Proc. Nati. Acad. Sci. USA Vol. 76, No. 9, pp. 4695-4699, September 1979 Neurobiology "Hybrid" synapses formed by foreign innervation of parasympathetic neurons: A model for selectivity during competitive reinnervation (regeneration/synaptic specificity/neuronal plasticity/cardiac ganglion) WILLIAM PROCTOR*, SAMY FRENK*, BARBARA TAYLOR*, AND STEPHEN ROPER*tt Departments of *Anatomy and tPhysiology, University of Colorado Health Sciences Center, Denver, Colorado 80262 Communicated by Stephen W. Kuffler, June 8, 1979 ABSTRACT Selectivity of synapse formation after nerve This report describes physiological and morphological regeneration was tested in the parasympathetic cardiac gan- characteristics of synaptic connections between glion of frogs (Rana pipiens). First, we tested the ability of so- parasympa- matic motor axons to establish synaptic connections with de- thetic neurons and regenerating preganglionic axons, and be- nervated ganglion cells by implanting the hypoglossus nerve into tween parasympathetic neurons and implanted somatic motor the vagotomized heart. After several weeks, stimulation of the axons. implanted hy oglossus mediated a parasympathetic-like inhi- bition of the heart rate, and synaptic responses produced by hypoglossal stimulation were recorded intracellularly in gan- METHODS glion cells. Light and electron microscopy indicated that im- planted hypoglossal nerve terminals contacted parasympathetic Surgical Procedures. To implant a somatic motor nerve into ganglion cells only on their axons and not on the cell body the parasympathetic cardiac ganglion in the frog; we anasto- (where most vagal synapses are found in control animals). Sec- mosed the central end of the first spinal nerve, the hypoglossus, ond, we tested whether regenerating vagal preganglionic axons to the distal stump of the vagus nerve leading to the heart as would compete with foreign (hypoglossal) terminals for inner- vation of cardiac ganglion cells. We allowed the vagus nerve to follows: Frogs (Rana pipiens, northern variety) were anesthe- regenerate in animals in which the implanted hypoglossus had tized with tricaine (MS 222) (Sigma, 100 ,g/g of body weight, established functional contacts with the cardiac ganglion. Vagal injected into the dorsal lymph sac) and the vagus nerves on both axons were able to reinnervate the heart and reestablish syn- sides were exposed. We sectioned the vagus nerves approxi- aptic connections on the cell bodies of ganglion cells. Further- mately 10 mm from their point of exit from the brain and su- more, functional transmission at the foreign (hypoglossal) ter- minals disappeared concomitant with vagal reinnervation. tured the central ends into the skin to retard regeneration. The left hypoglossus was then sectioned about 5 mm from its exit One of the more salient problems concerning repair of damage at the spinal cord and the central end was sutured to the distal in nervous tissue is the ability of regenerating axons to rees- stump of the left vagus nerve with 10-0 monofilament nylon. tablish synaptic connections with appropriate postsynaptic cells. In some animals, we reanesthetized the frog and sectioned the The mechanisms underlying the selectivity of synapse forma- vagus nerves central to the original cut 5 days to 6 weeks before tion during reinnervation are largely unknown, and as yet there characterizing foreign innervation to exclude any spurious vagal is no explanation why regenerating axons reestablish appro- reinnervation. Operated animals were returned to tanks with priate synaptic corrections in some cases but not in others. For running water for recovery and kept for 8 to 52 weeks. During example, we do not know why reinnervation of decentralized this time they were force-fed a slurry of liver, cooked lettuce, mammalian sympathetic ganglia accurately restores functional cod liver oil, and multivitamins. About half the animals survived connections (1, 2), whereas reinnervation of mammalian skel- the operation. etal muscle is largely nonselective (3, 4). Although attempts Electrophysiological Recordings. The techniques for iso- have been made to explain selectivity during reinnervation, lating the ganglion, impaling single cells, and measuring evoked such as the existence of a specific "chemoaffinity" between intracellular responses are discussed more fully by Dennis et nerve terminals and their appropriate targets (5), it has been al. (6) and Roper and Ko (7) and will be described here only difficult to test these ideas critically, and in brief, the cellular briefly. The heart, along with both vagus nerves, and the im- basis of selective synapse formation after nervous tissue damage planted left hypoglossal nerve were dissected free of the animal, still remains an enigma. placed in Ringer's solution (112 mM NaCl/5 mM CaCl2/2 mM We have been studying selectivity of synapse formation KC1/3 mM Hepes buffer, pH 7.2), and the interatrial septum during reinnervation of parasympathetic neurons in the am- containing the cardiac ganglion was exposed and pinned out phibian cardiac ganglion to extend our understanding of neu- in a shallow chamber. The vagal and implanted hypoglossal ronal interactions during regeneration. In particular, we have nerves were drawn into separate suction electrodes for stimu- asked (i) whether a somatic motor nerve (i.e., a nerve supply lation. Neurons were impaled with fine-tipped microelectrodes quite foreign to parasympathetic ganglia) will establish func- (100-300 MQ resistance). tional connections with denervated autonomic neurons in the In some experiments the heart rate was recorded in intact but heart of the frog, and (ii), if so, whether regenerating vagal pithed frogs by making a small incision in the skin overlying axons will successfully compete with foreign inputs to rein- the heart and inserting a fine stainless steel wire, insulated ex- nervate ganglion cells and thereby reveal specificity of synapse cept at its tip. Another stainless steel wire was inserted under formation during this process. the skin in one of the legs for a reference electrode. The elec- trodes were fed into a high gain ac amplifier and the heart rate The publication costs of this article were defrayed in part by page was recorded on a pen recorder. charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. f To whom reprint requests should be addressed. 4695 Downloaded by guest on September 27, 2021 4696 Neurobiology: Proctor et al. Proc. Natl. Acad. Sci. USA 76 (1979) k;I :I .I; ;; A A I I1 30r 20O/ I 0 V B TI 0 l C{ :; :i.. :..;.I;.., I.- I- !.. 1: ;::; 20 msec C E vI5 I( V GO sec 20 mV FIG. 1. Electrocardiograms from three frogs, showing the effect of stimulating vagal and hypoglossal nerve roots. Trains of stimuli (2.5 msec duration pulses, 10 Hz) were applied to the nerves with silver wire bipolar hook electrodes near the brain and spinal cord. The in- tensity (volts) and duration of stimulation are shown below the re- cordings. The animals were pithed prior to exposure ofthe nerve roots. (A) Control unoperated frog. Vagal stimulation immediately stopped the heart, whereas hypoglossal stimulation had no measurable effect. (B) Experimental animal in which the hypoglossus had been surgically FIG. 2. Intracellularly recorded synaptic responses from para- redirected to the denervated heart 12 weeks before. Hypoglossal sympathetic neurons in the isolated frog cardiac ganglion. Three stimulation produced a parasympathetic-like bradycardia. Vagal different preparations are shown. (A) Postsynaptic potentials evoked regeneration had been prevented in this animal (see text). (C) Ex- by stimulating the preganglionic (vagus) nerve in control unoperated perimental animal in which the hypoglossal nerve had been implanted animals. Two traces are superimposed. Increasing the stimulus in- in the heart and vagal axons had been allowed to regenerate (see text). tensity to the vagus produced two synaptic responses, a subthreshold The surgical operation had been performed 29 weeks before. and a suprathreshold one, indicating that at least two separate pre- ganglionic fibers innervated this neuron. The residual depolarization Morphological Techniques. For light microscopy, the tissue after the action potential is caused by the intense and prolonged action was stained with a modified zinc iodide/osmium technique (M. of the transmitter at vagal synapses and is typical for these neurons as amounts (see ref. 6). (B) Synaptic potentials produced by stimulating a hy- Letinsky, personal communication) follows: equal poglossal nerve that had been surgically redirected into the cardiac of zinc iodide solution (3 g of zinc powder plus 1 g of iodine ganglion 16 weeks prior to the experiment. Two traces are superim- crystals in 40 ml of distilled water) and 0.2 M sodium acetate posed. Both sub- and suprathreshold responses were evoked by buffer (pH 4.0) were mixed, and 0.5 ml of 4% osmium tetroxide varying the stimulus intensity to the implanted hypoglossus. (C) (Polysciences, Warrington, PA) was added to 2 ml of the buf- Postsynaptic responses in a neuron innervated by a regenerating vagal fered zinc iodide solution. The mixture was then immediately axon (upper trace) and an implanted hypoglossal axon (lower trace). The anastomosis had been performed 31 weeks before, and the vagus added to the tissue and left for 60-90 min. Last, the tissue was had been allowed to regenerate (see text). The conduction velocity rinsed