; Schmidt and Tieman, 1985), (2) the sharpening of diffuse retinotopic order in the regenerated retinotectal projection of goldfish (Schmidt, 1985), (3) the alignment of the indirect and direct retinotectal maps to allow binocular vision in Xenopus (Udin, 1985), (4) the segregation of on and off inputs in geniculate of the kitten (Archer et al., 1982) and the formation of receptivefield properties of cells in visual cortex (Hirsch, 1985). In the above cases, a block of visual activity (e.g., intraocular tetrodotoxin or, in some cases, rearing in the dark) prevents the rearrangement of synaptic connections. In general, it is the pattern of activity that is important. Thus, with visual activity blocked, synchronous stimulation of the two pathways from the eyes does not cause segregation into eye-specific stripes,

Activity-related changes in protein phosphorylation in an identified Aplysia neuron.

Abstract: 1. The relationship between long-term electrical activity and protein phosphorylation was investigated in single, identifiable neurons in the abdominal ganglion ofAplysia californica by the intracellular injection of radiolabeled ATP followed by sodium dodecyl sulfate (SDS) gel electrophoresis. 2. Natural and pharmacological treatments that alter the impulse activity of neurons L6 and R15 for prolonged periods did not appear to affect the phosphorylation of most of the 15 major phosphoproteins examined in these cells. 3. Long-term excitation of L6 induced by the phosphodiesterase inhibitor IBMX correlated with phosphorylation of a 29,000-dalton protein. Long-term inhibition of L6 induced by afterdischarge of peptidergic bag-cell neurons appeared to cause dephosphorylation of a 29,000-dalton protein. 4. Burst augmentation of R 15 induced by bag-cell afterdischarge did not cause detectable changes in the phosphorylation of the major proteins we examined. 5. These data are consistent with other studies of neural and nonneural tissues which have found a correlation between activity and the level of phosphorylation of a 29,000-dalton protein.