Giant Aplysia neuron R2 reliably forms strong chemical connections in vitro

The giant cholinergic neuron R2 of Aplysia was cultured in combination with identified neurons L11 and R15 and members of a group of left upper quadrant (LUQ) cells L2 to L6 from the abdominal ganglion. All of these neurons receive cholinergic input from other cells in vivo, but not from R2. In vitro, R2 reliably formed unidirectional chemical connections with these cells. Single action potentials in R2 produced a dual fast and slow inhibitory response in LUQ cells (L2 to L6), a dual fast inhibitory-slow excitatory response in L11, and a slow inhibitory response in R15. The connections formed on LUQ cells were characteristic of their cholinergic input, but the R2-L11 and the R2-R15 connections also had noncholinergic properties. Thus, unlike L10 which forms connections only with its normal targets in vitro, R2 forms strong chemical connections with other neurons which are not found in vivo. The properties of the R2 connections also suggest that it may release another neurotransmitter besides acetylcholine.     

Convergence of muscle spindle afferents on single neurons of the cat dorsal spino-cerebellar tract and their synaptic efficacy

By means of tungsten microelectrodes, action potentials from axons within the dorsal spino-cerebellar tract (DSCT) and from muscle spindle afferents were recorded. A quantitative study was performed in monomuscular DSCT neurons which were excited predominantly by Ia fibers originating in the gastrocnemius muscles. In some experiments single Ia fibers were stimulated electrically while the impulse sequence of a DSCT neuron postsynaptic to the respective afferent fiber was recorded. The gastrocnemius DSCT neurons receive excitatory inputs from 10-18 Ia muscle spindle afferents. The efficacy of each of these inputs is very similar. Thus the neuronal activation decreased approximately linearly with the number of the excitatory afferents cut. Cross-correlograms between the impulse sequence of a Ia gastrocnemius muscle spindle afferent and a DSCT neuron postsynaptic to it exhibited an increased discharge probability of the DSCT neuron from 3-4 ms to 10 ms after the Ia action potential. With increasing impulse rates of the Ia afferent fibers, the excitatory efficacy of the single action potential decreased, but the overall excitation increased with the presynaptic discharge frequency, according to a hyperbolic function. This effect was tested by electrical stimulation of a single Ia axon exciting the DSCT neuron recorded. Interval histograms computed from DSCT neuron impulse trains at steady stretch conditions were predominantly monomodal. They can be well approximated by a Gaussian distribution. The coefficient of variation was independent of the mean activity. At impulse rates above 25 imp X s-1 a negative correlation between successive intervals was observed in first order joint interval diagrams. With an increasing mean discharge rate this correlation (expressed as the serial linear correlation coefficient of the first order r1,2) became stronger up to--0.62 at 90 imp X s-1. Only in a few neurons did the higher order linear correlation coefficients deviate significantly from zero. In 15% of the observed histograms double discharging (mean interval 3-5 ms) produced bimodal distributions. Under steady-state conditions the response of Ia-activated DSCT cells are linearly related to muscle stretch within a middle range of extensions. The differences between Ia impulse pattern and DSCT neuron impulse pattern at steady stretch are discussed. The number of large dendrites of the principal cells in the nucleus dorsalis (Clarke's column) corresponds to the number of excitatory afferent muscle fibers. It is assumed that each excitatory Ia axon sends one axon collateral to the DSCT neuron, forming a climbing type terminal mainly on one of the large dendrites of a DSCT cell.     

Trophic dependence of fiber diameter in a crustacean muscle

Denervation or immobilization of crayfish opener muscles produces little or no atrophic change in fiber diameter within 250 days whereas tenotomy of the same muscle leads to significant fiber atrophy within 50 days. The observations that opener nerve terminals probably degenerated 40–90 days prior to sampling denervated muscles, and that opener muscle fibers are 10–30% shorter in tenotomized muscles than for muscles immobilized in the fully open position, lead to the conclusion that opener fiber diameter is much more dependent on muscle length (or passive tension) than upon neurotrophic factors or muscle activity. These results differ from those reported for most vertebrate twitch muscles which undergo significant atrophy after denervation, immobilization, or tenotomy. These data from a polyneuronal, multiterminal, tonic muscle of a crayfish closely resemble results obtained from avian muscles having similar innervation patterns and suggest that one or more of their common functional properties may determine their similar trophic dependencies.     

Suppression of proprioceptor—motor neuron interactions by proprioceptors in crayfish claw

Crayfish claw proprioceptors and slow closer exciter and opener inhibitor motor neurons were monitored simultaneously during imposed claw displacements. With increasing displacement velocity and decreasing joint angle, the activity of closing sensitive receptors increased, while dynamic-static opening sensitive receptor activity decreased during claw closing. Motor neuron activity evoked by claw opening varied inversely as a function of preceding closing velocity, and directly with preceding pause duration at the closed position. This dependence on closing history cannot be accounted for by changes in opening sensitive receptor activity. Data demonstrate that closing sensitive receptors can suppress excitatory interactions between claw proprioceptors and motor neurons.     

Modulation of arterial muscle contraction in Aplysia by glycine and neuron R14

Glycine and electrical activity in neuron R14 both enhance the contractility of the anterior aorta of the gastropod Aplysia californica. Glycine and R14 do not seem to cause contraction directly, change membrane permeabilities or alter junctional potentials occurring in the muscle fibers, yet they increase the force of contractions induced by other means. Modulation of muscle contraction is a new function for glycine.     

Synaptic actions of identified peptidergic neuron R15 in Aplysia. II. Contraction of pleuroabdominal connectives mediated by motoneuron L7

The purpose of this study was to determine the synaptic actions of the bursting peptidergic neuron R15 in Aplysia. R15 is known to be excited by the neuroendocrine bag cells, which trigger egg laying. In the two companion papers, we show that R15 mediates some of the effects of the bag cells on respiratory and reproductive organs. In this paper, we demonstrate that R15 excites L7, a multimodal motoneuron located in the abdominal ganglion. Although L7 excites several types of muscle fibers as well as neurons, the excitation of L7 by R15 is probably strong enough to cause contraction only of the sheath muscle of the pleuroabdominal connectives, which has an exceptionally low threshold for activation. The excitatory actions of R15 on L7, which desensitize profoundly, appear to be mediated by R15 alpha 1 peptide. The synaptic action of R15 on L7 and on the respiratory pumping system (Alevizos et al., 1991a) can be fully expressed only if R15 is first silenced for 2 hr by injection of hyperpolarizing current. A similar protocol for eliminating desensitization may prove to be generally useful for revealing the synaptic actions of other spontaneously active neurons that have rapidly desensitizing postsynaptic actions.     

Localization of myomodulin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia californica.

The distribution of myomodulin-like peptides in the nervous system of Aplysia californica was examined by using immunocytochemical techniques. Neurons and cell clusters containing immunoreactive material were located in each of the major central ganglia. Myomodulin-like immunoreactivity was also present in fibers in each of the connectives between the ganglia and in peripheral nerves. Varicosities containing immunoreactive material were located on specific regions of peripheral tissues associated with the feeding, digestive, cardiovascular, and reproductive systems. Double-labeling experiments were used to demonstrate myomodulin-like immunoreactivity in two identified neurons, the motor neuron B16 in the buccal ganglion and the widely acting interneuron L10 in the abdominal ganglion. Structures in the eye and cerebral ganglion that may correspond to the optic circadian pacemaker system were also stained. The central and peripheral distribution of myomodulin-like immunoreactivity indicates that this family of neuropeptides is present in specific efferent, afferent, and interneuronal elements that participate in a diversity of neural circuits in Aplysia.     

Peptidergic and serotonergic facilitation of a neuromuscular synapse in Aplysia

(1) A buccal muscle motor neuron which synthesizes the neuromodulatory small cardioactive peptides (SCPs) was identified in the buccal ganglion of Aplysia by using a combination of electrophysiological and single cell biochemical experiments. This neuron was designated B38. (2) Exogenous SCPb enhanced B38-induced contractions when perfused over the target muscle, the rostral portion of the buccal I3 muscle. SCPB potentiation of muscle contraction was associated with an increase in the excitatory junction potential (EJP) amplitude recorded from the muscle fibers, increased muscle cyclic AMP (cAMP) content, hyperpolarization of the muscle fibers, and an increase in the muscle fiber membrane conductance. Exogenous SCPB also depolarized the cell body of B38 and increased electrical coupling between the symmetrically paired B38 neurons. (3) These results suggest that the SCPs may be co-released from B38 along with an unidentified conventional neurotransmitter to homosynaptically facilitate B38 synaptic transmission by modulating presynaptic and postsynaptic components. (4) Stimulation of the identified serotonergic metacerebral neuron or perfusion of exogenous serotonin (5-HT) over the 13 muscle also potentiated B38-induced muscle contractions and EJP amplitude. Thus the B38 neuromuscular synapse represents a peripheral site of serotonergic heterosynaptic facilitation in Aplysia. (5) Presynaptic and postsynaptic serotonergic effects were qualitatively similar to those of SCPB. Serotonergic effects on muscle fiber hyperpolarization and increase in muscle fiber membrane conductance were similar in magnitude to those of SCPB but 5-HT induced a much larger increase in the EJP amplitude which was additive with that of SCPB. (6) The effect of 5-HT on the EJP amplitude was associated with inhibition of a slowly decaying component of synaptic facilitation. Concentrations of SCPB that increased the EJP were much less effective at inhibiting the slow component of facilitation. These observations indicate that 5-HT also exerted a presynaptic effect on B38 transmitter release. (7) Both 5-HT and SCPB increased muscle cAMP levels and application of forskolin mimicked many of their effects. suggesting that at least some of the postsynaptic effects were mediated by increased cAMP levels in the 13 muscle.     

Cellular and synaptic morphology of a feeding motor circuit in Aplysia californica

The cellular and synaptic morphology of a component of the feeding motor circuit in Aplysia californica was examined with light and electron microscopic techniques. The circuit consists of a pair of inhibitory premotor interneurons, B4 and B5, as well as two motoneurons, B15 and B16, which innervate the accessory radula closer muscle. The neurons have wide, varicose arborizations in the buccal ganglion neuropil. All four of these neurons are cholinergic, and in addition, B15 contains immunoreactivity to sera raised against small cardioactive peptide B. Varicose processes in the accessory radula closer muscle are immunoreactive with antisera against several neuropeptides. We identified specific neuromuscular junctions by visualizing horseradish peroxidase uptake in recycled synaptic vesicles. Direct innervation of the accessory radula closer muscle by B15 and B16 is demonstrated by experiments in which horseradish peroxidase is transported from motoneuronal soma to the terminals on muscle fibers. In addition, specific synaptic contacts between B4 and B5 and each of the motoneurons are observed in the buccal ganglion neuropil. Finally, multiple contacts consistent with peptidergic, serotoninergic, and cholinergic synapses are made onto the neurons, suggesting that a variety of transmitters modulate motor output at each level of the hierarchical circuit. These results support the physiological evidence suggesting the involvement of neuropeptides as well as "classical" transmitters in the modulation of circuitry governing feeding behavior in Aplysia.     

On the occurrence of striated muscle within the spinal leptomeninges

A clinically normal adult male rabbit was found to possess fascicles of muscle fibers within the leptomeninges over the spinal cord at L6, L7, and S1. The finding is believed to be purely incidental and unrelated to any experimental procedure. Morphologically, the fascicles had the characteristics of striated muscle; and, while most were of normal dimensions, several were made up of small fibers 4–10 μm in diameter. Motor endplates, capillaries, and endomysial and perimysial connective tissue were also present. Axons myelinated by Schwann cells were associated with the normal bundles but were absent from the bundles of small fibers. In such bundles, large, unmyelinated axons predominated, some of which lacked ensheathment while others were invested by perineurial-type cells. The observation of incidental intraleptomeningeal skeletal muscle probably reflects a rare developmental anomaly. Its occurrence in humans is also discussed.     

Myoblasts, myosins, MyoDs, and the diversification of muscle fibers

Distinct types of muscle fibers form and become innervated by appropriate motor neurons during development. Though the activity pattern of the innervating motor neuron affects fiber type in the adult, it is now clear that innervation is not required for the initial formation of fast and slow muscle fibers during embryonic and fetal development. In addition, multiple types of intrinsically different myoblasts are found at different stages of development and motor neurons may preferentially innervate specific types of muscle fibers at relatively early stages of myogenesis. Thus, at least some of the information required for the formation of specific motor units must be carried by muscle cells. Cellular and molecular analyses of the multiple types of myoblasts, myosin heavy chain isoforms, and myogenesis regulating proteins of the MyoD family are leading to a new understanding of the events that choreograph the formation of fast and slow motor units.     

Trophic reactions of crayfish muscle fibers and neuromuscular synapses after denervation,tenotomy, and immobilization

Severed motor nerve terminals remain morphologically intact and functionally competent for several months following transection of the motor nerve to the claw opener muscle of crayfish (Procambarus clarki). However, severed motor axons are not entirely normal in that excitatory synapses produce junctional potentials that are somewhat smaller than control values. Tenotomy or immobilization often produce little change in synaptic potentials for at least 180 days. Opener muscle fibers with severed motor axons or immobilized muscle fibers show little ultrastructural change for at least 100 days, provided the nerve terminals remain intact. However, after tenotomy, muscle fibers atrophy within 20 to 30 days. This atrophy is more rapid and more severe if nerve terminals on that muscle fiber have degenerated. These atrophic changes in muscle fine structure include disorganization of myofibrils and disruption or loss of the sarcomere Z bands. In summary, our data show that drastic changes in crayfish muscle structure can proceed after tentomy without concomitant changes in function of the motor nerve terminals; and conversely, although transecting the motor nerve has some effect on motor nerve terminals, decentralization has little effect on muscle fiber structure as long as nerve terminals remain intact. These data are in agreement with the hypothesis that crustacean muscle fibers are trophically dependent on the presence of functional nerve terminals and on passive fiber tension (or resting length).     

Nonsynaptic characteristics of neurotransmission mediated by egg-laying hormone in the abdominal ganglion of Aplysia

The bag cell neurons of the marine mollusk Aplysia are a putative multitransmitter system which utilizes two or more neuropeptides that are enzymatically cleaved from a common precursor protein. It has been proposed that one of the neuropeptides, egg-laying hormone (ELH), acts nonsynaptically as a neurotransmitter in the abdominal ganglion by diffusing long distances to target neurons compared to conventional transmitters acting at synapses. To test this idea further, we investigated the physiological properties of neurotransmission mediated by ELH. We found that ELH acts directly to duplicate two types of responses produced by a burst discharge of the bag cells: prolonged excitation of LB and LC cells, and the previously described effect of ELH, burst augmentation of cell R15. Analysis of perfusate collected after electrical stimulation of the bag cells showed that the peptide is released in sufficient quantity to diffuse long distances within the ganglion without being completely inactivated. To mimic the way the peptide is thought to be released physiologically, ELH was arterially perfused into the ganglion. The response normally produced by bag cell activity was duplicated by 0.5 to 1.0 microM concentrations of ELH and showed no rapid desensitization. ELH had no effect on cells that are unaffected by bag cell activity and no effect on cells that are inhibited (LUQ cells) or transiently excited (cells L1 and R1) by bag cell activity. Acidic peptide, another peptide encoded on the ELH precursor protein, was found to be synthesized and released by the bag cells, but it had no effect on the cells we tested. We conclude that the combined properties of ELH neurotransmission resemble the properties of transmission at autonomic nerve endings on cardiac and smooth muscle rather than those of conventional synaptic transmission. ELH released from bag cells is dispersed throughout the interstitial and vascular spaces of the ganglion to produce responses in the cells that have receptors for the peptide. The results also suggest that ELH mediates only a subset of the responses induced by bag cell activity; they are consistent with data indicating that the other responses are mediated by other bag cell peptides derived from the same precursor protein as ELH.     

SD大鼠动眼神经不同部位损伤后对靶器官特异性支配的实验研究

目的研究不同部位损伤对Sprague-Dawley（SD）大鼠动眼神经功能修复的影响及可能机制。方法实验Ⅰ组大鼠（n=24）经幕下、实验Ⅱ组（n=24）大鼠经眶上裂干预动眼神经，术后通过前庭眼反射评估眼外肌在垂直、水平方向的恢复程度。经右侧上直肌注射辣根过氧化酶（HRP），逆行追踪中脑动眼神经核团内神经元分布；48h后行动眼神经组织学、解剖学研究。结果实验Ⅰ组大鼠支配上直肌的神经纤维有45％～51％由对侧中脑运动神经元发出：实验Ⅱ组81％～87％由对侧中脑运动神经元发出，神经元在中脑的分布更接近正常大鼠。实验Ⅱ组大鼠眼外肌功能恢复程度明显优于实验Ⅰ组大鼠。结论动眼神经损伤部位距离眼外肌越近，最终的神经功能恢复水平越好，这可能与再生神经纤维通过损伤部位时的迷行程度相关。     

Identification of Aplysia neurons containing immunoreactive FMRFamide

Electrophysiological and immunocytochemical techniques were used in the abdominal ganglion of Aplysia to identify neurons containing immunoreactive FMRFamide. Large numbers of neurons were immunoreactive for FMRFamide, including R2, L2, L3, L4, L5, L6, 2 cells tentatively identified as L12 and L13, and a previously unidentified cluster on the ventral surface of the right lower quadrant. There was also heavy labelling of fibers, often with beaded varicosities, throughout the neuropil, the cell layers, and the sheath overlying the ganglion. This data provides further evidence that FMRFamide is an important neurotransmitter in Aplysia. The demonstration of immunoreactive FMRFamide in the giant cholinergic neurons R2 and LP1(1) suggests that these well-studied and experimentally convenient cells use acetylcholine and an FMRFamide-like peptide as cotransmitters.     

Autoradiographic analysis of ascending projections from the pontine and mesencephalic reticular formation and the median raphe nucleus in the rat

Ascending projections from the medial pontine reticular formation, the mesencephalic reticular formation, and the median raphe nucleus were examined using the autoradiographic technique. The majority of the ascending fibers labeled after injections of [3H]-leucine into the nucleus pontis caudalis (RPC) course through the brainstem within the tracts of Forel (tractus fasciculorum tegmenti of Forel) and directly ventral to them. At the caudal diencephalon, Forel's bundle divides into dorsal and ventral components bound primarily for the dorsal thalamus and the subthalamus, respectively. RPC fibers project to several regions involved in oculomotor/visual functions. These include the abducens nucleus, the intermediate gray layer of the superior colliculus (SCi), the anterior pretectal nucleus (APN), the ventral lateral geniculate nucleus (LGNv), and regions of the central gray directly bordering the oculomotor nucleus, the interstitial nucleus of Cajal, and the nucleus of Darkschewitsch. Few, if any, fibers from RPC (or from nucleus pontis oralis-RPO) terminate within the oculomotor nucleus proper. Other sites receiving heavy projections from the RPC include adjacent regions of the pontomesencephalic reticular formation (RF), the parafascicular (PF) and central lateral (CL) nuclei of the thalamus and the fields of Forel/zona incerta (FF-ZI). RPO fibers also ascend predominantly in Forel's bundle. Other ascending tracts for these fibers are the medial longitudinal fasciculus and the central tegmental tract (CTT). RPO fibers distribute significantly to the same structures of the oculomotor/visual system receiving projections from RPC. The RPO projections to the SCi and the APN are particularly pronounced. RPO fibers terminate heavily in several nuclei located ventrally within the rostral midbrain/caudal diencephalon. These include major dopamine-containing cell groups (the retrorubral nucleus, the ventral tegmental area, and the substantia nigra-pars compacta) as well as the interpeduncular nucleus, the lateral mammillary nucleus, and the supramammillary nucleus. Other prominent targets for RPO fibers include the mesencephalic RF, specific regions of the central gray, the PF, the CL, the paracentral and central medial nuclei of the thalamus, and the FF/ZI. The major bundle of the ascending fibers labeled after injections of the mesencephalic reticular formation (MRF) travels within the CTT in a position just lateral to the central gray, but a significant number of labeled axons also course in Forel's bundle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anatomy and innervation of the anterior aorta of Aplysia and the ultrastructure of specialized neuromuscular junctions on vascular smooth muscle

The fine structure of the cellular layers and innervation of smooth muscle in the anterior aorta of Aplysia were examined. The inner layer of circular muscle is not innervated but its fibers may be electrically coupled. In contrast, longitudinal fibers in the outer layer are well separated and richly innervated by highly specialized neuromuscular junctions (NMJ). Three distinct types of NMJ are present on this smooth muscle, each identifiable by a set of quantitatively described morphological features including size, degree of contact with sarcolemma, density of active zones, number of mitochondria and vesicular content. The three types of NMJ are likely to arise from the identified serotonergic (RDAAE), cholinergic (RDAAI), and glycinergic (R14) neurons that provide the major known excitatory, inhibitory, and modulatory inputs to this vessel. Each longitudinal muscle fiber is separately innervated by one or more NMJ of each type. Since there are no intercellular junctions between longitudinal fibers, coordination of contractility is clearly a function of the pattern of neural activity. This report further characterizes the rapid and fine control of the vasculature in Aplysia and demonstrates the utility of this preparation for cellular-level studies on the neural control of smooth muscle and neurochemical messengers mediating its activity.     

In situ hybridization of whole-mounts of Aplysia ganglia using non-radioactive probes.

A protocol for carrying out in situ hybridization with non-radioactive, digoxigenin-labelled probes has been developed for whole-mounts of Aplysia ganglia. Whole-mount preparations preserve the anatomical relationships of neurons within intact ganglia and facilitate the precise identification of a particular neuron in live preparations so that functional studies can be correlated with biochemical attributes of an identified neuron. The protocol was developed with the use of probes to messenger RNAs that are abundant in Aplysia neurons. In situ hybridization with a cDNA probe to a neuronal form of beta-actin stained all neurons, including their processes, whereas use of a cDNA probe for the neuropeptide FMRFamide resulted in staining of a select group of Aplysia neurons.     

Development of the trochlear nucleus in quail and comparative study of the trochlear nucleus, nerve, and innervation of the superior oblique muscle in quail, chick, and duck

The present study was undertaken to examine the development of the trochlear nucleus in quail and to compare the mature trochlear nucleus, nerve, and their sole target of innervation, the superior oblique muscle, in quail, chick, and duck. Study of the trochlear nucleus in quail from embryonic day 5 through hatching shows a maximum of 1,248 neurons on embryonic day 10 followed by spontaneous degeneration of 40% of the neurons between days 10 and 16. Previous studies have shown that although the initial and final number of neurons is different in chick and duck, the magnitude of trochlear cell loss in both species is about 40%. This study shows the average number of neurons in the nucleus of quail, chick, and duck, 2 weeks post-hatching, to be 658, 743 and 1,459, respectively. Fiber counts in the trochlear nerve from electron micrograph montages at the same period indicated a ratio of about 1:1 between neurons and axons. While a majority of the fibers in these nerves are myelinated, an average of 3-6% of the fibers are unmyelinated. The nucleus in the quail not only contains the smallest number of neurons but it also innervates the smallest muscle in terms of total number of muscle cells and endplates. However, the opposite relationship does not hold true. The nucleus in duck contains the largest number of neurons, yet the largest number of muscle cells and endplates were found in the chick. The ratios between the neurons and muscle cells as well as between neurons and endplates are about the same in quail and duck. These ratios are much higher in the chick, reflecting the relatively small neuron pool destined for a relatively large target. In spite of variations in the number of neurons, muscle fibers, and endplates the average number of endplates per muscle fiber is relatively constant among the three species.