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Identified neurons implanted into leech ganglia sprout and form synaptic connections
Developmental Brain Research, 8 (1983) 131 - 135
13 I
Elsevier Biomedical Press
Short Communications
Identified neurons Implanted into leech ganglia sprout and form synaptic connections REN-JI ZHANG* and JOHN NICHOLLS
Department of Neurobiologv, Stanford University School of Medicine, Stanford, CA 94305 ( U.S.A .) (Accepted December 21st, 1982)
Key words: culture - synapse formation - sprouting - plasticity - regeneration
Individual neurons of known function, have been dissected out of leech ganglia and implanted into other leech ganglia maintained in culture. Such 'extra' cells survived within the ganglion, sprouted to enter the roots and connectives, maintained their membrane properties and formed synaptic connections. Thus when two extra Retzius cells were implanted into a ganglion, they became electrically coupled to each other and received synaptic inputs from other neurons.
Individual identified neurons in the CNS of the leech have remarkable powers of regeneration t. After a lesion, axons can grow back and reform synapses on appropriate target cells with a high degree of precision, in the animal and in organ culture. Recently it has been shown that selected pairs of isolated neurons maintained in tissue culture can also form specific chemical and electrical connections2,3. In the present experiments we have removed individual nerve cells from the CNS and implanted them surgically into normal leech ganglia. Under such conditions the neurons are cultured not on polylysine, but on a more normal substrate, their membranes in contact with glial and neuronal processes. Questions that arise are: will such cells survive and maintain their membrane properties, as in culture? Will their processes grow randomly or in specific directions? And will they make synaptic connections with each other and with cells in the ganglion? In contrast to experiments in which specific nerve cells are removed from the CNS by killing them with pronase injection 4, these preparations offer the possibility for exploring how extra cells can be fitted into a
ganglion containing its normal complement of nerve cells. The surgical procedures for removing cells were similar to those described for preparing cultures2.3: a loop was tied around a particular P cell (a sensory neuron that responds to pressure) or a Retzius cell (the large 5-HT-containing neuron that causes mucus secretion and plays a part in the swimming of the animal), so that it could be pulled out of the ganglion. Another leech ganglion into which the cells were to be inserted was pinned on Sylgard in a dish containing sterile Leibowitz-15 medium with 2% fetal calf serum, antibiotics (gentamycin 100 /tg/ml) and mycostatin (100 U/ml). A small slit was made in the capsule of the ganglion on the dorsal or the ventral aspect, thereby exposing the neuronal and glial tissue onto which the isolated cells were placed by a transfer pipette. (The incisions in the capsule did not involve damage to neuronal cell bodies or processes but of necessity did disrupt the glial cytoplasm.) In some experiments the cell was inserted from the transfer pipette into the region still covered by capsule, a technically difficult procedure; in others it was placed on
* Present address: Dept of Biology, Peking University, Beijing, China. 0165-3806/83/00~ 0000/$03.00 ;": 1983 Elsevier Science Publishers
132
Retzius cell ,
B
(
1
,,,,J
2
C
P cell
/
i
(10 days)
\
/
,:, I" :
i~ . I
" ; :i \
h.
"
rools
~ ~"
•
conneclives
~.¥
Fig. I. Sprouting by individual identified neurons implanted into the dorsal surface of leech ganglia, maintained in culture and injected with HRP. A: two Retzius cells in a ganglion for 9 days. Abundant processes are apparent. B:camera lucida drawing of a Retzius cell implanted for 12 days. C: P sensory neuron implanted for 10 days. Note the processes entering roots and connectives. The diameter of an implanted Retzius cell is approximately 60 ttm.
the exposed surface of the ganglia. Cells were implanted into freshly dissected ganglion. After a few days at room temperature (20 °C), intracellular recordings were made with microelectrodes and the cells filled with horseradish peroxidase (HRP) or Lucifer yellow ~. The properties of the implanted cells were in many respects similar to those grown on polylysine-coated tissue culture dishes: they retained their normal appearance, sprouted and formed connections. Examples of extra Retzius and P cells injected with HRP are shown in Fig. 1. After 9 days Retzius cells placed onto the dorsal side of a ganglion show large numbers of sprouts forming a dense neuropil (Fig. IA, B). Occasional swellings or varicosities presumably corresponding to release sites for 5-HT are apparent 2.
Certain processes clearly run through the neuropil of the ganglion to enter the root. P cells (Fig. I C) also showed extensive arborizations with processes extending over long distances with obvious processes entering roots and connectives. This pattern of axons finding their way through the ganglion to enter roots and connectives was observed consistently in preparation after preparation. The implanted cells continued to exhibit membrane resting and action potentials as well as passive electrical properties resembling those seen in their normal counterparts in situ or in culture. Thus, P cells gave large overshooting action potentials with a pronounced undershoot and displayed rectification with subthreshold pulses. In contrast. Retzius cells gave longer las-
133
A
B
Implanted P cell
(7 days)
[4 days)
2
SL_
-20 -40
Lf ,
-60
k,.__,
20
C
Implanted Retzius cell
40 msec
I
I
I
20
40
60
,:•...:..,
Coupling between Lmplanted Retzius cells (4 days)
msec
%°"~~ ,° I
mV
,,,,f
mY 25
I
50
I
I
25
I
50 msec
Fig. 2. Electrical properties and connections of implanted cells. A: action potential from a P cell implanted 4 days beforehand. B: action potential from a Retzius cell (7 days). Both of these potentials appear identical to those recorded from their normal counterparts in situ. C: electrical coupling between two Retzius cells implanted into a ganglion 4 days beforehand. These cells were coupled by non-rectifying junctions. They were not coupled to the normal Retzius cells in situ on the other side of the ganglion (shown by dotted outlines).
134
Synaptic potentials in implanted Retzius cells
A
epsp's
7 days
B
ipsp's
5 days
-46mY
mv[ -76 mV
I
I
10
I
20 msec
I
I
I
50
100
msec
Fig. 3. Synaptic potentials evoked by stimulating roots or connectives with extracellular suction electrodes. A: an implanted Retzius cell (7 days) in which stimuli of different strengths evoked depolarizing EPSPs. B: implanted Retzius cell (5 days) in which stimuli evoked hyperpolarizing IPSPs that were reversed by hyperpolarizing the cell with current.
ting but smaller impulses and did not show rectification (Fig. 2). Fig. 2 also shows that when two individual Retzius cells were inserted onto the dorsal surface of a ganglion, they became electrically coupled. Such cells in situ are similarly coupled by non-rectifying junctions. Tests made to ascertain whether the extra Retzius cells became coupled to the original Retzius cells or to other identified neurons in the ganglion have so far failed to reveal such connections. At the same time the added Retzius cells did receive synaptic inputs.
This is shown in Fig. 3A in which stimulation of a root by a suction electrode resulted in a depolarizing synaptic potential. This could not represent an invasion failure since its amplitude varied in a graded manner with the strength of the stimulus. In another preparation (Fig. 3B) an IPSP evoked by root stimulation was reversed by hyperpolarizing the implanted Retzius cell with current injected through the microelectrode. Together these results indicate that in the environment provided by a ganglion, additional cells
135 tribute to functional reflex connections like the ' e x t r a ' or s u p r a n u m e r a r y cells that h a v e on occasion been f o u n d in ganglia o f a b n o r m a l leeches 5.
can survive a n d grow new processes as in culture. M o r e o v e r , the g r o w t h was not r a n d o m : fibers s o m e h o w f o u n d their way to a n d entered the roots a n d connectives and i m p l a n t e d Retzius cells b e c a m e c o u p l e d to one another, as well as acting as p o s t s y n a p t i c targets for n e u r o n s within the ganglion. It will be o f interest to e x a m i n e in detail the fine structure and properties o f s y n a p ses f o r m e d u n d e r these conditions a n d to assess further w h e t h e r such additional cells can con-
W e t h a n k Drs. L. H e n d e r s o n and M. Pellegrino for m a n y helpful discussions a n d Ms. Lyn L a z a r for unfailing technical assistance. This work was s u p p o r t e d by U S P H S G r a n t 11544-09 and by a grant f r o m the M a r c h o f D i m e s for which we are most grateful.
I Muller, K. J. and Nicholls, J. G., Regeneration and plasticity. In K. J. Muller, J. G. Nicholls and G. Stent (Eds.), Neurobiology of the Leech, Cold Spring Harbor, New York, 1981. 2 Fuchs, P. A., Nicholls, J. G. and Ready, D. F., Membrane properties and selective connexions of identified leech neurones in culture, J. Physiol. (Lond), 316 (1981) 203223. 3 Fuchs, P. A., Henderson, L. P. and Nicholls, J. G., Chemical transmission between individual Retzius and sensory
neurones of the leech in culture, J. Physiol. (Lond.), 32i (1982) 195-210. Bowling, D., NichoUs, J. G. and Parnas, 1., Destruction of a single cell in the C.N.S. of the leech as a means of analyzing its connexions and functional role. J. Physiol. fLorid.), 282 (1978) 169- 180. Kuffler, D. P. and Muller, K. J., The properties and connections of supernumerary sensory and motor nerve cells in the central nervous system of an abnormal leech, J. NeurobioL, 5 (1974) 331-348.
13 I
Elsevier Biomedical Press
Short Communications
Identified neurons Implanted into leech ganglia sprout and form synaptic connections REN-JI ZHANG* and JOHN NICHOLLS
Department of Neurobiologv, Stanford University School of Medicine, Stanford, CA 94305 ( U.S.A .) (Accepted December 21st, 1982)
Key words: culture - synapse formation - sprouting - plasticity - regeneration
Individual neurons of known function, have been dissected out of leech ganglia and implanted into other leech ganglia maintained in culture. Such 'extra' cells survived within the ganglion, sprouted to enter the roots and connectives, maintained their membrane properties and formed synaptic connections. Thus when two extra Retzius cells were implanted into a ganglion, they became electrically coupled to each other and received synaptic inputs from other neurons.
Individual identified neurons in the CNS of the leech have remarkable powers of regeneration t. After a lesion, axons can grow back and reform synapses on appropriate target cells with a high degree of precision, in the animal and in organ culture. Recently it has been shown that selected pairs of isolated neurons maintained in tissue culture can also form specific chemical and electrical connections2,3. In the present experiments we have removed individual nerve cells from the CNS and implanted them surgically into normal leech ganglia. Under such conditions the neurons are cultured not on polylysine, but on a more normal substrate, their membranes in contact with glial and neuronal processes. Questions that arise are: will such cells survive and maintain their membrane properties, as in culture? Will their processes grow randomly or in specific directions? And will they make synaptic connections with each other and with cells in the ganglion? In contrast to experiments in which specific nerve cells are removed from the CNS by killing them with pronase injection 4, these preparations offer the possibility for exploring how extra cells can be fitted into a
ganglion containing its normal complement of nerve cells. The surgical procedures for removing cells were similar to those described for preparing cultures2.3: a loop was tied around a particular P cell (a sensory neuron that responds to pressure) or a Retzius cell (the large 5-HT-containing neuron that causes mucus secretion and plays a part in the swimming of the animal), so that it could be pulled out of the ganglion. Another leech ganglion into which the cells were to be inserted was pinned on Sylgard in a dish containing sterile Leibowitz-15 medium with 2% fetal calf serum, antibiotics (gentamycin 100 /tg/ml) and mycostatin (100 U/ml). A small slit was made in the capsule of the ganglion on the dorsal or the ventral aspect, thereby exposing the neuronal and glial tissue onto which the isolated cells were placed by a transfer pipette. (The incisions in the capsule did not involve damage to neuronal cell bodies or processes but of necessity did disrupt the glial cytoplasm.) In some experiments the cell was inserted from the transfer pipette into the region still covered by capsule, a technically difficult procedure; in others it was placed on
* Present address: Dept of Biology, Peking University, Beijing, China. 0165-3806/83/00~ 0000/$03.00 ;": 1983 Elsevier Science Publishers
132
Retzius cell ,
B
(
1
,,,,J
2
C
P cell
/
i
(10 days)
\
/
,:, I" :
i~ . I
" ; :i \
h.
"
rools
~ ~"
•
conneclives
~.¥
Fig. I. Sprouting by individual identified neurons implanted into the dorsal surface of leech ganglia, maintained in culture and injected with HRP. A: two Retzius cells in a ganglion for 9 days. Abundant processes are apparent. B:camera lucida drawing of a Retzius cell implanted for 12 days. C: P sensory neuron implanted for 10 days. Note the processes entering roots and connectives. The diameter of an implanted Retzius cell is approximately 60 ttm.
the exposed surface of the ganglia. Cells were implanted into freshly dissected ganglion. After a few days at room temperature (20 °C), intracellular recordings were made with microelectrodes and the cells filled with horseradish peroxidase (HRP) or Lucifer yellow ~. The properties of the implanted cells were in many respects similar to those grown on polylysine-coated tissue culture dishes: they retained their normal appearance, sprouted and formed connections. Examples of extra Retzius and P cells injected with HRP are shown in Fig. 1. After 9 days Retzius cells placed onto the dorsal side of a ganglion show large numbers of sprouts forming a dense neuropil (Fig. IA, B). Occasional swellings or varicosities presumably corresponding to release sites for 5-HT are apparent 2.
Certain processes clearly run through the neuropil of the ganglion to enter the root. P cells (Fig. I C) also showed extensive arborizations with processes extending over long distances with obvious processes entering roots and connectives. This pattern of axons finding their way through the ganglion to enter roots and connectives was observed consistently in preparation after preparation. The implanted cells continued to exhibit membrane resting and action potentials as well as passive electrical properties resembling those seen in their normal counterparts in situ or in culture. Thus, P cells gave large overshooting action potentials with a pronounced undershoot and displayed rectification with subthreshold pulses. In contrast. Retzius cells gave longer las-
133
A
B
Implanted P cell
(7 days)
[4 days)
2
SL_
-20 -40
Lf ,
-60
k,.__,
20
C
Implanted Retzius cell
40 msec
I
I
I
20
40
60
,:•...:..,
Coupling between Lmplanted Retzius cells (4 days)
msec
%°"~~ ,° I
mV
,,,,f
mY 25
I
50
I
I
25
I
50 msec
Fig. 2. Electrical properties and connections of implanted cells. A: action potential from a P cell implanted 4 days beforehand. B: action potential from a Retzius cell (7 days). Both of these potentials appear identical to those recorded from their normal counterparts in situ. C: electrical coupling between two Retzius cells implanted into a ganglion 4 days beforehand. These cells were coupled by non-rectifying junctions. They were not coupled to the normal Retzius cells in situ on the other side of the ganglion (shown by dotted outlines).
134
Synaptic potentials in implanted Retzius cells
A
epsp's
7 days
B
ipsp's
5 days
-46mY
mv[ -76 mV
I
I
10
I
20 msec
I
I
I
50
100
msec
Fig. 3. Synaptic potentials evoked by stimulating roots or connectives with extracellular suction electrodes. A: an implanted Retzius cell (7 days) in which stimuli of different strengths evoked depolarizing EPSPs. B: implanted Retzius cell (5 days) in which stimuli evoked hyperpolarizing IPSPs that were reversed by hyperpolarizing the cell with current.
ting but smaller impulses and did not show rectification (Fig. 2). Fig. 2 also shows that when two individual Retzius cells were inserted onto the dorsal surface of a ganglion, they became electrically coupled. Such cells in situ are similarly coupled by non-rectifying junctions. Tests made to ascertain whether the extra Retzius cells became coupled to the original Retzius cells or to other identified neurons in the ganglion have so far failed to reveal such connections. At the same time the added Retzius cells did receive synaptic inputs.
This is shown in Fig. 3A in which stimulation of a root by a suction electrode resulted in a depolarizing synaptic potential. This could not represent an invasion failure since its amplitude varied in a graded manner with the strength of the stimulus. In another preparation (Fig. 3B) an IPSP evoked by root stimulation was reversed by hyperpolarizing the implanted Retzius cell with current injected through the microelectrode. Together these results indicate that in the environment provided by a ganglion, additional cells
135 tribute to functional reflex connections like the ' e x t r a ' or s u p r a n u m e r a r y cells that h a v e on occasion been f o u n d in ganglia o f a b n o r m a l leeches 5.
can survive a n d grow new processes as in culture. M o r e o v e r , the g r o w t h was not r a n d o m : fibers s o m e h o w f o u n d their way to a n d entered the roots a n d connectives and i m p l a n t e d Retzius cells b e c a m e c o u p l e d to one another, as well as acting as p o s t s y n a p t i c targets for n e u r o n s within the ganglion. It will be o f interest to e x a m i n e in detail the fine structure and properties o f s y n a p ses f o r m e d u n d e r these conditions a n d to assess further w h e t h e r such additional cells can con-
W e t h a n k Drs. L. H e n d e r s o n and M. Pellegrino for m a n y helpful discussions a n d Ms. Lyn L a z a r for unfailing technical assistance. This work was s u p p o r t e d by U S P H S G r a n t 11544-09 and by a grant f r o m the M a r c h o f D i m e s for which we are most grateful.
I Muller, K. J. and Nicholls, J. G., Regeneration and plasticity. In K. J. Muller, J. G. Nicholls and G. Stent (Eds.), Neurobiology of the Leech, Cold Spring Harbor, New York, 1981. 2 Fuchs, P. A., Nicholls, J. G. and Ready, D. F., Membrane properties and selective connexions of identified leech neurones in culture, J. Physiol. (Lond), 316 (1981) 203223. 3 Fuchs, P. A., Henderson, L. P. and Nicholls, J. G., Chemical transmission between individual Retzius and sensory
neurones of the leech in culture, J. Physiol. (Lond.), 32i (1982) 195-210. Bowling, D., NichoUs, J. G. and Parnas, 1., Destruction of a single cell in the C.N.S. of the leech as a means of analyzing its connexions and functional role. J. Physiol. fLorid.), 282 (1978) 169- 180. Kuffler, D. P. and Muller, K. J., The properties and connections of supernumerary sensory and motor nerve cells in the central nervous system of an abnormal leech, J. NeurobioL, 5 (1974) 331-348.