Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.     

Motor innervation of the pharynx levator muscle of the snail, Helix pomatia: physiological and histological properties

(1) Motor innervation of the pharynx levator muscle of Helix pomatia was investigated with intracellular recording and axonal iontophoresis of cobalt chloride. (2) Muscle fibers respond to direct electrical stimulation of the muscle with active graded responses or non-overshooting spike potentials. (3) Each fiber is innervated via the external and internal lip nerves by several (mostly 3) excitatory nerve fibers each. Two types of EPSPs can be distinguished according to amplitude, duration, and facilitation. (4) Axonal CoCl2-staining via an external lip nerve branch revealed many nerve fibers entering the muscle and branching there into a rich network of blebbed fibers of various diameters.     

Catecholaminergic innervation of the rat adrenal cortex

Summary The zona glomerulosa of the rat adrenal gland is innervated by catecholaminergic nerves. Using histofluorescence techniques, we observed catecholaminergic plexuses surrounding adrenal capsular and subcapsular blood vessels. Individual varicose nerve fibers that branched off these plexuses were distributed among adrenal glomerulosa cells. This innervation was permanently eliminated after neonatal sympathectomy with guanethidine or 6-hydroxydopamine, but was not affected by ligation of the splanchnic nerve or extirpation of the suprarenal ganglion. At the ultrastructural level, axonal varicosities were commonly observed in close proximity to glomerulosa cells and blood vessels. Nerve fibers and varicosities were found to contain small (30–60 nm) clear vesicles as well as large (60–110 nm) and small (30–60 nm) dense-cored vesicles. In tissue fixed for the dichromate reaction with or without pretreatment with the false transmitter 5-hydroxydopamine, many nerve terminals contained numerous small dense-cored vesicles which are thought to contain catecholamines. These results establish the anatomical substrate for the catecholaminergic innervation of the rat adrenal cortex.     

The nervous environment of individual smooth muscle cells of the guinea pig vas deferens

Smooth muscle cells of the external longitudinal coat of the guinea pig vas deferens were followed for 480 mu at 4.5-mu intervals. Muscle bundles and fibers interwove, facilitating intermuscular and neuromuscular contacts. The ribbon- or rodlike muscle cells were about 450 mu long, 3,000 mu3 in volume, and 4,500 mu2 in area. The thickened nuclear zone day anywhere along the middle one-third of the cell. Intercellular distances were 500-800 A. Intrusions were rare, and tight-junctions absent. At any level in a field of 80 muscle fibers there were 10-15 nerve bundles, each containing several varicose axons. Bundles and axons divided. Axons, en passage, were frequently within 500-1,000 A of a muscle fiber. En passage close contacts were rate. Axon terminations were bare, and bare axons invariably terminated. Bare terminations had scattered vesicle-laden varicosities and were from 10-60 mu in length, and all ended within 500 A of muscle fibers. Some made close contact with muscle fibers. Less than half of the muscle cells received this close contact, but some cells were approached by more than one termination. Most terminations involved more than one cell. Some cells had little or no innervation. Some groups of cells had a rich innervation. There was very little evidence of sensory innervation. These conclusions are not valid for other smooth muscles.     

The efferent innervation of the genital chamber by an identified serotonergic neuron in the female cricket Acheta domestica

Summary The serotonergic innervation of the genital chamber of the female cricket, Acheta domestica, has been investigated applying anti-serotonin (5-HT) immunocyto-chemistry at both light- and electron-microscopic levels as well as using conventional electron microscopy. Whole mount and pre-embedding chopper techniques of immuno-cytochemistry reveal a dense 5-HT-immunoreactive network of varicose fibers in the musculature of the genital chamber. All of these immunoreactive fibers originate from the efferent serotonergic neuron projecting through the nerve 8v to the genital chamber (Hustert and Topel 1986; Elekes et al. 1987). At the electron-microscopic level, 5-HT-immunoreactive nerve terminals, which contain small (50–60 nm) and large ( 100 nm) agranular vesicles as well as granular vesicles (100nm), contact the muscle fibers or the sarcoplasmic processes without establishing specialized neuromuscular connections. In addition to the 5-HT-immunoreactive axons, two types of immunonegative axons can also be found in the musculature. By use of conventional electron microscopy, three ultrastructurally distinct types of axon processes can be observed, one of which resembles 5-HT-immunoreactive axons. While the majority of the varicosities do not synapse on the muscle fibers, terminals containing small (50–60 nm) agranular vesicles occasionally form specialized neuromuscular contacts. It is suggested that the 5-HTergic innervation plays a non-synaptic modulatory role in the regulation circular musculature in the genital chamber of the cricket, while the musculature as a whole may be influenced by both synaptic and modulatory mechanisms.Fellow of the Alexander von Humboldt-Stiftung     

The innervation pattern of crustacean skeletal muscle

Summary The innervation pattern of distal muscle fibers of the opener muscle of walking legs of crayfish (Astacus leptodactylus) was investigated using methylene-blue staining, cobalt infiltration, and electron microscopy. A quantitative analysis of the entire innervation of single muscle fibers was attempted.It was found that instead of the generally assumed parallel array of numerous excitatory and inhibitory terminals, innervation consists of only a few branched terminals. The branches of excitatory and inhibitory terminals lie side-by-side. Both types are characterized by numerous varicosities (see Fig. 9B). The aggregate length of excitatory as well as inhibitory terminals on one muscle fiber is, on the average, about 1,500 m with a total of 152 varicosities spaced about 10 m apart. The average diameter of the varicosities is 4.26 m, that of the connecting thin segments about 0.5 m. Total terminal surface of motor or inhibitory terminals amounts to about 10,000 m² per muscle fiber. There are approximately 2,000 motor synapses on each muscle fiber, but their average total area is only about 6% of the terminal membrane area, or 0.06% of the (idealized) muscle fiber surface.There are conspicuous differences in the postsynaptic specializations associated with excitatory and inhibitory terminals; these are described in detail.The results are discussed in a functional context and with regard to design and results of electrophysiological experiments.Supported by Sonderforschungsbereich 138 of the Deutsche Forschungsgemeinschaft     

Serotonergic innervation of the foot of the pond snail Lymnaea stagnalis (L.)

The aminergic innervation of the foot of Lymnaea stagnalis was investigated using electron microscopy, immunocytochemistry, and HPLC. The foot was found to contain large amounts of serotonin and dopamine, though at lower concentrations than are found in nervous tissue. Serotonin containing tissue was concentrated in the ventral surface of the foot, under ciliated areas of the epidermis where it occurred in varicosities, with fine tracts joining these varicosities. Varicosities also occurred in deeper tissues, probably adjacent to mucus cells. Positive fluorescence for serotonin in axons was found in nerves innervating the foot, but few neuronal cell bodies containing serotonin were detected, indicating that most of the innervation was coming from the central ganglia. Axon varicosities were found using TEM on ciliated cells, mucus cells, and muscle cells as well as interaxonal junctions (possibly non-synaptic) within nerves. The neuronal varicosities contacting the ciliated cells and mucus cells contained mostly dense-cored vesicles of between 60 and 100 nm in diameter. Smaller, lucent vesicles also occurred in these terminals. The origin and significance of this innervation is discussed. It is suggested that both serotonin and dopamine may play a large role in controlling ciliary gliding by the foot.     

Deficiency of intramuscular ICC increases fundic muscle excitability but does not impede nitrergic innervation

The motility of the gastrointestinal tract is generated by smooth muscle cells and is controlled to a large extent by an intrinsic neural network. A gap of approximately 200 nm usually separates nerve varicosities from smooth muscle cells, which suggests that direct innervation of the smooth muscle by synapses does not occur. Enteric nerves do make synapse-like contact with proposed regulatory cells, the interstitial cells of Cajal (ICC), which in turn may be in gap junction contact with smooth muscle cells. The role played by ICC in enteric innervation is controversial. Experimental evidence has been presented in vitro for the hypothesis that nitrergic inhibitory innervation is strongly reduced in the absence of ICC. However, in vivo data appear to dispute that. The present report provides evidence that explains the discrepancy between in vivo and in vitro data and provides evidence that inhibitory neurotransmitters can reach smooth muscle cells without hindrance when ICC are absent. The fundic musculature shows increased responses to substance P-mediated innervation and shows marked spontaneous activity, which is consistent with increased muscle excitability.     

The relationship between nerves and smooth muscle cells in the rat iris

Summary The dilatator muscle cells form short projections into the stroma of the iris. Close to these projections run several nerve bundles. The unmyelinated axons often show enlargements (varicosities) containing mitochondria and vesicles. Several of the varicosities are partly denuded of the Schwann cell and are covered only by a basement membrane. The varicosities are then separated from the muscle cells only by basement membranes and a 0.1–1 stromal space. The ultrastructure of the iris dilatator muscle thus also fits the view that the autonomic ground plexus with its varicosities forms the real innervation apparatus.The smallest space between axon and muscle has a width of 700–900 Å and is cemented with basement membrane material. It is suggested that the main function of these contact sites is not to transmit a nerve impulse but to anchor the nerves to their effector organ.This study has been supported by grants from the Swedish State Research Council (U 267) and the United States Public Health Service (N B 2854-04).     

Autonomic innervation of receptors and muscle fibres in cat skeletal muscle

Cat hindlimb muscles, deprived of their somatic innervation, have been examined with fluorescence and electron microscopy and in teased, silver preparations; normal diaphragm muscles have been examined with electron microscopy only. An autonomic innervation was found to be supplied to both intra- and extrafusal muscle fibres. It is not present in all muscle spindles and is not supplied at all to tendon organs. Fluorescence microscopy revealed a noradrenergic innervation distributed to extrafusal muscle fibres and some spindles. On the basis of the vesicle content of varicosities the extrafusal innervation was identified as noradrenergic (32 axons traced), and the spindle innervation as involving noradrenergic, cholinergic and non-adrenergic axons (14 traced). Some of the noradrenergic axons that innervate spindles and extrafusal muscle fibres are branches of axons that also innervate blood vessels. We cannot say whether there are any noradrenergic axons that are exclusively distributed to intra- or extrafusal muscle fibres. The varicosities themselves may be in neuroeffective association with striated muscle fibres only, or with both striated fibres and the smooth muscle cells in the walls of blood vessels. The functional implications of this direct autonomic innervation of muscle spindles and skeletal muscle fibres are discussed and past work on the subject is evaluated.     

Synapse elimination by fiber type and maturational state in rabbit soleus muscle

We have compared the development of fast and slow motor innervation in the neonatal rabbit soleus, a muscle which contains two distinct motor unit types during the early period of polyneuronal innervation. The innervation state of individual muscle fibers was ascertained using an intracellular electrode; a fluorescent dye was then injected into particular fibers to permit subsequent identification of histochemical type. We found no significant difference in the time course of synapse elimination for fast and slow motor units as judged by the percentage of fibers remaining polyneuronally innervated at two ages: 7-8 days, when most fibers are multiply innervated, and 10-11 days, when the level of polyinnervation is low. In a second experiment, we examined a phenomenon in which compound end-plate potentials were occasionally seen in muscle fibers at an age (17-23 days) well past the major episode of synapse elimination. We present evidence that this apparent polyinnervation in fact derives from an electrode-induced electrical coupling artifact and that genuinely polyinnervated fibers are very rare at this stage, if present at all.     

The abdominal muscle receptor organ in Astacus leptodactylus (Crustacea)

The structure of both the slow- and the fast-adapting abdominal muscle receptor organ of Astacus leptodactylus is described with particular reference to differences between the two systems. The receptors are composed of a thin muscle that extends from the front edge of one segment to the front edge of the following and a sensory cell connected with this muscle. In the zone where the sensory cells enter their respective muscle, muscle fibers are reduced (zone of relative muscle exclusion = ZRME) and partly replaced by connective tissue. The occurrence of dendritic processes of both the slow and the fast neurons is confined to this zone. The following differences between the two receptor types are established: (1) The fast receptor muscle reveals a smaller sarcomere length than the slow receptor muscle and a higher myosin/actin filament ratio. (2) Muscle fibers that pass the ZRME are always found at its periphery in the fast system, separated from dendritic processes by layers of connective tissue, while in the slow system muscle fibers frequently are intermingled with the sensory elements. (3) The ZRME of the slow receptor is 20-30% longer than that of the fast receptor. (4) The dendritic varicosities of the slow neuron, on an average, contain many more mitochondria than those of the fast neuron. (5) Dendritic processes (fine twigs as well as varicosities) are juxtaposed to the sarcolemma of the muscle fibers only in the slow system; in the fast system dendrites and muscle are spatially separated by connective tissue. It is assumed that these differences between the two receptor types are at least in part responsible for the different thresholds observed in physiological experiments.     

The 5-HT immunoreactive innervation of the Helix procerebrum

In the procerebrum of terrestrial snails, 5-HT is a key modulatory substance of the generation of synchronous oscillatory activity and odor learning capability. In this study, we have analyzed the characteristics of the 5-HT-immunoreactive (5-HT-IR) innervation of the distinct anatomical regions of the procerebrum of Helix pomatia, applying correlative light- and electron microscopic immunocytochemistry. A dense network of 5-HT-IR innervation was demonstrated in the cell body layer, meanwhile a varicose fiber system of different density occurred in the different neuropil regions. At the ultrastructural level, labeled varicosities were found to contact both procerebral cell bodies, and different unlabeled axon profiles in the neuropils. The labeled structures established mostly close non-specialized membrane contacts with the postsynaptic profiles. The overall dense distribution of 5-HT-IR innervation supports a general modulatory role of 5-HT in processing different olfactory events.     

An anatomical and electrophysiological study of synapse elimination at the developing frog neuromuscular junction

Synapse elimination was examined in the developing frog cutaneous pectoris muscle using histological and electrophysiological techniques. Morphological synapse elimination occurred in two phases. The first phase, which began at the time of metamorphosis and continued until the second to third postmetamorphic week, was characterized by a rapid decline in the number of endplates receiving greater than or equal to 3 synaptic inputs. However, 50% of the muscle fibers still remained dually innervated. This dual innervation decreased with a much slower time course; approximately 20% of the muscle fibers were dually innervated in 1- to 2-year-old frogs. During the first phase of synapse elimination no difference was noted between the distribution of acetylcholine receptors or acetylcholinesterase activity associated with the terminal arborizations formed by separate axons at one synaptic site. However, terminal arborizations formed by small diameter axons and consisting of varicosities separated by thin interconnectives became apparent during this period. Such varicose arborizations responded to nerve stimulation and released acetylcholine in proportion to their terminal length as did the nonvaricose arborizations. In addition, the number of morphological and physiological inputs at one endplate site was well correlated throughout the first phase of synapse elimination.     

Die Doppelbrechung der glatten Muskelfasern des Penisretraktors von Helix pomatia

Zusammenfassung Die Eigendoppelbrechung der glatten Muskelfasern des Penisretraktors der Schnecke Helix pomatia nimmt mit steigendem Faserdurchmesser ab. Bei konstanter Länge des Muskels und gleichbleibendem Faserdurchmesser wächst die Eigendoppelbrechung mit zunehmender Anzahl der Dehnungscyclen, die der Muskel vor der Fixierung durchlief.

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The birefringence of the smooth muscle fibers of the penis retractor muscle of Helix pomatia

Summary The intrinsic birefringence of smooth muscle fibers of the penis retractor muscle of the snail Helix pomatia decreases with increasing diameter of the fibers. At constant length of the muscle and at constant diameter of the fibers the intrinsic birefringence of the fibers increases with growing number of stretch-and-release cycles the muscle was treated with before fixation.

     

The monoamines in molluscs. II. Dopamine and neurotransmission. Cardiac dopaminergic innervation in Helix pomatia (author's transl)]

In the molluscs, dopamine is very probably a chemical transmitter at the level of both the central nervous system and certain peripheral structures. The heart of Helix pomatia does not have any intrinsic innervation, but it receives extrinsic innervation from fibres coming from the visceral nerve. Formaldehyde fluorescence histochemistry localizes the cardiac catecholamines in some of these fibres and in their endings. However, dopamine, which dominates, does not seem to be a transmitter involved in cardioregulation in the same way as 5-hydroxytryptamine. The quantities of active dopamine (stimulants) cannot be compared with those required for a neurotransmitter. This is also true for noradrenaline. Dopamine more certainly plays a role at the metabolic and tropic level by acting within a more or less short period as a regulator of cellular activity and contractility. The Helix heart is a suitable model for future research in this field.     

Neurotensin. Immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone

The present study attempts to compile information on the possible physiologic role of the endogenous peptide neurotensin (NT) as a hormone and/or neurotransmitter. The methodological approach is immunohistochemical localization of NT in the entero-endocrine system as well as in the central and peripheral nervous systems. The results found in the three systems are first related to the pharmalogical and physiological findings in the literature. Subsequently their significance is discussed for each organ separately before attempting a final overall interpretation. Briefly, the present study reveals the following essential findings: The occurrence and distribution of NT-IR entero-endocrine cells (N-cells) in different mammals including man, as well as in representative members of all classes of vertebrates and higher invertebrates, are analyzed and evaluated morphometrically. The NT-IR cells in all investigated species are demonstrated to be of the open type. The innervation of paravertebral and prevertebral ganglia by NT-IR fibers is described; at least a portion of these fibers is thought to originate in NT-IR perikarya of the substantia intermedia of the spinal cord. The involvement of these NT-IR fibers in the regulation of systemic blood flow (hypertension) is suggested. The existence of NT-IR innervation of the gastro-intestinal tract is considered to be a general phenomenon. This notion is reaffirmed by phylogenetic investigation of the NT-IR enteric nerves. The pharmacological effects of NT in different portions of the gastro-intestinal tract, reported in the literature are related to the immunohistochemical localization of NT. In light of the present results, some of the effects of NT which were previously considered to be of an endocrine or paracrine nature - such as contraction of the guinea-pig ileum - are interpreted as effects of NT of neuronal origin. The specific NT-IR innervation of target cells in the exocrine pancreas (vascular smooth muscle, acinar cells) is demonstrated, and participation of NT-IR nerve fibers in regulation of the secretion of pancreatic juice is postulated. The innervation of the heart (coronary vasculature, myocardium, conduction system) by NT-IR fibers is demonstrated in various mammals and for the first time also in man. The cardiac NT-IR nerve fibers are thought to be the cytological substrate for different NT effects on heart action (coronary vasoconstriction, positive inotropy and chronotropy) reported in the literature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excitatory neurotransmitters in the tentacle flexor muscles responsible for space positioning of the snail olfactory organ

Recently, three novel flexor muscles (M1, M2 and M3) in the posterior tentacles of the snail have been described, which are responsible for the patterned movements of the tentacles of the snail, Helix pomatia. In this study, we have demonstrated that the muscles received a complex innervation pattern via the peritentacular and olfactory nerves originating from different clusters of motoneurons of the cerebral ganglia. The innervating axons displayed a number of varicosities and established neuromuscular contacts of different ultrastructural forms. Contractions evoked by nerve stimulation could be mimicked by external acetylcholine (ACh) and glutamate (Glu), suggesting that ACh and Glu are excitatory transmitters at the neuromuscular contacts. Choline acetyltransferase and vesicular glutamate transporter immunolabeled axons innervating flexor muscles were demonstrated by immunohistochemistry and in Western blot experiments. Nerve- and transmitter-evoked contractions were similarly attenuated by cholinergic and glutamatergic antagonists supporting the dual excitatory innervation. Dopamine (DA, 10^?5 M) oppositely modulated thin (M1/M2) and thick (M3) muscle responses evoked by stimulation of the olfactory nerve, decreasing the contractions of the M1/M2 and increasing those of M3. In both cases, the modulation site was presynaptic. Serotonin (5-HT) at high concentration (10^?5 M) increased the amplitude of both the nerve- and the ACh-evoked contractions in all muscles. The relaxation rate was facilitated suggesting pre- and postsynaptic site of action. Our data provided evidence for a DAergic and 5-HTergic modulation of cholinergic nerves innervating flexor muscles of the tentacles as well as the muscles itself. These effects of DA and 5-HT may contribute to the regulation of sophisticated movements of tentacle muscles lacking inhibitory innervation.     

Cholinergic innervation of the mouse superior cervical ganglion: light-and electron-microscopic immunocytochemistry for choline acetyltransferase

Summary The cholinergic innervation of the mouse superior cervical ganglion was investigated by means of immunocytochemistry using a well-characterized monoclonal antibody against choline acetyltransferase (ChAT). Immunopositive nerve fibers entered the superior cervical ganglion from the cervical sympathetic trunk. Light-microscopically, these fibers appeared to be heterogeneously distributed among the principal ganglion cells. The rostral part of the ganglion contained more ChAT-positive fibers then the middle or the caudal one. The axons branched several times before forming numerous varicosities. Most of the ChAT-stained fibers and varicosities aggregated in glomerula-like neuropil structures that were surrounded by principal ganglion cell bodies, whereas others were isolated or formed little bundles among principal neurons. None of the neurons or other cell types in the ganglion exhibited ChAT-positivity. ChAT-immunoreactive fibers disappeared from the ganglion 5 or 13 days after transection of the cervical sympathetic trunk. At the ultrastructural level, most axon terminals and synapses showed ChAT-immunoreactivity. An ultrastructural analysis indicated that immunostained synapses occurred directly on the surface of neuronal soma (1.8%) and dendritic shafts (17.6%). Synapses were often seen on soma spines (18.4%) and on dendritic spines (62.2%). All immunoreactive synapses were of the asymmetric type. The results provide immunocytochemical evidence for a heterogeneous cholinergic innervation of the ganglion and the principal neurons.     

The rat olivocerebellar system visualized in detail with anterograde PHA-L tracing technique, and sprouting of climbing fibers demonstrated after subtotal olivary lesions

The rat olivocerebellar climbing fiber system has been investigated at the light and electron microscopic level with anterograde Phaseolus vulgaris leucoagglutinin (PHA-L) tracing. From PHA-L Injections in different parts of the inferior olive labelled axons could be traced to the contralateral cerebellum. Arriving in the deep cerebellar white matter, the olivocerebellar axons ran around and through the cerebellar nuclei. Plexuses of labelled terminal fibers appeared in the cerebellar nuclei, and the density of this innervation was estimated to 1-4 million varicosities per mm3. Ultrastructurally, these boutons engaged in asymmetric synapses with small dendrites. Bundles of labelled fibers continued into the folial white matter, and terminated as climbing fibers in sagittal zones of the cerebellar cortex. Both the cortical and nuclear terminations of the olivocerebellar system are strictly topographically organized. The plasticity of climbing fibers was studied after partial lesions of the inferior olive induced by 3-acetylpyridine. One to 6 months after the lesion, surviving climbing fibers demonstrated extensive sprouting. The newly formed axons originated from parent climbing fiber plexuses, grew in the direction of parallel fibers, and formed terminal plexuses around several neighbouring Purkinje cells. As normal climbing fiber terminals, these terminals formed asymmetric synapses with spines of proximal Purkinje cell dendrites, and evidence by Benedetti et al. (1983) shows that the regenerated innervation is electrophysiologically functional. It is suggested that denervated Purkinje cells release a trophic substance, which stimulate surviving climbing fibers to sprouting, axonal growth and synapse formation.