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Altogether there are 3462 chemical synapses and 754 gap
junctions between the 183 neurons in the H series database. The
two different types of connection, gap junctions and chemical
synapses, are distributed rather differently in the nervous system,
as can be seen from table 7.1. Table 7.1 rows a) and b) show the
proportion of pairs of adjacent processes that are connected by
chemical synapses or by gap junctions.
Although there are
4.5 times as many chemical synapses as gap junctions, the
proportion of pairs of adjacent cells connected by chemical
synapses is similar to that connected by gap junctions (9% and 7%
respectively). This is because a pair of cells is often connected
by several chemical synapses, but rarely by more than one or two
gap junctions. In other organisms there may be hundreds or
thousands of synapses between a given pair of neurons (e.g. a
cerebellar basket cell make many synapses on a Purkinje cell). The
small number of synapses in C. elegans is probably due to the
extremely small size of the entire nervous system.
It is immediately obvious from table 7.1 (rows a, b) that
both the probability of being connected by a chemical synapse, and
the number of synapses actually formed, are highly correlated with
adjacency. There is a 14 fold increase in the proportion of
connections formed when pairs of cells that have a high adjacency
are compared with pairs that touch only briefly. This correlation
is far less marked for gap junctions, for which the corresponding
increase is only a factor of two. While the bast majority of
chemical synapses are between pairs of cells with adjacency greater
than 10, nearly half the gap junctions are between cells with
adjacency less than or equal to 5.
It might be thought that
the increase in the average number of synapses formed with higher
adjacencies provides evidence that synapse formation is dependant
on the area of contact. However, for any given pair of neurons it
is possible to show that the number of synapses made between them
does not substantially change with their adjacency. This proposal
can be tested by considering pairs A, B which have contralateral
homologues A', B'. In general A', B' will have a different
adjacency from that of A, B, and they will often also form a
different number of synapses. If we assume that the symaptic
formation mechanisms for symmetrical pairs on the two sides are
equivalent then we can test statistically whether the number of
synapses formed tends to vary proportionally with the adjacency, or
remains independent of adjacency. The details of the test are
given in the appendix, but the results are as follows. There were
391 pairs of processes which formed synapses on both sides and
showed different adjacencies on the two sides, and the value of the
test statistic was 7103 with an expected standard error of around
1330. If the number of synapses varied proportionally with
adjacency then the value should ideally be 0, while if it was
independent the value should be around 7655. It is clearly many
standard error values from 0, and only about half a standard error
distance from 7655. Therefore there is strong evidence against a
proportional system, and the data are consistent with the number of
synapses between a given pair of neurons being determined
independently of the adjacency. The slightly lower value of the
calculated test statistic than that expected by a wholly
independent model can be explained by the fact that there must be
some effect at very low adjacencies. If the adjacency is 0 then
clearly no synapses can be made, and when it is only 1 or 2 there
are spatial limitations preventing a large number of synapses being
formed.
It is important to consider the correlation between synapse
formation and adjacency when looking for statistical interactions
between the different types of synapse. For instance, the
frequency of reciprocal chemical synapses is consistent with
synapses being independently specified in each direction. Although
the overall probability of a connection from neuron B to neuron A
given one from A to B is high (14% as opposed to 9%, see table
7.1 row c), this is because most of the pairs with a synapse from A
to B have high adjacency, and so are more likely than normal to
have a reverse synapse from B to A. When the probability of a
reverse synapse is shown separately for each adjacency range, as in
table 7.1, it can be seen to be essentially the same, or possibly
slightly lower, as that for an unconditional synapse.
The
same approach can be used to consider whether there is any
statistical interaction between chemical synapses and gap
junctions. In this case there is a slight negative correlation,
since the probability of a gap junction between a pair of cells
that are linked by chemical synapses is reduced by 1 or 2 percent
from the unconditional probability in each adjacency range (table
7.1 row d). This difference can be partly explained by the fact
that a significant proportion (66/284) of gap junctional
connections are between members of the same neuronal class, while
it is rare for there to be intraclass chemical synapses (discussed
below).
Therefore the presence of one type of connection
between cells does not provide any indication of whether another
type, either gap junctional or chemical in the opposite direction,
will also be present. The only exception to this is when the cells
are members of the same class, in which case if there are chemical
synapses (only 5 examples) then they tend to be reciprocal (4/5),
as would be expected. Together with the overall dependence of
synapse formation on adjacency these results mean that there is a
remarkably high probability of some form of connection between
processes that have a high adjacency. If the adjacency is greater
than 10 then the proportion of pairs forming either a gap junction
or a chemical synapse one way or the other is 47% (478/1020).
Gap junctions are far more frequent than normal when both
cells are members of the same neuronal class. The 302 neurons in
C. elegans have been put into 118 different classes on the base of
similar morphology and connectivity (White et al., 1986). Many of
these classes, particularly those with processes in the nerve ring,
consist of a pair of bilateral, symmetry related neurons. There
are 61 such pairs in which the two cells make physical contact and
35 (57%) of these have gap junctions while only 5 (8%) have
chemical synapses.
It is common for there to be a gap
junction where the ends of two processes from different members of
the same neuronal class abut, such as when left and right symmetry
related processes meet at the top of the nerve ring. This abuttal
is interesting in itself, since it suggests that the processes
might stop growing when they establish contact with the tips of
their contralateral homologues. A similar phenomenon is seen in
the ventral and dorsal nerve cords, where the processes from
consecutive neurons of two classes of motor neuron (VD and DD) abut
and make gap junctions but do not overlap; the other classes
overlap (except AS), and sometimes make gap junctions. In the ring
there are 24 pairs of cells whose processes end where they meet at
the dorsal midline, and a gap junction is present in 22 of these
cases. Of the 37 other classes of cells that have interclass cell
contacts, but which do not abut, only 13 interclass gap junctions.
None of the pairs that form internal chemical synapses abut, nor do
any of interneurons that send adjacent processes back along the
ventral cord; perhaps if they had inhibited each other's growth
they could not have grown out together along the cord.
There are two sources of synaptic reproducibility that might
be expected in the database. The first is from animal to animal,
and the second is due to internal bilateral symmetry within one
animal. Most neuronal classes that are associated with the nerve
ring, and so represented in the database, consist of one or more
bilateral pairs of homologous neurons. One problem with the
comparison between animals is that the U series animal was an
adult, while the H series animal was an L4 larva. Although almost
all neural development in the nerve ring is embryonic the
sex-specific circuitry concerned with egg laying is incomplete in
the H series, and there may be other less obvious differences due
to age.
The probability that a given pair of H series
neurons A, B will be connected by chemical synapses if their
contralateral homologues !' and B' are connected is 87%, while the
probability that they will be connected if their U series
homologues Au and Bu are connected is only 75% (table 7.2 rows a,
b). The corresponding figures for gap junctions are 89% and 79%
(table 7.2 rows c, d). It therefore appears that gap junctional
connections are slightly more reproducible than synaptic ones and
that the two sides of the same animal are more similar than the two
different animals. Part of the difference in chemical synaptic
connectivity between the two animals is that there are more
chemical synapse connections in the U series than the H series (916
as opposed to 819), and the extra connections are certain to be
unmatched. However the difference between the animals is still
probably significant because there are fewer U series gap junction
connections than ones in the H series (260 against 284) but a lower
proportion of U series gap junctions are matched in the H series
than ones of the opposite side of the H series (above).
The
difference between the "within animals" (left/right) comparison and
the "between animals" (H/U) comparison can be further illustrated
by considering the pairs that form connections in only two of the
four possible places (each side of each animal). There are six
ways that this can happen: both sides of the H series but not at
all in the U series, both sides of the U series but not in the H
series, and four different ways that there could be one synapse in
the U series and one in the H series. If the similarity between
animals were the same as that between sides of the same animal then
the size of all these classes should be the same: the number of
pairs synapsing only in the H series, and the number synapsing only
in the U series, should be a quarter of the number that synapse
just once in the H series and once in the U series. In fact there
are 23 pairs that synapse only in the H series, 51 only in the U
series, and 64 that synapse once in each series. For gap junctions
these numbers are 41, 22 and 14. In both cases there are more
connections than expected that are U series specific or H series
specific, indicating that there are significant differences between
the two animals.
Similarly, a test can be performed to
detect whether there is also a reproducible difference between the
right and left sides of the nerve ring. If there was such a
difference then one would expect that the connection between a pair
of Au,Bu in the U series would resemble more closely that between
their exact equivalents in the H series, A,B, than that between the
corresponding cells on the contralateral side, A' and B'. In fact
the figures are 75% reproducibility to A,B, 74% to A'B' for
chemical synapses, and 79% to A,B, 77% to A',B' for gap
junctions. Therefore if there is consistent difference between the
two sides it is very slight (1 or 2%). It would not be possible
to identify the source of any consistent difference that may be
reflected in this small change of reproducibility, because there is
too much noise from the difference between the two animals.
Overall the observed levels of
reproducibility suggest that there is an underlying regular
pattern. In particular the two sides of the same animal appear to
be sufficiently similar to allow us to assume that they were
subject to the same synaptic specification procedure during
development. This allows us to consider some aspects of the
question of how neural connectivity is determined. By identifying
the sources of inaccuracy of synaptic reproducibility it is
possible to obtain information about, and thus to make suggestions
about, the mechanisms for establishment of specific circuitry.
This approach has already been used to indicate the independence of
the number of synapses made between a pair of neurons from their
adjacency. All the effects we will consider would not be altered
significantly by one or two percent change due to possible slight
genuine differences between the two sides.
Although it
appeared at first sight that there was an unexpectedly high
variation in the formation of chemical synapses, almost all the
inconsistencies are due to the unreliability of weak connections,
i.e. connections with only a small number of synapses. For pairs
connected by three or more synapses (which is true for about two
thirds of connected pairs) the probability that their homologues
are connected is greater than 90%, whereas if there is only a
single synapse then the probability that their homologues will be
connected is only around a half. This difference exists both
between and within animals (for exact numbers see table 7.2).
Clearly one cause of a mismatch in which a connection seen
is present on one side but missing on the other might be that the
neurons are not in contact in the case where the synapse is
missing. However if they do touch each other then the dependence
of reproducibility on the number of synapses made is practically
independent of adjacency. If we consider A, B such that A' and B'
are connected by only one synapse then the probability of
connection between A and B is around 60% in all adjacency ranges.
At the other extreme if A' and B' are connected by three or more
synapses then the probability that A and B are connected is 79%
(11/14) if their adjacency is one or two, and more than 95% in any
adjacency range greater than two (table 71 rows e, f). It is
necessary to consider the data this way, because a direct
comparison of reproducibility at different adjacencies would
suggest that adjacency is an important factor (table 7.2 rows a, b,
final columns). In fact this effect is mostly secondary, caused by
the fact that connections with higher adjacency tend to have a
larger number of synapses. If we combine this result with the
earlier observation that the number of synapses made between a
given pair of neurons is essentially independent of adjacency, then
it appears that, except at very low adjacencies, the probability
that two given processes will form a synapse is also practically
independent of their adjacency. The overall correlation between
synapse formation and adjacency (Table 1, row a) therefore implies
that the physical organisation of processes is such that neurons
that repoducibly synapse tend to have higher adjacencies. This is
considered further in the discussion.
The picture that
emerges is that pairs of neurons are programmed to make an
approximately predetermined number of synapses, and that the
connection will be more reliable if this number is greater than one
or two. There is no significant similar set of results for gap
junctions, because, as far as numbers of junctions are concerned,
more than three quarters of gap junctional connections involve only
one gap junction, and as regards adjacency, there is very little
correlation between the presence of gap junctions and adjacency.
It is however true that in the cases where there are two or more
gap junctions on one side the chance of there being at least one on
the other side is higher than normal (94%).
If we
assume that there is an underlying "normal" pattern of connectivity
then there are essentially two ways that a mismatch can occur.
Either an additional abnormal connection can be made, or a normal
connection can be missing. For any given connection involving a
bilaterally represented neuronal class there are four equivalent
possible locations that the connection could occur in the database,
one on each side of each animal. If a mismatch exists in one
animal then one can look in the other animal to get some idea of
whether the connection is normally there or not. For chemical
synapses there are over four times as many connections that occur
in only one of the four cases as in three out of four cases (137
and 32 respectively). This implies that four times as many
mismatches are due to a single extra connection being present as
are due to a single connection being missing. The same is not true
of gap junctional connections, for which there are 74 present in
only one case and 58 in three cases.
These observations
concerning unmatched chemical synaptic connections suggest that
there are a set of extra synapses in addition to a fairly
consistent set of basic connections. We can attempt to estimate
the consistent set of synapses by counting only those synapses in
addition to a fairly consistent set of basic connections. We can
attempt to estimate the consistent set of synapses by counting only
those synapses that are seen in three or four of the possible cases
(both cases if both members of the pair of neurons are unique).
There are 2890 of these synapses per animal, leaving 1184 extra
synapses altogether in both animals, associated with 647 different
cell pair types. Some of these synapses are between cells that are
already connected by consistent synapses. However less than half
(498) fall into this class; those that do so add fairly evenly to
consistent connections with both more and fewer consistent
synapses. The remainder (686) are formed between pairs that are
not consistently connected. There is a tendency for these to be
between processes that have higher adjacency; 437 are between
processes with average adjacency 6 or greater.
White (1983)
suggested that mismatches might arise because synaptic connections
were only specified between processes that are normally adjacent,
so that if a nerve process was misplaced and acquired new
unexpected neighbours it might make additional, incorrect
connections to some of them. There is quite a large variation in
the set of cells contacted by any neuron, since if A is adjacent to
B then the probability of A' being adjacent to B' is only about
75% (7028/9214). However this is not the predominant source of
mismatches. If A' is not adjacent to B' the probability of a
connection from A to B is far smaller than normal (table 7.1 row g
compared to row a). Only about a quarter of all mismatches can be
explained in this fashion (24/108 for the bilateral comparison and
65/224 for the H/U comparison - 5th column of table 7.2). The
proportions are similar for gap junctions (8/32 and 10/55
respectively). These results suggest first that specificity is
probably determined for at least an extension of the normal set of
neighbours, covering processes with which a neuron is likely to
stray into contact, and second that physical contact is not a major
limiting factor in determining synapse formation, since most of the
marginal synapses are made with processes with which the neuron is
normally in contact. Instead the earlier observations suggest that
additional synapses are mostly made between processes that normally
have reasonable adjacency.
So far we have compared pairs of neurons that are directly
symmetry related, such as A, B to A' B'. There are also many
situations where a neuron B contacts both A and its contralateral
homologue A'. These contacts are not symmetry related, but if
connections are determined purely by the classes of the two
neurons, then one would expect a connection between A and B
whenever there was one between A' and B. In fact the overall
measures of this type of reproducibility are 67% for chemical
synapses and 57% for gap junctions (table 7.2 rows e and f
respectively), significantly less than the levels of
reproducibility for are 67% for chemical synapses and 57% for gap
junctions (table 7.2 rows e and f respectively), significantly less
than the levels of reproducibility for symmetry related connections
(rows a, c). The given frequencies of a matching connection from A
to B are conditional on A and B being adjacent, since there is no
automatic reason why A and B should be adjacent whenever A' and B
are, ecause there is no geometrical symmetry relating to the two
pairs. This difference is dreproducible in the symmetric
left/right comparison. The only set of conditions under which
there is a respectable degree of reproducibility is when the
adjacency of A to B is high (column 7).
What might cause
the difference between A and A'' in synaptic specificity with
respect to B? If the difference is cell intrinsic, as opposed to
being activity related, then either A and A' must be inherently
distinguishable or else either they or B must be regionally
specialised. Regional specialisation would account for the
observations because in general different parts of A and A' contact
B, and they contact it in different places (see figure 7.1 for an
example concerning cells all of the same class). There are several
indications that suggest that this might be a correct explanation.
Although few neurons in the C. elegans nerve ring have a classical
bipolar morphology, with a presynaptic dendritic structure and a
predominantly postsynaptic axonal structure, there are many neurons
whose processes show regional differences (White et al., 1983,
White et al., 1986), and most of the cases of differential
specificity that we are considering, such as that between the RMD
neurons shown in figure 7.1, involve these cells. In addition, if
the differences were due to regional specialisation, then there
would be the highest chance of a mismatch if the cells only touched
in one place, and much less chance if they were in contact over a
large proportion of their axonal structure. This suggests that the
mismatch frequency would be lower when the adjacency of A and B was
high, which is indeed the case (table 7.2 column 7). Such an
effect would not be expected if the difference between A'B and AB
synapses was due to distinct identificatory labelling of A and A'.
Similar results are obtained when comparing synaptic
specificity of dorsal and ventral members of the same neuronal
class. Once again their interactions with other neurons are not
symmetry related, and so the lower levels of reproducibility that
are observed (69% for chemical synapses and 52% for gap
junctions) can be explained by regional specialisation of some of
the neurons. ue neither to the number of connections from A' to B
tending to be low nor to the adjacency of A and B being low, since
there is still a difference when the frequency is tabulated with
respect to either of these categories (columns 2 and 6 of table 2).
Indeed the reproducibility is down to 70% even in the cases where
there are 3 or more synapses, which are 95%
Figure 7.1
A schematic diagram showing the interconnections between the 6 members of
the RMD class of neurons. The circle represents the nerve ring. At each
radial position around the ring one of the RMD neurons has synaptic output
(arrows), both onto muscle and onto other neurons, including the
diametrically opposite RMD neuron, which „intercepts¾ the neuromuscular
junctions (as with the DD process in figure 3.6). In general the RMD
neurons are monopolar, with their proximal regions showing this
intercepting behaviour, and their distal regions being synaptically active.
Gap junctions (thin bars) link neighbouring neuromuscular regions but are
not formed in general even where processes are close, sich as for instance
where chemical synapses are made. Some variability was seen in this
general pattern, since in the U series RMDL had output (both to muscle and
other neurons) from the proximal as well as the distal part of its
process).
The main conclusions of this set of investigations
are:
1. The overall pattern of connectivity between neurons is
fairly reproducible. However at a detailed level there are
differences. There is a greater difference between the two
animals than between the two sides of one animal.
2. Although chemical synapse formation is correlated with
adjacency when all pairs of neurons are considered, between any
particular pair both the probability of forming a connection,
and the number of synapses made, are essentially independent of
adjacency.
3. Synaptic reproducibility is very high (over
95%) if several synapses are normally made. However there is
a substantial number of unmatched single synapses, present in
only one of two symmetrical cases. A significant majority of
unmatched chemical synapses appear to be due to the formation
of abnormal or infrequently made connections, rather than the
loss of a normal connection.
4. The formation of a
connection between two neurons is not purely dependent on the
classes of the neurons involved and whether they are adjacent.
A possible additional factor is that in many cases synaptic
specificity is regionally localised on neurons.
I shall discuss these points in turn.
There are two significant questions concerning the overall
reproducibility of the synaptic connections in the nerve ring. The
first is whether the differences seen are systematic and
functional, or merely random variation due to looking at an
essentially fixed pattern at too fine a level of detail. The
second is whether the left and right sides of the nerve ring are
developmentally equivalent in terms of synaptic circuitry. Some
parts of the nervous system show significant reproducible
left/right asymmetry (e.g. the ventral nerve cord, see Chapter 5),
but at a gross level the two sides of the ring are symmetrical.
These questions could be addressed by considering the four examples
of each cell pair interaction present in the database, one for each
side of each animal.
To consider the second question first,
a comparison was made to search for consistent synaptic differences
between the left and right sides across the two animals. This
indicated that they were very similar, although there may be a
slightly greater similarity overall between the same sides of the
two animals than between their opposite sides (approximately 1%,
the effect for instance of a single possible asymmetric neuron
class). This possible minor difference was ignored for the rest of
the analysis in order to consider the two sides as developmentally
equivalent when investigating their differences. There was a
certain amount of unsystematic variation between the two sides.
Clearly it is not easy to assess the functional significance of
anatomical changes in synaptic connectivity in a circuit whose
function is largely unknown. However there was no obvious
observable pattern to the differences that might have suggested a
rewiring of any piece of the circuitry, such as loss of one
connection but a gain of a compensating connection to a parallel
interneuron, and the vast majority of differences involved
connections with just one or two synapses, or a single gap
junction. It seems reasonable to suggest that the variation seen
here is mostly due to random fluctuation in process positioning and
synapse formation.
The equivalent comparison between the
two difference animals, however, revealed that the two sides of
each animal were more similar than the same sides of different
animals, suggesting possible real differences between the two
animals. The U series is from an older animal but again, except
for the introduction of the major egg-laying motor neurons (HSNL/HSNR)
and a generally raised number of connections from the labial
receptors (IL1, IL2 classe), the differences appear to be scattered
randomly throughout the circuitry, and to consist mostly of only
one or two synapses for any particular pair of cells. Since the
animals are isogenic, the systematic differences must be either age
related, or a consequence of environmental differences during
development. Laboratory specimens of C. elegans are cultured on
bacterial lawns grown on agar plates; their environment varies only
in the level of food supply and the degree of dessication! There
have been no studies to determine whether any of the C. elegans
neural circuitry shows variation under different conditions. A
possible approach would be to compare the ventral nerve cord
circuitry of animals that have been raised swimming freely in
liquid culture with the standard nerve cord of animals raised on
agar plates, which has been reconstructed many times.
A simple model for formation of synapses between two neurons
would be that there is a cell-cell recognition event where the
cells contact, and that synapse formation is a local event, so that
the probability of forming a synapse, and the number formed, would
increase with increasing length of mutual contact. However there
is clear evidence from the variation in adjacency and synapse
formation in equivalent pairs of cells in the database that this is
not the case. Both the probability of forming a synapse, and the
number of synapses formed, are essentially independent of the
degree of adjacency, providing that some contact is made. This
implies that there is internal regulation of synapse formation at a
cell-wide level. Such regulation is biologically reasonable,
because it would be asking a lot of a process placement mechanism
to finely control the exact amount of contact between different
neurons, and the circuitry is reasonably fixed. Indeed the
adjacency of a particular pair of neurons is much more variable
than their connectivity. The next chapter pursues further the
question of process placement in the nerve ring.
Although
there is no apparent link at the level of individual process pairs,
there is a strong general correlation between synapse formation and
adjacency. Neuron pairs that are synaptically connected tend to
have high adjacencies, and the more synapses they form the higher
the adjacency tends to be. The amount of contact is in general
much larger than that needed to make the synapses in. In a sense
adjacency seems to depend on the likelihood of forming synapses,
rather than synapse formation on adjacency. This also makes
biological sense, since the goal of positioning nerve processes is
to place in contact with each other those neurons that will form
connections. However the same correlation is not seen for gap
junctions, for which this post hoc biological rationale is just as
relevant. This seems to suggest an effect on neuronal placement,
and thus possibly process guidance, of either chemical synapses or
gap junctions (or something involved in forming them). One might
postulate either process attraction linked with synapse formation,
or a repulsion, or stopping of growth, associated with gap
junctions. When considering connections within the same class it
was observed that bilaterally symmetric processes that gap junction
with each other often terminate when they meet at the midline,
while those few classes that form intraclass synapses all
substantially overlap. Elsewhere in C. elegans the muscle arms in
the body are clearly attracted to the presynaptic processes, since
when motor neuron axons are displaced the muscle arms still go to
them and receive neuromuscular input (Chapter 4, and J White, S
Brenner, unpublished observations). However, although muscle arms
resemble postsynaptic processes, they are phylogenetically
anomalous and possibly a special example.
The average number of chemical synapses between a pair of
synaptically connected neurons 4.2, and there are many cases with
more than 10 synapses, whereas there are rarely more than one or
two gap junctions between a pair of neurons. We have seen that in
individual cases the level of adjacency between two processes does
not appear to be important to synaptic reproducibility. Instead
the most significant general factor as an indicator of
reproducibility is the number of synapses made. Connections with
only one or two synapses are unreliable, while those normally
containing many synapses are nearly all present in all cases. This
result is statistical; there may be individual pairs of neurons
that always connect but only form one synapse (e.g. OLQV and RICL/RICR
only contact briefly but in each case seen make a single very large
synapse full of vesicles). However overall there is evidence that
the probability of forming a connection and the number of synapses
are linked.
If we consider mismatches in which a connection
is present on one side but not the other, there are four times as
many cases where the connection is seen on neither side of the
other animal as ones in which it is seen on both sides. An
explanation of this result would be provided by the hypothesis that
there is an underlying pattern of circuitry that is consistently
present, but there are also always a number of additional synapses
that are selected from a wide range of possibilities, and which
therefore do not generate reproducible connections. The
distribution of this set of additional synapses was estimated by
subtracting away consistently seen synapses. Since this operation
was performed with synapses rather than connections it leaves some
additional synapses between processes which have a consistent
connection, for example where in one of the four cases 10 synapses
were made, and in others only 6. However the majority of
additional synapses are between processes that are not reproducibly
connected, which implies that the variation observed in the
circuitry is not simply due to modulation in level of a small
restricted set of possible connections. The new connections
normally contain only one or two synapses. This would provide an
explanation for the low reproducibility of synaptic connections
containing only one or two synapses. Both the adjacency
distribution of the additional synapses, and a direct count of the
proportion of mismatches for which the unconnected pair of neurons
are not adjacent, indicate that most of the additional connections
are between pairs of neurons that normally have a reasonable
adjacency, rather than being due to a process wandering away from
its normal circle of connections.
One possible alternative
explanation for the extra "random" connections is that they are due
to errors in scoring synapses during the reconstructions. It is
sometimes hard to unambiguously identify whether or not a process
is postsynaptic to a particular synapse. However the fact that the
comparison between the U and H series contains many differences
with similar characteristics to those described above, but many of
which are reproducible from side to side of each animal, suggests
that they are real.
There are a significant number of cases where synapses or gap junctions are formed between members of two classes of neurons when they make contact in one place on their processes, but not when equivalent cells touch in other places. It seems more likely that this is due to regional localistion of the potential to form the relevant type of synapse in the same way for all neurons in a class, than to all the different members of each class having different recognition properties. A more direct example of clear localisation is seen in the adult ventral cord, where the VA and VB motor neurons are bipolar, sending out processes in one direction that only receive input, and only have neuromuscular output from their other process, a classical axon/dendrite morphology. The interneurons are closely available to both processes. However many of the ring neurons are not bipolar, and it would appear that many of the specialisations, presumably involving differential localisation of surface components, would have to be between distal and proximal regions of the same nerve process, as in figure 1. The following neuronal classes can be tentatively implicated as candidates for regional specialisation on the grounds that they are involved in more than one mismatch and have a suggestive axonal structure: RMD, SMD, SAA, SIB, SMB, RICL/RICR. There are many others with apparently discontinuous axonal structure (White et al., 1986). An example of a different type of change between proximal and distal regions of the same nerve process is given by the single neurite of the major interneuron AIBL/AIBR, which changes relative position in the neuropil, running for the first half of its length with chemical receptor axons, and th distal half with motor neurons and motor circuitry interneurons (White et al., 1983).
Two types of information can be inferred from the foregoing
analysis. First a picture can be drawn of the degree of overall
synaptic rigidity in the C. elegans nerve ring, and how the
connectivity might vary or be modified. Second there are a number
of observations suggesting factors that might be important for the
formation of synapses between individual pairs of neurons.
It is apparent that synaptic circuitry in C. elegans is not so
rigidly reproducible as the positioning and lineal origin of cells,
which are practically identical from animal to animal. However the
data support the suggestion that there is a broad core of
connections that are constant, including most of the strong
synaptic connections containing many synapses. This core is
subject to a reasonably low level of variation itself (at the level
of one or two synapses per connection), but there are also a number
of additional chemical synapses (about 10% of the number in the
core pattern) connecting other pairs of processes, which are not
reproducible. Gap junctions show less variation, and no indication
of a strong additional component.
There is some evidence
that the changes and additions might not be random, since there are
many consistent ones, each small in itself, between the H series
animal and the U series animal. Since these two animals are
isogenic they must be due to either environmental or age-related
differences. If the additions are functional then there are two
possible ways in which they could be used. The first is to
"tinker" with a standard pattern, slightly altering the influence
of various parts of the nervous system on each other but not
changing the functional roles of cells. The second is to introduce
a new behaviour, or to flexibly wire a particular task so that it
is performed in a different way in different animals. This is
different from the question of functional flexibility in a single
neuron taking part in more than one task, for which there is plenty
of evidence: the IL1 and URA classes are for instance both sensory
and motor neurons. Although no data are available on the
variability of function of nerve ring neurons in different animals,
I believe that two observations point towards the tinkering rather
than the respecification theory. First, in no case is a systematic
pattern of connectivity change discernible (other than the
introduction of the egg-laying circuitry in the sexually mature U
series animal). Second, the great majority of changes consist of
only one or two synapses per connection.
There are several
observ ations concerning the formation of connections between pairs
of neurons. It seems likely that the potential to form synapses
and gap junctions may be localisable to particular parts of a
single process. The probability of a chemical synaptic connection
being formed, which may depend on environmental as well as genetic
factors, and the number of synapses formed, appear to be regulated
so as not to depend on the amount of contact between a pair of
cells. However the factors involved in synapse formation may
influence adjacency, because synapse formation, but not gap
junction formation, is correlated with adjacency, suggesting that
one or the other of the two processes may have an effect on nerve
process placement. The evidence for these properties generated by
this study is statistical and indirect, and therefore inappropriate
as a basis for a series of direct intrusive testing experiments.
However it is not susceptible to the bias that might be generated
by studying intensively a single connection. Also, since a very
large number of cell pairs were considered, it is statistically
significant; the conclusions do almost certainly reflect pervasive
properties of one simple nervous system, and they may also be
relevant to other organisms.