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Proc. R. Soc.Lond. B. 194, 295-311 (1976)

Printed in Great Britain

The properties and connections of nerve cells in

leech ganglia maintained in culture
B y S. MlYAZAKlf and J. G. N i c h o l l s
Department ofNeurobiology,
Stanford University School of
Stanford, California 94305

( < Communi cat edby S. W.Kuffl

Received 27 January 1976)

[P la te s 1 a n d 2]

Segmental ganglia of the central nervous system of the leech were

maintained in culture medium outside the animal for several weeks at
16 °C, and electrical recordings made from identified sensory and motor
nerve cells. Ganglia were isolated and cultured singly, in chains and
connected to the skin and muscles they normally innervate. Such
preparations are suitable for a study of relatively long-term changes
that occur as a result of denervation. The changes resemble those seen
in isolated ganglia th at had been kept in situ in the animal.
(1) Resting and action potentials in sensory and motor neurones of
isolated ganglia appear normal for up to ten weeks. The same cells can
be tested at intervals of a few days. (2) Sensory cells, classified as
touch (T), pressure (P) or nociceptive (N) according to their morphology
and electrical properties, continue to respond selectively to stimuli of
the appropriate modality applied to their receptive fields in the skin;
action potentials in motor cells cause contractions in the appropriate
muscles. (3 ) Chemical synaptic transmission between sensory and
motor nerve cells changes markedly over the first three weeks; excitatory
and inhibitory synaptic potentials can double in size and duration
over a period of about two weeks in culture. (4 ) The balance between
excitatory and inhibitory synaptic influences changes sequentially after
isolation in culture. For example, P or N cells innervating a cell which
erects skin annuli normally cause small depolarizing excitatory potentials.
After 5 days in culture they initiate large inhibitory synaptic potentials,
while by 15 days the balance between excitation and inhibition changes
again, so that the predominant synaptic action is an abnormally large
prolonged excitatory potential. (5 ) After more than three weeks synaptic
potentials disappear and ganglia lose transparency. (6) The morpho­
logical appearance of T, P and N sensory cells has been compared in
cultured and normal ganglia after injection of horseradish peroxidase.
In cultured ganglia the major branching pattern appears normal but
presumed sites of transmitter release become more conspicuous.
t P re sen t address: D e p a rtm e n t of Physiology, School of M edicine, U .C .L .A ., Los A ngeles,
C alifornia 90024.
[ 295 ]
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296 S. Miyazaki and J. G. Nicholls

I ntroduction
There are certain obvious advantages to be gained by m aintaining vitro, for
prolonged periods, an organized group of identified nerve cells w ith known
functions and properties. In culture, the ionic composition of th e m edium can
be altered, drugs applied in known concentrations and neurones stim ulated for
prolonged periods. The present series of experim ents has been made on segm ental
ganglia of the leech, where the basis of certain simple reflexes is now understood
in term s of the individual sensory cells and th eir connections to m otor neurones
(Nicholls & Purves 1972; Muller & Nicholls 1974). Furtherm ore, it is known th a t
a series of characteristic and striking changes develop slowly over days or weeks
in the synaptic interactions between sensory and m otor cells when ganglia are
isolated from the rest of the nervous system by surgical lesions. Such ganglia
are in effect m aintained by ‘organ c u ltu re’ w ithin th eir norm al environm ent in
the animal (Jansen & Nicholls 1972; Jansen, Muller & Nicholls 1974).
Our first aim has been to keep a variety of preparations alive outside the
anim al and to compare the properties of neurones and synapses w ith those seen
in normal uncultured preparations. Accordingly, we have kept in culture m edium
single ganglia, pairs of interconnected ganglia and th e entire nerve cord. In
addition, we have m aintained ganglia together w ith the skin and muscles th ey
normally supply to see whether individual sensory cells would continue to respond
to mechanical stimuli and w hether m otor cells would still produce muscular
contractions. A second, m ajor aim was to te st w hether synaptic transm ission
would change in cultured ganglia as in ganglia isolated w ithin the animal. I t will
be shown th a t cultured preparations survive for several weeks, undergo pro­
gressive changes and provide a basis for experim ental m anipulations which would
be quite impractical in the animal.

Methods
Isolation of ganglia
A leech was dipped in 7 0 % m ethanol for a few seconds to clean its skin and
then pinned to a sterile dish in an Edgegard Baker Lam inar Flow Hood. During
the dissection, normal precautions were taken to prevent contam ination. As each
ganglion was dissected out, it was placed in a few millilitres of culture medium
(see below) until about 12 ganglia had been removed. The ganglia were then
pinned out by sterile pins through their connectives and roots, into petri dishes
containing a shallow layer of transparent resin (Dow Corning Sylgard resin).
Three to five ganglia were pinned in each dish with enough medium to cover
them. Chains of two or more ganglia were prepared in the same way. When a
piece of skin was removed together with the ganglion innervating it, the procedure
was similar except th a t the leech was kept in w ater containing 1000 units/m l
penicillin and streptomycin for one day before the dissection. To record from
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Proc. R. Soc. Lond. B, volume 194 Miyazaki 6c Nicholls, plate 1

F igure 1. P h o to g ra p h s of th e v e n tra l (above) an d dorsal (below) aspects of a ganglion m a in ­

ta in e d in cu ltu re for^22 days. L e tte rs in d icate sensory cells resp o n d in g to to u c h (T), pressu re
(P) an d noxious stim u la tio n (N), th e an n u lu s erecto r (AE) and th e large lo n g itu d in al
(L) m otoneurones, a n d th e R e tziu s cell (R). A p a rt from som e fuzziness of th e sh e ath
aro u n d th e ganglion a n d th e connectives (conn.), th e ap p e aran c e is n o rm al a n d in d iv id u al
cells can be clearly distinguished.
F igure 2. G anglion to g e th er w ith skin a n d m uscle of b o d y w all m a in ta in ed in cu ltu re for
31 days. T he p igm ent m arkings a n d th e circu m feren tial an n u li can be d istin g u ish ed
a n d provide convenient la n d m ark s for m apping recep tiv e fields.

(Facing p. 296)
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Cultured leech ganglia 297

cells on repeated occasions a ganglion was removed from culture, and the cells
impaled as usual in normal Ringer fluid (see below). After washing in medium
the ganglion was pinned out on a fresh dish. It was possible to avoid contami­
nation by using conventional precautions.

Culture media
We routinely used Leibovitz 15 medium (L 15) with 10% foetal calf serum.
Each 100 ml contained in addition: 2 ml 3 0 % glucose; 100 units/ml penicillin
and streptomycin and 5000 units of mycostatin. Other media we tried were
Eagle’s and minimum essential medium. They appeared to be less effective
than L 15 but extensive tests were not made. The medium was changed every
three days. Preparations were kept at 16 °C.

Experimental observations
Electrical recordings were made from leech ganglia in Ringer fluid of the
following composition: NaC1 1 1 5 mM; K C l 4 mM; CaCl2 7 .5 mM; Tris maleate
buffered to pH 7.4 with NaOH, 10 mM; glucose, 11.7 mM. The raised concentration
of CaCl2 (7.5 mM instead of 1.8 mM) makes it easier to record synaptic potentials
(Jansen & Nicholls 1972). The intracellular recording techniques have been
described elsewhere (Nicholls & Baylor 1968; Muller & Nicholls 1974). For the
injection of horseradish peroxidase (Type VI Sigma), a microelectrode was
inserted into a cell and the enzyme extruded by pressure, according to methods
described by Muller & McMahan (1976) and Yau (unpublished). In brief, a cell
Was injected under visual inspection for a few seconds until the volume had
obviously increased. The ganglion was then kept for a variable period of 1-10 h.
After fixation, the ganglion was incubated with 0 .4 % benzidine and 0 . 15 %
hydrogen peroxide. The reaction product was stabilized with 15 % Na2Fe(CN)5NO.
The processes of cells were examined in whole mounts.
The segmental ganglia which make up the ventral nerve cord each contain
about 350 nerve cells and 8 glial cells; they are all virtually identical and are
small enough to allow rapid diffusion (Nicholls & Kuffler 1964).

R esults
Appearance of cultured preparations
For the first three weeks or so after removal from the animal, the ganglia
appeared almost indistinguishable from ones th at had been freshly dissected.
Figure 1, plate 1, shows the dorsal and ventral surfaces of a ganglion th at had
been maintained in medium for 22 days at 16 °C. The outlines of nerve cells
and the ‘packet’ margins that mark the boundaries of the large glial cells
(Coggeshall & Fawcett 1964) were sharply delineated and clearly visible. Situated
in their characteristic positions are the sensory cells responding to touch (T),
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298 S. Miyazaki and J. G. Nicholls

pressure (P) and noxious (N) mechanical stim ulation, th e large R etzius cells (R)
and the m otor cells th a t erect the annuli of the skin (AE) and innervate longi­
tudinal muscles (L).
A minor difference from norm al was th a t th e connective tissue sheath su r­
rounding cultured ganglia appeared fuzzy. More drastic changes in the appearance
of cultured ganglia occurred from the th ird week onwards. In particular, such
ganglia became less transparent, which made it more difficult to identify and
impale nerve cells (see also below). The changes developed between days 21 and
28 and thereafter progressed only gradually, so th a t ganglia looked much the
same after 4 or 10 weeks.
Figure 2 shows a ganglion kept in culture together w ith a portion of skin
th a t it innervated in the animal. For the first 20-25 days th e ganglion appeared
norm al b u t then became opaque. The skin itself appeared unchanged after
10 weeks (the longest tim e we waited) except for a slight flattening of the ridges
and an increase in its fragility.

mV
54 d a y s

40 ms
F ig u r e 3. Im p u ls e s in n o c io c e p tiv e (N ), to u c h (T) a n d p re s s u re (P ) se n s o ry cells e v o k e d b y
c u r r e n t p u lse s in a g a n g lio n m a in ta in e d in c u ltu r e fo r 54 d a y s . C h a ra c te r is tic a lly , a s
in a n o rm a l g a n g lio n , th e im p u lse a n d u n d e r s h o o t a r e la rg e s t in t h e N cell a n d s m a lle s t
in th e T cell.

Electrical properties of sensory and motor nerve cells

A feature of normal leech ganglia is th a t cells w ith different functions give
characteristic and distinctive impulses by which they can be reliably identified.
The cells in cultured ganglia behaved in a similar way. Figure 3 shows recordings
from sensory cells classified as nociceptive (N), touch (T) or pressure (P), in
ganglia m aintained for several weeks. The action potentials of these sensory cells
resembled those seen in normal ganglia, freshly removed from a leech. Moreover,
it was possible to record from the same cell repeatedly a t intervals a few days
apart. There was no evidence th a t impalement by a microelectrode damaged the
cell.
The electrical properties of sensory cells remained distinctive; thus delayed
rectification was marked in T and P cells. As in normal ganglia, a prolonged
after-hyperpolarization appeared in sensory cells following trains of impulses;
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Cultured leech ganglia 299

its magnitude and duration depended on the frequency and duration of the train
and it was largest in N cells (see, for example, figure 4 ; also Jansen & Nicholls
J973)-
In view of the progressive changes we observed in synaptic potentials it was
important to estimate the time constants and input resistances of neurones. Such
estimates were made by current pulses injected from a single electrode. The
values obtained in cultured cells were close to the normal values, ± about 20%.

F igure 4. S ensory im pulses evoked b y m echanical stim u li ap p lied to th e sk in in T a n d P

cells of ganglia m a in ta in e d for 21 or 54 d ays. I n (a) th e T cell w as a c tiv a te d b y lig h t
to u c h , an in d e n ta tio n o f th e skin o f ap p ro x . 20 pm b y a piezo-electric cry sta l. T he
skin w as lig h tly stro k e d in (b) a n d pressed in (c). As in n o rm al g an g lia th e P cell a d a p ts
slow ly w hile th e T cell a d a p ts rap id ly .

Such changes could not account for the prolonged decay of the synaptic potentials
in cultured cells which was far too long (tens or hundreds of milliseconds) to be
caused by changes in time constant which was approximately 5 ms. (See below,
for example, and figure 5 which shows electrotonic potentials of cells in cultured
ganglia.)
When a ganglion was cultured with skin, the sensory cells continued to respond
for at least 10 weeks to the normal adequate stimulus with characteristic and
appropriate discharge patterns. Figure 4 shows impulses from T and P cells
in response to light touch and heavier pressure applied to the skin. The touch
cell discharge was initiated by a 20 jim indentation of the skin applied by a piezo­
electric crystal or by gentle stroking. Adaptation was rapid and the cell was
able to follow two touches at an interval of 10 ms. In contrast, P cells responded
to maintained pressure with a slowly adapting discharge while N cells required
more drastic pinching or squeezing to be activated. In all these respects the
sensory cells in cultured and normal preparations behaved alike.
The resting potentials and the shapes of impulses recorded in motor cells were
also within the normal range. Thus, the annulus erector (AE) and large longi­
tudinal (L) motor neurones gave small impulses th at did not invade the cell
body but which were nevertheless recognizable and different from those of other
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300 S. Miyazaki and J. G. Nicholls
cells in their vicinity. The appropriate movem ents of th e body wall were still
elicited by stim ulation of these and other m otor cells.

Electrical synapses
In leech ganglia, m any types of cells have been shown to be electrically coupled
to one another. For example, the tw o R etzius cells are coupled by non-rectifying
junctions and so are the two L m otor neurones (Hagiwara & M orita 1962; S tu a rt
1970); in contrast, the T and P sensory cells are coupled to th e L cell by rectifying

19 days

40 ms
F ig u r e 5. C h a ra c te ris tic e le c tric a l c o u p lin g o f tw o R e tz iu s cells (R ) in a g a n g lio n m a in ta in e d
in c u ltu r e fo r 19 d a y s . T h e u p p e r tr a c e sh o w s t h e v o lta g e in th e R e tz iu s cell in to w h ic h
c u r r e n t w as in je c te d ; th e lo w e r tr a c e is fro m th e o th e r R e tz iu s cell. D e p o la riz in g p u lse s
(rig h t) g iv e rise to im p u lse s ( c u r r e n t in je c te d in to lo w e r cell).

junctions (Nicholls & Purves 1970). In cultured ganglia similar electrical in te r­

actions were seen between the appropriate cells. Current spread in both directions
between the Retzius cells and between L cells, b u t only depolarizing current
spread from T or P sensory cells to the L cell. An example of current spread
between Retzius cells in a ganglion th a t had been m aintained in culture for
19 days is shown in figure 5 .

Changes at chemical synapses

Unlike electrical synapses which underw ent no discernible change in cultured
ganglia during the first four weeks a t least, certain chemical synaptic interactions
became drastically modified. A t different times after removal of ganglia from
the animal, excitatory potentials, inhibitory potentials or both became increased
in magnitude and greatly prolonged. We studied, in particular, changes in the
interactions between N, P and T sensory cells and the L and AE m otor neurones
because the properties of these synapses are well known in norm al ganglia, as
well as in ganglia maintained or ‘cultured ’ in the animal after section of the
connectives and roots (Nicholls & Purves 1970; Muller & Nicholls 1974; Jansen
et al. 1974).
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Cultured leech ganglia 301

Synaptic potentials in the annulus erector motor neurone

For the first 5-7 days after placing ganglia in culture the synaptic potentials
recorded in the AE cell were normal. P and N cell impulses gave rise to small
(less than 1 mV) monosynaptic excitatory potentials (figure 6 see also Muller
& Nicholls 1974). Thereafter a sequence of changes occurred. First, from about
days 5 to 15 the sign of the response changed from excitation to inhibition.
Figure 66 shows a typical result obtained from a ganglion cultured for 10 days.

F igure 6. P rogressive changes in th e sy n a p tic in te ractio n s o f sensory (P) a n d m o to r (AE)

cells in c u ltu re d ganglia. I n n o rm al ganglia (a), a n im pulse in a P cell gives rise to
a sm all e x c ita to ry p o te n tia l in th e A E cell (m arked b y arrow ), (b) I n a ganglion m a in ta in e d
in cu ltu re for 10 d ay s th e e x c ita to ry p o te n tia l is follow ed b y a larg e in h ib ito ry p o te n tia l,
(c) B y 18 d ay s th e response consists of a large e x c ita to ry p o te n tia l (note th e change
in v o ltage scale). N cells evoke sim ilarly chang ed responses in A E cells in cu ltu red
ganglia.

Following the P cell impulse, the initial excitatory potential could still on occasion
be observed but the predominant postsynaptic potential was hyperpolarization
of relatively long duration often consisting of multiple potentials, as though
interneurones were firing. We emphasize th at such inhibitory potentials masking
the excitation are not usually seen in normal ganglia but were consistently evoked
by each presynaptic impulse in a P or N cell at the appropriate stage in culture.
By the end of the second week a further change had occurred. The inhibitory
potentials became smaller as the excitatory potential increased. Figure 6c
shows a recording made from a ganglion that had been in culture for 18 days. The
excitatory synaptic potential was by now about 10 mV at its peak and persisted
for more than 200 ms.
Several lines of evidence indicated th at the unusual inhibitory potentials
observed in 5-15 day cultured ganglia arose by way of a polysynaptic pathway
rather than from a direct effect of P cell synapses upon the AE cell: (1) The
latency was variable and always longer than 2 ms, the delay for a monosynaptic
connection in the leech c.n.s. (Nicholls & Purves 1970). (2) The inhibitory
potentials were reversibly abolished by bathing the cultured ganglion in a solution
containing high concentrations of Ca (20 m M ) and Mg (15 m M ). The rationale for
this experiment is as follows: raised Ca increases the release of transm itter by
presynaptic terminals and Mg decreases it. But, both ions raise the threshold
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302 S. Miyazaki and J. G. Nicholls

for the initiation of impulses. Jansen et al. (1
synaptic potential is still observed in solution containing appropriate Ca and
Mg concentrations, but polysynaptic potentials are not, because the interneurones
do not fire. Figure 7 shows this effect on the inhibitory potentials in the AE cell
of ganglia th a t had been in culture for 13 days. In high Ca/high Mg fluid the
excitatory potential was unm asked and seen to be larger in am plitude and of
considerably longer duration th a n norm al (cf. figure 6a). This enlarged excitatory
potential conformed to criteria of a m onosynaptic potential: (1) the latency was
less th a n 2 ms, (2) the potential persisted in high Ca/high Mg solution, (3 ) in
high Mg solution, the potential became decreased in size, as expected, in a
continuously graded manner.

(a) (b) (c)

20 mM Ca
. 13 days 15 mM Mg
1

p % ■
AE A

F ig u r e 7. E ffe c t o f h ig h C a /h ig h M g flu id o n m o d ifie d s y n a p tic p o te n tia ls in A E m o to r cell

in a g a n g lio n c u ltu r e d fo r 13 d a y s , (a) I n n o rm a l R in g e r flu id a n im p u ls e in t h e P cell
ev o k e s b o th e x c ita to r y a n d in h ib ito r y p o te n tia ls in th e A E cell (as in fig u re 6). In
h ig h C a /h ig h M g flu id th e in h ib ito r y p o te n tia l is a b o lis h e d b u t th e e x c ita to r y p o te n tia l
re m a in s a n d is seen to b e la rg e r a n d lo n g e r t h a n n o rm a l (cf. fig u re 6). (c) O n r e tu r n in g
to n o rm a l R in g e r flu id th e in h ib ito r y p o te n tia l is re s to r e d . T h e se r e s u lts s u g g e st t h a t
th e in h ib ito r y p o te n tia l a rise s b y w a y o f a p o ly s y n a p tic p a th w a y w h ic h is b lo c k e d in
h ig h C a /h ig h M g flu id .

A t later stages in culture when the inhibitory potential had disappeared

(figure 6c), some reduction in the excitatory potential occurred in high Ca/high
Mg solution indicating the possible contribution of a polysynaptic component.
N cells, like P cells, evoked inhibitory potentials in most AE cells a t early
stages and later evoked enlarged excitatory potentials. In AE cells of cultured
ganglia as in other cells, normal sized ‘spontaneous’ synaptic potentials were
observed and the input impedances during the first 3 weeks were w ithin 2 0 %
of the values found in normal cells.

Connections of sensory cells to L motor neurone in cultured ganglia

The L motor cell innervates the longitudinal body muscle and receives synaptic
input from T, P and N cells. As in AE cells, for the first week or so, the synaptic
potentials in L cells appeared normal. An impulse in the N cell produced a
chemical synaptic potential of about 5-10 mV in am plitude th a t appeared after
a latency of less than 2 ms and was abolished by 20 mM Mg fluid. The T cell
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Cultured leech ganglia 303

gave rise to a short-latency coupling potential, with a peak amplitude of about
1 mV or less, th at persisted in fluid containing 20 mM Mg and was mediated
electrically. And the P cell gave rise to a mixed electrical-chemical response,
the chemical synaptic potential being about 3 mV in amplitude (see also
Nicholls & Purves 1970, 1972).
By about ten days these synaptic interactions had become permanently and
obviously changed. One of the most conspicuous changes was in the chemical
synaptic potential in the L cell following an impulse in the N cell. This showed

40
mV

10

40 ms
F igure 8. C hanged sy n a p tic in te ra c tio n s of N" sensory a n d L m o to r cells in a ganglion
cu ltu red for 17 days. T he increase in b o th a m p litu d e a n d d u ra tio n are a p p a re n t. T hese
p o te n tia ls are m o n o sy n ap tic according to a v a rie ty of c riteria (see te x t).

an increase in amplitude and time course from the normal peak value of 5-10 mV
and half time of about 20 ms. In 15-21 day cultured ganglia, the synaptic potential
was usually about 15 mV and could reach 25-30 mV, declining with a half time
of about 50 ms as in figure 8. Several lines of evidence indicated th at an exag­
geration of the normal monosynaptic excitatory potential had occurred: (1) the
potentials followed presynaptic impulses in a 1 to 1 manner at a constant latency.
Their general form was similar to the normal monosynaptic potential but larger
and longer (figure 8). (2) In fluid containing raised concentrations of Mg the
synaptic potential decreased in a graded continuous manner. (3 ) The synaptic
potentials were still present, enlarged and of long duration in solution containing
both high Mg and high Ca. From these experiments one can therefore conclude
th at transmission in the monosynaptic pathway from the N cell to the L cell
was enhanced in culture. In addition, however, irregular polysynaptically mediated
pathways also appeared to contribute to the late falling phase of the synaptic
potential in the L cell. These were abolished reversibly by bathing in solutions
containing high Ca and Mg (see also figure 7 ).
Increases in amplitude and duration also occurred in the synaptic potentials
evoked by the P cell in the L cell. Between 10 and 21 days, the chemical excitatory
potential in the L cell became more than doubled in amplitude and duration
following a single impulse in a P cell. In addition, however, inhibitory potentials
became obvious; these were not driven at constant latency and appeared to be
polysynaptically mediated.
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304 S. Miyazaki and J. G. Nicholls

Figure 9 shows a comparison of the effects of an impulse in a T sensory cell
on the L cell in norm al and cultured ganglia. In th e cultured ganglion a single
impulse in the T cell gave rise to a large excitatory synaptic potential in addition
to the small electrical component, which it overshadowed. This excitatory
potential was increased by hyperpolarization of the L cell and disappeared
reversibly in high Mg, indicating th a t it was chemically m ediated. The pathw ay
is probably polysynaptic because the latency was variable and only the electrical
coupling potential rem ained when the ganglion was bathed in solutions containing
high concentrations of Ca and Mg. A fter returning to norm al Ringer fluid th e
large chemical excitatory potential reappeared.
(a) 14 days
40 m V

4 mV

40 ms
F ig u r e 9. C h a n g e d s y n a p tic in te r a c tio n s o f T se n so ry a n d L m o to r cells. I n n o r m a l g a n g lia
(a) a n im p u lse in th e T cell e v o k e s a sm a ll e le c tro to n ic a lly m e d ia te d c o u p lin g p o te n tia l
in th e L cell. I n th e g a n g lio n c u ltu r e d fo r 14 d a y s , (b) a la rg e , p ro lo n g e d , c h e m ic a lly
m e d ia te d s y n a p tic p o te n tia l is a p p a r e n t. T h is d e la y e d p o te n tia l d o e s n o t c o n fo rm to
c r ite r ia fo r a m o n o s y n a p tic p a th w a y (see te x t) .

In the same L cells th a t showed the enlarged potentials following N, P or

T cell stim ulation, ‘spontaneous’ synaptic potentials of norm al shape and size
were always observed. These occur a t variable frequency and arise from tra n s­
m itter release by unidentified cells. The input impedances and tim e constants
of L cells were little if a t all different from normal (see above) and certainly not
enough to account for the changes observed.

Consistency of results in culture: silent ganglia and changes after three weeks
Under the conditions of our experiments the recordings obtained from different
ganglia were reproducible. As mentioned previously, for the first 5-7 days the
synaptic potentials evoked in AE and L cells were normal. N ext, during the
second week, inhibitory potentials appeared in the AE cell and an exaggeration
of the excitatory potentials in the L cell. During the third week the AE cell
showed increased excitation instead of inhibition. The reproducibility was such
th a t one could accurately predict the type of response th a t would occur by
knowing the days in culture. N ot all ganglia were changed to the same extent
and one P or N cell might be more effective than another in producing inhibitory
potentials on the AE cell. B ut, in spite of minor variability, the ganglia recorded
from in the first 3 weeks conformed to this general description.
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C ultured leech ganglia 305

The exceptions consisted of a few ganglia th at behaved in a qualitatively
different manner. From the first few days in such ganglia, the resting potential
of sensory and motor cells showed no fluctuations corresponding to spontaneous
excitatory or inhibitory bombardment. Moreover these ‘silent’ ganglia appeared
to be less transparent than usual. Of the 50 batches of cultures we examined in
this study, only 3 were silent during the first 3 weeks. Moreover, all the ganglia
taken from an animal were either active with a high background of synaptic
activity or silent. At present we have no explanation for these differences.
After 3 weeks in culture, a further change occurred in those ganglia th at had
shown activity. They became silent; it was rare to see any cells th at fired
spontaneously and synaptic potentials were not observed either at rest or as
a result of stimulating presynaptic cells. Cells in these ganglia did continue to
give normal impulses upon direct electrical stimulation or with natural activation
of their receptive fields in the periphery (see figure 4 ). The disappearance of
synaptic potentials occurred at about the same time as ganglia became less
transparent. We have at present no explanation for these changes.

Effects of culture on ganglia in media with raised

Ca or Mg concentration
An advantage of ganglia maintained in vitro is th at one can manipulate the
environment in a controlled manner. An initial experiment of this type was to
use L l 5 medium with raised Ca, 15 m M instead of 1.2 m M , or raised Mg, 20 m M
instead of 1.0 m M . One would expect the continued, ongoing release of trans­
mitter by active neurones to be increased in the Ca fluid and decreased in Mg.
After ganglia had been cultured in high Ca medium for 4-7 days, the cells
became silent and it was not possible to record synaptic potentials in the usual
Ringer fluid (7.5mM Ca); on the sixteenth day, at the stage when excitatory
potentials are enhanced in normal L l 5 culture, small synaptic potentials appeared
after stimulation of a presynaptic cell.
In contrast, cells of ganglia cultured in L l 5 medium containing 20 m M Mg
behaved normally once the Mg was washed out and replaced by normal Ringer.
The synaptic potentials recorded in L and AE cells underwent similar changes
(enhancement and increased inhibition) to those observed in ganglia maintained
for comparable periods in normal L 15 medium. No synaptic potentials could be
evoked after 3 weeks in culture even in ganglia th at had been maintained in high
Mg medium.

Chains of ganglia maintained in culture

The changes described in single cultured ganglia in many respects resembled
those th at had been observed earlier in ganglia maintained within the animal
after their roots and connectives had been cut. For example, in such ganglia the
excitatory effects of P and N cells on the AE motor neurone changed to inhibition
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300 S. Miyazaki and J. G. Nicholls
and impulses in T cells gave rise to chemically m ediated potentials in th e L m otor
neurone instead of electrical coupling potentials (Jansen & Nicholls 1972; Jan sen
et al. 1974)- Some of these changes were also observed in neighbouring ganglia or
ganglia a t a distance from the isolated single ganglion, b u t only after a considerably
longer delay of several m onths. To te st w hether chains of ganglia m aintained in
culture might behave differently from single ganglia we cultured chains of two,
three or five ganglia. In tw o experim ents we cultured the entire nerve cord,
including the head ganglia, all 21 segm ental ganglia and th e tail ganglia. In these
preparations, the roots were cut as usual.
The cells and synapses in chains of ganglia behaved like those in single ganglia.
A t present we cannot say if there are subtle differences of degree which m ay be
detected w ith further studies.
Morphology of cells cultured ganglia
Tests were made in cultured ganglia to see w hether th e morphology of cells
changed as synaptic transm ission became modified and after ganglia had become
silent. The arborizations of T, P and N sensory cells were exam ined by injecting
neurones w ith horseradish peroxidase according to th e methods described by
Muller & McMahan (1976) and Y au (unpublished). The precipitate of the reaction
product allows their processes to be followed by light microscopy. The results
are shown in figures 10 and 11, plate 2, which show camera lucida drawings and
photographs. The cells in cultured ganglia retained m any of th eir original
characteristic features. Thus the sensory cells sent axons through th e roots on
the same side of the animal b u t m otor cell axons crossed the midline to leave
by the contralateral root. The differences in the arborization p attern s of the
various sensory cells in normal ganglia have been observed earlier by Nicholls &
Purves (1970), using procion yellow and by Muller & McMahan (1976), using
peroxidase. The branches of the T cells are less abundant th a n those of P and
N cells and are restricted to the ipsilateral side of the ganglion. On all three
types of cell one can discern small swellings or varicosities. These are fewer in
num ber on the T cells. By electron microscopy, Muller & McMahan (1976) have
shown th a t these varicosities contain vesicles and have both pre- and post-
synaptic specializations. Varicosities were still observed in cells in ganglia cultured
for 26 days which had become silent.
In all, a to tal of more th an 100 sensory cells in norm al and cultured ganglia
m aintained for various periods of from 12 to 21 days were injected successfully.
Certain differences from normal were consistently observed in cultured cells. In
the photomicrographs of figures 10 and 11, plate 2, the varicosities in cultured
ganglia appear larger, coarser and more conspicuous. Although norm al injected
cells show some variability, the difference in the varicosities of cultured ganglia
was obvious from preparation to preparation. Thus, one could readily distinguish
cultured from normal cells by simple inspection of varicosities under the micro­
scope, even when the stain was weak or the full arborization not filled.
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Proc. R. Soc.Lond. B, volume 194 M iyazaki & Nicholls, plate 2

{Facing p. 307)
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Cultured leech ganglia 307

T cells cu ltu red 15 days

norm al

0.1 m m

cultured
norm al N cells
19 days

0.1 mm
GURE 10. C am era lucid a draw ings a n d photo m icro g rap h s of T cells in je cted w ith h o rserad ish p ero x id ase in
n o rm al a n d cu ltu red (15 days) ganglia. A xons leave th ro u g h th e ro o ts a n d w ith in th e n eu ropile th e re are
n u m ero u s sm all processes stu d d e d w ith varicosities. T hese v arico sities are m ore conspicuous in th e T
cell o f th e cu ltu red ganglion.
:g ure 11. C am era lucid a draw ings a n d photom icro g rap h s of N cells in je cted w ith h o rserad ish p ero x id ase in a
n o rm al a n d a cu ltu red (19 days) ganglion. T he arb o riza tio n in th e neuropile is far m ore profuse th a n t h a t
of T cells a n d th e v aricosities are m ore a b u n d a n t. T h ey are m ore conspicuous in th e N coll o f th e
cu ltu red ganglion.
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308 S. Miyazaki and J. G. Nicholls

D iscussion
Survival of ganglia in culture
The experiments we have made so far indicate th a t leech ganglia survive for
several weeks in an appropriate medium. In sensory cells the action potentials,
resting potentials, in put impedance, after-hyperpolarization and responses to
cutaneous stim ulation all appeared norm al; m otor cells still evoked contractions
of muscles in the body wall. Moreover an individual nerve cell upon repenetration
on a second occasion several days later showed no residual effects. In the present
series of experim ents we would not have detected small changes in m em brane
properties of the order of 20 %.
A puzzling feature was the disappearance of synaptic potentials and the loss
of transparency which occurred abruptly a t about 3 weeks. Morphological studies
on nerve cells injected w ith horseradish peroxidase failed to reveal any novel
changes a t 21 days. One possible reason for the disappearance of synaptic
potentials m ight be depletion of tran sm itter in cultured ganglia. However, when
we used medium containing high concentrations of Mg in order to minimize
tran sm itter released by the ‘spontaneous’ firing of presynaptic neurones, the
preparations still became silent after about 3 weeks in culture. A t the same tim e
sensory transduction and impulse propagation still appeared to be norm al in
such ganglia. I t remains to be seen w hat mechanisms are responsible for the
failure of synaptic transm ission and whether glial cells are also involved, as
suggested by the loss of transparency.
Changes in synaptic transmission in culture and in operated animals
In m any respects the changes th a t occur in a cultured ganglion resemble those
th a t occur within an animal in a ganglion th a t has had its connections w ith the
rest of the nervous system severed (Jansen et al. 1974). The most obvious charac­
teristic changes in both types of ganglia consisted of a marked increase in the
size and duration of synaptic potentials which were often more th a n doubled a t
certain synapses (see figure 8).
Two examples of changes in polysynaptic pathw ays th a t were almost identical
in culture and in operated animals are the large chemical excitatory potentials
in the L cell with T cell stim ulation and the inhibitory potentials in the AE cell
with P or N cell stimulation. On the rare occasions when such potentials are
seen in normal ganglia they are small (less th a n 2 mV in amplitude) and driven
only interm ittently. W hat a t first appeared to be an original change in culture
was the greatly enhanced monosynaptic excitatory potential in the L cell produced
by an impulse in the N cell. This conformed to the criteria of being an increase
in a monosynaptic connection possibly with additional polysynaptically mediated
contributions to the late phase. F urther inspection of unpublished records by
Jansen et al. (1974) has revealed th a t similar changes also occur in isolated
ganglia within the animal. In th a t work, however, the emphasis was on qualitative
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C ultured leech ganglia 309

unambiguous changes such as the appearance of inhibition or a chemical component
when none had been obvious beforehand. Similarly ganglia isolated in the animal
also showed prolonged excitatory potentials in the AE cells resembling those
of figure 7 ; but, they could be revealed only after the polysynaptic inhibitory
pathways were blocked (see figure 10 in Jansen et al. 1974). In any event, so far
no striking difference has appeared between consequences of isolating ganglia
in situ or keeping them in culture.
The monosynaptic pathways in cultured ganglia provide favourable preparations
for future studies of the mechanisms th at are responsible for producing the gross
overemphasis of synaptic potentials. One possibility is th at the changes might
be physiological. For instance, following prolonged inactivity of the sensory
cells in culture, enhanced release of transm itter might occur at their endings
(see, for example, Geffen & Hughes 1972). Alternatively, like cultured sympathetic
and dorsal root ganglion cells studied by Mains & Patterson (1973), they might
begin to synthesize new transm itter substances. A further possibility is super­
sensitivity of the postsynaptic AE or L cells to transm itter as a result of sub­
normal activity (see Lomo & Rosenthal 1972). On the other hand, the changes
might be primarily related to structural changes; for example, if presynaptic
inputs to the AE and L motor neurones degenerate (see Frank, Jansen & Rinvik
1975) the processes of T, P and N cells or of interneurones driven by them might
sprout and form additional presynaptic endings.
Of particular interest is the finding of an obvious change in the appearance of
varicosities on the processes of sensory cells within the neuropile of cultured
ganglia, where the varicosities were more conspicuous and appeared coarser.
Recent electron microscopic studies by Muller & McMahan (1976) on normal
sensory cells injected with horseradish peroxidase indicate th at the varicosities
are sites of synaptic transmission. This in turn raises questions about what
mechanism is responsible for the changed appearance in culture, and whether
release sites for transmitters are structurally altered. Our experiments suggest
th at the changes observed are not simply artefacts produced by injections for
the following reasons. First, similar changes were seen in both understained and
Well-stained cells in over 100 cultured and normal ganglia injected in the same
way. The differences were quite obvious under the microscope and in micro­
graphs. They are, however, less clear in low-power camera lucida drawings (such
as those in figures 10 and 11, plate 2) which cannot accurately represent the
varicosities or the cellular details evident in micrographs or in drawings made
at higher magnification (such as figures 3 and 4 in Muller & McMahan 1976).
It also appears unlikely th at increased leakage from fine processes could cause
the changes seen in cultured ganglia, since if one injects peroxidase extracellularly
(as often happens inadvertently) the extracellular deposits can be clearly
distinguished. It remains for electron microscopic studies to reveal whether the
varicosities in cultured ganglia contain synaptic vesicles and if so whether they
still contain them after 21 days when the cells become ‘silent’.
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310 S. Miyazaki and J. G. Nicholls

A t present our results seem more or less in accord w ith th e idea th a t presynaptic
nerve cells release more tran sm itter th a n usual in cultured ganglia b u t we do
not have experim ental evidence to enable us to distinguish between this and
various other possible alternatives.

Advantages of cultured ganglia

Invertebrate ganglia represent an interm ediate level of com plexity when one
considers the variety of neuronal tissues th a t can be k ep t alive outside an animal
in culture. A t one end of the spectrum are neuroblastom a cells (Peacock & Nelson
1973) and dissociated cells tak en from embryonic central nervous system (Garber
& Moscona 1972) and sym pathetic ganglia (Mains & P atterso n 1973). A t the
other extrem e are explants of organized tissues of vertebrate central nervous
system from structures as complex as spinal cord, hippocam pus, cerebellum and
retina (see Nelson 1975).
While ganglia from the leech or aplysia (Dewhurst & W einreich 1974) are
still complex, they are in some respects more manageable because in each
experim ent one cultures the same cells which are packaged in an orderly m an n er;
the individual neurones can still be recognized and th eir morphology, electrical
properties, biochemistry, and connections can be compared w ith those of th eir
counterparts in their norm al environm ents w ithin the animal. Cultured ganglia
are far easier to work w ith th a n ganglia from operated animals. Most im portant,
the supply is not limited and one can keep on hand an array of ganglia a t various
stages of change w ithout the m ortality th a t limits the supply from operated
animals. This in tu rn enables one to make more experim ents of the ty p e th a t
require large num bers of ganglia. For example, the injection of horseradish
peroxidase is not always successful for technical reasons and requires m any trials
before one can ascertain w hether the morphology of a particular cell has changed.
Furtherm ore, the experiments w ith high Ca/high Mg illustrate the potential
usefulness of cultured ganglia for a variety of other purposes. Here one can
hope to examine directly the effects of repeated cutaneous stimuli, to test the
maintained application of drugs and metabolites, to remove individual cells, and
to see whether regeneration of connections can occur vitro.

This work was supported by U.S.P.H.S. G rant No NS 11544 . We are greatly

indebted to Dr Susan Friedm an who participated in early experiments and
helped to devise the culture techniques. We also wish to th an k Dr K. W. Yau,
Dr K. J. Muller and Dr U. J. McMahan, who showed us how to inject peroxidase,
and Dr P. B. Sargent and Dr B. Wallace for m any valuable discussions.
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Cultured leech ganglia 311

R eferences
Coggeshall, R . E . & F a w c e tt, D. W . 1964 T he fine stru c tu re of th e c e n tra l n erv o u s sy stem
of th e leech. J . Neurophysiol. 27 , 229-289.
D ew h u rst, S. A. & W einreich, D. 1974 E ffects of long te rm organ c u ltu re on n e u ro tra n s m itte r
m etabolism in th e ganglia of A p lysia californica. J . Neurobiol. 5 , 21-31.
F ra n k , E ., Ja n se n , J . K . S. & R in v ik , E . 1975 A m u ltiso m a tic ax o n in th e ce n tra l n erv o u s
sy stem of th e leech. J . comp. Neurol. 159, 1-14.
G arber, B. B. & M oscona, A. A. 1972 R e co n stru c tio n of b ra in tissu e from cell suspensions.
Dev. Biol. 27 , 217-234.
Geffen, L. B. & H ughes, C. C. 1972 D egeneration of sy m p a th e tic n erv es in vitro a n d
developm ent of sm ooth m uscle su p e rse n sitiv ity to n o rad ren alin e. J . P h ysio l., Loud.
221 , 71-84.
H ag iw ara, S. & M orita, H . 1962 E le ctro to n ic tran sm issio n b etw een tw o n erv e cells in
leech ganglion. J . Neurophysiol. 25 , 721-731.
Ja n se n , J . K . S., M uller, K . J . & N icholls, J . G. 1974 P e rsiste n t m odification o f sy n a p tic
in te ractio n s betw een sensory a n d m o to r nerv e cells follow ing d iscrete lesions in th e
ce n tral nervous sy stem of th e leech. J . P h ysio l., Loud. 2 42 , 289-305.
Ja n se n , J . K . S. & N icholls, J . G. 1972 R e g en e ra tio n a n d changes in sy n a p tic connections
betw een in d iv id u al nerv e cells in th e ce n tral nerv o u s sy stem of th e leech. Proc. natn.
Acad. Sci. U .S .A . 69, 636-639.
Ja n sen , J . K . S. & N icholls, J . G. 1973 C onductance changes, a n electrogenic p u m p a n d
th e h y p erp o la riza tio n of leech neurones follow ing im pulses. J . P h ysio l., Loud. 229 ,
635-655.
Lom o, T. & R o sen th a l, J . 1972 C ontrol of ACh se n sitiv ity b y m uscle a c tiv ity in th e r a t.
J . P h ysio l., Loud. 221 , 493-513.
M ains, R . E . & P a tte rso n , P . H . 1973 P rim a ry cu ltu res of d isso ciated sy m p a th e tic neu ro n s.
I I I . Changes in m etabolism w ith age in cu ltu re. J . Cell Biol. 59 , 361-366.
M uller, K . J . & M cM ahan, U . J . 1976 S ynapses o f specific sensory a n d m o to r neu ro n es in
th e leech: A lig h t a n d electron m icroscope stu d y a fte r in tra cellu la r in jectio n o f h o rse ­
rad ish peroxidase. Proc. R. Soc. Bond. B 194 , 481-499.
M uller, K . J . & N icholls, J . G. 1974 D ifferent p ro p ertie s o f syn ap ses b etw een a single
sensory n eurone a n d tw o different m o to r cells in th e leech C.N .S. J . P h ysio l., Loud.
3 3 8 , 357-369.
N elson, P . G. 1975 N erve a n d m uscle cells in cultu re. Physiol. Rev 55 , 1-61.
N icholls, J . G. & B aylor, D. A. 1968 Specific m o d alities a n d recep tiv e fields of sensory
n eurons in th e C.N.S. of th e leech. J . Neurophysiol. 3 1 , 740-756.
N icholls, J . G. & K uffler, S. W . 1964 E x tra c e llu la r space as a p a th w a y for ex change
b etw een blood an d neurons in th e ce n tral nervous sy stem of th e le e c h : ionic com position
o f glial cells an d neurons. J . N europhysiol. 27 , 645-671.
N icholls, J . G. & P u rv e s, D. 1970 M onosynaptic chem ical a n d electrical connexions b etw een
sensory a n d m o to r cells in th e ce n tral nervous sy stem o f th e leech. J . P h ysio l., Loud.
209 , 647-667.
N icholls, J . G. & P u rv e s, D. 1972 A com parison of chem ical a n d electrical sy n a p tic tr a n s ­
m ission betw een single sensory cells a n d a m oto n eu ro n e in th e ce n tral n erv o u s sy stem
of th e leech. J . P h ysiol., Loud. 225 , 637-656.
Peacock, J . & N elson, P . G. 1973 C hem osensitivity of n eu ro b la sto m a cells. J . Neurobiol.
4 , 363-374.
S tu a rt, A. E . 1970 P hysiological a n d m orphological p ro p erties o f m o to n eu ro n es in th e
ce n tral nervous system of th e leech. J . P h ysiol.. Loud. 209 , 627-747.