Journal of Comparative Physiology A

, Volume 191, Issue 12, pp 1157–1171

A dye mixture (Neurobiotin and Alexa 488) reveals extensive dye-coupling among neurons in leeches; physiology confirms the connections

  • Ruey-Jane Fan
  • Antonia Marin-Burgin
  • Kathleen A. French
  • W. Otto Friesen
Original Paper


Although the neuronal circuits that generate leech movements have been studied for over 30 years, the list of interneurons (INs) in these circuits remains incomplete. Previous studies showed that some motor neurons (MNs) are electrically coupled to swim-related INs, e.g., rectifying junctions connect IN 28 to MN DI-1 (dorsal inhibitor), so we searched for additional neurons in these behavioral circuits by co-injecting Neurobiotin and Alexa Fluor 488 into segmental MNs DI–1, VI–2, DE–3 and VE–4. The high molecular weight Alexa dye is confined to the injected cell, whereas the smaller Neurobiotin molecules diffuse through gap junctions to reveal electrical coupling. We found that MNs were each dye-coupled to approximately 25 neurons, about half of which are likely to be INs. We also found that (1) dye-coupling was reliably correlated with physiologically confirmed electrical connections, (2) dye-coupling is unidirectional between MNs that are linked by rectifying connections, and (3) there are novel electrical connections between excitatory and inhibitory MNs, e.g. between excitatory MN VE-4 and inhibitory MN DI-1. The INs found in this study provide a pool of novel candidate neurons for future studies of behavioral circuits, including those underlying swimming, crawling, shortening, and bending movements.


Hirudo Circuits Gap junctions Diode Motoneurons 


  1. Antonsen BL, Edwards DH (2003) Differential dye-coupling reveals lateral giant escape circuit in crayfish. J Comp Neurol 466:1–13CrossRefPubMedGoogle Scholar
  2. Bacskai T, Matesz C (2002) Primary afferent fibers establish dye-coupled connections in the frog central nervous system. Brain Res Bull 57:317–319CrossRefPubMedGoogle Scholar
  3. Brodfuehrer PD, Debski EA, O’Gara BA, Friesen WO (1995) Neuronal control of leech swimming. J Neurobiol 27:403–418CrossRefPubMedGoogle Scholar
  4. Cacciatore TW, Brodfuehrer PD, Gonzalez JE, Jiang T, Adams SR, Tsien RY, Kristan WB Jr, Kleinfeld D (1999) Identification of neural circuits by imaging coherent electrical activity with FRET-based dyes. Neuron 23:449–459CrossRefPubMedGoogle Scholar
  5. Coggeshall RE, Fawcell (1964) The fine structure of the central nervous system of the leech, Hirudo medicinalis. J Neurophysiol 27:229–289PubMedGoogle Scholar
  6. Colombaioni L, Brunelli M (1988) Neurotransmitter-induced modulation of an electrotonic synapse in the CNS of Hirudo medicinalis. Exp Biol 47:139–44PubMedGoogle Scholar
  7. Connors BW, Long MA (2004) Electrical synapses in the mammalian brain. Annu Rev Neurosci 27:393–418CrossRefPubMedGoogle Scholar
  8. Devor A, Yarom Y (2002) Electrotonic coupling in the inferior olivary nucleus revealed by simultaneous double patch recordings. J Neurophysiol 87:3048–3058PubMedGoogle Scholar
  9. Dykes IM, Freeman FM, Bacon JP, Davies JA (2004) Molecular basis of gap junctional communication in the CNS of the leech Hirudo medicinalis. J Neurosci 24:886–894CrossRefPubMedGoogle Scholar
  10. Edwards DH, Yeh SR, Krasne FB (1998) Neuronal coincidence detection by voltage-sensitive electrical synapses. Proc Natl Acad Sci USA 95:7145–7150CrossRefPubMedGoogle Scholar
  11. Eisenhart FJ, Cacciatore TW, Kristan WB Jr (2000) A central pattern generator underlies crawling in the medicinal leech. J Comp Physiol [A] 186:631–643CrossRefGoogle Scholar
  12. Ewadinger N, Syed N, Lukowiak K, Bulloch A (1994) Differential tracer coupling between pairs of identified neruones of the mollusc Lymnaea Stagnalis. J Exp Biol 192:291–297PubMedGoogle Scholar
  13. Fan RJ, Friesen WO (2004) Dye mixtures (Neurobiotin and Alexa 488) reveal extensive dye coupling among swim-related neurons in leeches. Program No. 658.5 2004 Abstract Viewer/Itinerary Planner. Society for Neuroscience, Washington, DCGoogle Scholar
  14. Friesen WO (1981) Physiology of water motion detection in the medicinal leech. J Exp Biol 92:255–275Google Scholar
  15. Friesen WO (1989a) Neuronal control of leech swimming movements. I. Inhibitory interactions between motor neurons. J Comp Physiol A 166:195–203CrossRefGoogle Scholar
  16. Friesen WO (1989b) Neuronal control of leech swimming movements. II. Motor neuron feedback to oscillator cells 115 and 28. J Comp Physiol A 166:205–215Google Scholar
  17. Friesen WO (1994) Reciprocal inhibition: a mechanism underlying oscillatory animal movements. Neurosci Biobehav Rev 18:547–553CrossRefPubMedGoogle Scholar
  18. Gaskell JF (1914) The chromaffine system of annelids and the relation of this system to the contractile vascular system in the leech, Hirudo medicinalis. Philos Trans R Soc Lond B 205:153–212CrossRefGoogle Scholar
  19. Granzow B, Friesen WO, Kristan WB Jr (1985) Physiological and morphological analysis of synaptic transmission between leech motor neurons. J Neurosci 5:2035–2050PubMedGoogle Scholar
  20. Haag J, Borst A (2005) Dye-coupling visualizes networks of large-field motion-sensitive neurons in the fly. J Comp Physiol A 191:445–454CrossRefGoogle Scholar
  21. Kita H, Armstrong W (1991) A biotin-containing compound N-(2-aminoethyl) biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin. J Neurosci Methods 37:141–50CrossRefPubMedGoogle Scholar
  22. Kristan WB Jr, Stent GS, Ort CA (1974) Neuronal control of swimming in the medicinal leech. I. Dynamics of the swimming rhythm. J Comp Physiol 94:97–119CrossRefGoogle Scholar
  23. Kristan WB Jr, Calabrese RL, Friesen WO (2005) Neuronal control of leech behavior. Prog in Neurobiol, in pressGoogle Scholar
  24. Lee SC, Krasne FB (1993) Ultrastructure of the circuit providing input to the crayfish lateral giant neurons. J Comp Neurol 327:271–288CrossRefPubMedGoogle Scholar
  25. Macagno ER (1980) Number and distribution of neurons in leech segmental ganglia. J Comp Neurol 15:283–302CrossRefGoogle Scholar
  26. Macagno ER, Muller KJ, Kristan WB, Deriemer SA, Stewart R, Granzow B (1981) Mapping of neuronal contacts with intracellular injection of horseradish peroxidase and Lucifer yellow in combination. Brain Res 217:143–149CrossRefPubMedGoogle Scholar
  27. Marder E, Calabrese RL (1996) Principles of rhythmic motor pattern generation. Physiol Rev 76:687–717PubMedGoogle Scholar
  28. Marin-Burgin A, Kristan W, French KA (2003) Development of sensory-motor circuits associated with behavior in the leech. Program No. 500.3. Abstract Viewer/Itinerary Planner. Society for Neuroscience, Washington, DCGoogle Scholar
  29. Martinez AD, Hayrapetyan V, Moreno AP, Beyer EC (2002) Connexin43 and Connexin45 form heteromeric gap junction channels in which individual components determine permeability and regulation. Circ Res 90:1100–1107CrossRefPubMedGoogle Scholar
  30. Muller KJ, Nicholls JG, Stent GS (1981) Neurobiology of the leech. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  31. Nicholls JG, Baylor DA (1968) Specific modalities and receptive fields of sensory neurons in CNS of the leech. J Neurophysiol 31:740–756PubMedGoogle Scholar
  32. Nicholls JG, Purves D (1970) Monosynaptic chemical and electrical connextions between sensory and motor cells in the central nervous system of the leech. J Physiol 209:647–667PubMedGoogle Scholar
  33. Norris BJ, Calabrese RL (1987) Identification of motor neurons that contain a FMRFamide-like peptide and the effects of FMRFamide on longitudinal muscle in the medicinal leech, Hirudo medicinalis. J Comp Neurobiol 266:95–111CrossRefGoogle Scholar
  34. O’Gara BA, Chae H, Latham LB, Friesen WO (1991) Modification of leech behavior patterns by reserpine-induced amine depletion. J Neurosci 11:96–110PubMedGoogle Scholar
  35. Onn SP, Grace AA (1999) Alterations in electrophysiological activity and dye coupling of striatal spiny and aspiny neurons in dopamine-denervated rat striatum recorded in vivo. Synapse 33:1–15CrossRefPubMedGoogle Scholar
  36. Ort CA, Kristan WB Jr, Stent GS (1974) Neuronal control of swimming in the medicinal leech. II. Identification and connections of motor neurons. J Comp Physiol 94:121–154CrossRefGoogle Scholar
  37. Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY, Haugland RP (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem 47:1179–1188PubMedGoogle Scholar
  38. Peinado A, Yuste R, Katz LC (1993) Extensive dye coupling between rat neocortical neurons during the period of circuit formation. Neuron 10:103–114CrossRefPubMedGoogle Scholar
  39. Poon M, Friesen WO, Stent GS (1978) Neuronal control of swimming in the medicinal leech. V. Connexions between the oscillatory interneurones and the motor neurons. J Exp Biol 75:45–63PubMedGoogle Scholar
  40. Prinz AA, Thirumalai V, Marder E (2003) The functional consequences of changes in the strength and duration of synaptic inputs to oscillatory neurons. J Neurosci 23:943–954PubMedGoogle Scholar
  41. Sparks DL, Kristan WB Jr, Shaw BK (1997) The role of population coding in the control of movement. In: Stein PSG, Grillner SS, Selverston AI, Stuart DG (eds) Neurons, networks, and motor behavior. MIT Press, Cambridge, MAGoogle Scholar
  42. Stuart AE (1970) Physiological and morphological properties of motoneurons in the central nervous system of the leech. J Physiol Lond 209:627–646PubMedGoogle Scholar
  43. Taylor, AL, Cottrell, GW, Kristan WB Jr (2000) A model of the leech segmental swim central pattern generator. Neurocomputing pp 32–33, 573–584Google Scholar
  44. Taylor AL, Cottrell GW, Kleinfeld D, Kristan WB Jr (2003) Imaging reveals synaptic targets of a swim-terminating neuron in the leech CNS. J Neurosci 23:11402–11410PubMedGoogle Scholar
  45. Vaney DI (1991) Many diverse types of retinal neurons show tracer coupling when injected with biocytin or Neurobiotin. Neurosci Lett 125:187–190CrossRefPubMedGoogle Scholar
  46. Wolpert S, Friesen WO, Laffely AJ (2000) A silicon model of the Hirudo swim oscillator. IEEE Eng Med Biol Mag 19:64–75CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Ruey-Jane Fan
    • 1
  • Antonia Marin-Burgin
    • 2
  • Kathleen A. French
    • 2
  • W. Otto Friesen
    • 1
  1. 1.Department of BiologyUniversity of VirginiaCharlottesvilleUSA
  2. 2.Division of Biological Sciences, Neurobiology SectionUniversity of CaliforniaSan Diego La JollaUSA

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