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AM. ZOOLOGIST, 2:79-96 (1962).
ORGANIZATION OF NEUROPIL
DONALD M. MAYNARD
Department of Zoology, The University of Michigan
Most complex nervous systems consist of
central ganglia connected with outlying
sensory organs and effectors by peripheral
nerves. Information moves to and from the
ganglia in the nerves in coded form, as a
spatial-temporal pattern of all-or-none impulses. Decoding of the incoming message
and recoding of the appropriate outgoing
response occur somewhere in the ganglia.
Many of the fundamental problems of
neurophysiology revolve around the related
questions of the functional significance of
impulse patterns in the central ganglia
(codes), and of the mechanisms of central
neural integration (coding and decoding).
Information about impulse patterns and
the properties of integrative systems can be
derived from input-output analyses (Wiersraa, 1962), but problems regarding detailed
mechanism eventually require penetration
into the ganglion and dissection of the integrating system itself.
Let us look, then, at the organization of
a central invertebrate ganglion. The emphasis will be on arthropod material, for I
am most familiar with this group; most of
the comments should apply with little
modification to many of the annelids, molluscs, and lower vertebrates.
NEUROPIL
In the central ganglia of most higher invertebrates four histological divisions are
evident (Fig. 1): 1) An outermost sheath
of non-neural connective tissue cells or glial
elements separates the central neural tissue
from general body cavities. 2) Just within
the sheath tissue are the cell bodies of the
monopolar neurons. 3) Tracts of fibers
form the third division. These run from
Original work on Panulirus reported in this paper
was carried out at the Bermuda Biological Station.
It was supported by Rackham Grants from the University of Michigan, an ONR grant to the Biological
Station, and U.S.P.H.S. grant B 3271. Miss C. Goodrich collaborated on the experiments with the stomatogastric ganglion.
cell bodies into the neuropil, from the neuropil to the origin of peripheral nerves, and
from neuropil to neuropil as connectives or
commissures. The fibers of the tracts are
either axons or extended, unbranched portions of the dendritic arborizations. 4) The
fourth and final division is the central neuropil, the neuron feltwork. In many cases
it represents the major portion of the ganglion.
The term neuropil, however, has beef,
used in different ways by a number of authors, and is not a precisely defined concept
(see Herrick, 1948; Dempsey and Luse,
1958; Friede, 1960). Classically it is an anatomical term referring to regions of the central nervous system composed of tangles or
meshworks of nerve processes and non-neural elements such as glia and blood vessels.
By extension it also refers to such tangles of
collaterals, dendrites, and axon terminations in peripheral ganglia. Neuropil has
also been considered synonymous with synaptic field, however, and thus has acquired
functional connotations as the primary site
of integrative neuron activity (Herrick,
1948). The structural limits of neuropil are
apparent in forms such as the crayfish or
lobster where the majority of the centra'
neurons are monopolar with cell bodies located in the periphery of the ganglion. As
one moves either to such non-vertebrates as
the Coelenterata or Platyhelminthes, however, or to the higher vertebrates, bipolar or
multipolar neurons in the central nervous
system increase and the cell body becomes
increasingly mixed with dendritic fields.
Finally the original distinction between cell
body regions and fibrous regions is lost;
there is a continual gradation from the classical neuropil, a synaptic field which included processes only, to the synaptic field
which places the ce.ll body in the center and
surrounds it with dendrites and terminal
arborizations, as for example in the vertebrate olivary nucleus (Fig. 2). In organisms
with the latter kind of synaptic field the
(79)
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DONALD M. MAYNARD
BIG. 1. Brain of craylish, Astacus fliwiatilis, as seen
from above and behind. Methylene blue stain showing peripheral cell bodies, fiber tracts, and neuropil.
Peripheral nerves from top to bottom: anterior me-
dian nerve, optic tract, tegumentary nerve, antennal
nerve, and circumesophageal connectives. (From
Retzius, 1890, Biol. Untersuch. N.F.)
cell body seems more intimately involved
with electrical activity of the neuron, and
it is reasonable to consider the entire integrating portion of the nervous system as
functional neuropil.
Many authors have been impressed with
the apparent structural disorder of neuropil (Dempsey and Luse, 1958; Horridge,
1961). It is indeed heterogeneous and complex, and because of this has resisted most
attempts at a systematic analysis. It has
been called the terra incognita of neuroanatomy and neurophysiology. Nevertheless, neuropil often seems to conform to
some underlying structural pattern, even
when showing random connectivity. We
shall begin this discussion, therefore, with
a preliminary structural classification of
neuropil. The recurrence of similar patterns in regions subserving similar functions in widely divergent organisms suggests that much of the structure may have
unique functional significance. The direct
measurement of neural activity within neuropil has just begun, but electrical recordings have revealed several functional properties that in some cases correlate closely
with neuropil structure.
l i d . 2. (ells of inferior olivary nucleus of new
born human, a, axon: b, collateral. (From Cajal,
1904, Textura del sistcma nervioso del hombre y rle
los vertebrados. II. Part 1.)
ORGANIZATION OF NEUROPIL
FIG. 3. Plexiform neuropil. Nerve net of bipolar
cells in mesentary of Metridium. Whole mount,
Holmes' silver. (From Pantin, 1952, Proc. Roy. Soc.
London, B.)
STRUCTURE OF NEUROPIL
Microscopic Morphology
On the basis of gross fiber configurations,
all neuropil can be placed in either of two
general classes, structured or unstructured
neuropil. These in turn can be subdivided:
the unstructured into plexiform and diffuse
neuropil, the structured into glomerular
and stratified neuropil. The groupings are
not sharply bounded or exhaustive, but
they do emphasize significant differences in
organization and, without undue stretching, seem to include most kinds of neuropil.
M
tic junctions seem to be lacking, and the
nerve net has been considered syncytial
(Mackie, 1960).
Plexiform neuropil may form the major
portion of the nervous system in primitive,
sessile, or burrowing organisms. In the Coelenterata, for example, the entire nervous
system is a two-dimensional nerve net (or
nets) and cell bodies are continuous with
processes. In Balanoglossids, on the other
hand, the cell bodies are separated from a
thicker plexiform fiber mat (Bullock,
1945a). Plexiform neuropil also forms limited regions of more complex nervous systems or serves as a component of structured
neuropil. It is the common pattern of innervation for hollow organs such as the digestive tract.
Diffuse neuropil presents the classical picture of "a tangled confusion" (Horridge,
1961) (Fig. 4). Neurons contributing to it
in most invertebrates are either monopolar
elements of average or large size, or peripheral bipolar sense cells whose axon terminations ramify centrally. Many of the
monopolar cells are motor neurons or interneurons connecting several ganglia or segments of the organism (Fig. 5). Processes
are characteristically tortuous, extensively
Unstructured neuropil
Unstructured neuropil is characterized
by ill-defined fiber configurations. Areas of
fiber interaction are not sharply differentiated within the general fiber network, and
the domain of a single neuron may be extensive. The neuropil may take either of
two forms.
Plexiform neuropil is homogeneous and
net-like (Fig. 3). The neurons forming it
are usually simple mono-, bi- or multipolar
structures. The processes are often unbranched, straight, and of constant diameter. Synaptic junctions appear simple, of
few varieties, and may occur at almost any
point on the process. Many appear to be
unpolarized, and transmit impulses in
either direction. In some forms, the synap-
FIG. 4. Diffuse neuropil. Central region of brain
of spiny lobster, Panulirus argus. Thin section,
Masson's trichrome stain. Ramifying dendrites and
fiber tracts form a "haystack" with regions of varying texture. Width of field, about 1.5 millimeters.
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DONALD M. MAYNARD
FIG. 5. Diffuse neuropil. Last thoracic and abdominal ganglia of Carcinus maenas. Whole mount,
methylene blue stain. Note extensive branching of
motor unit in right lobe of thoracic ganglion.
(From Jiethe, 1895, Arch. Mikroscop. Anat. u. Entwitklungsinech.)
branched, and of widely varying diameters.
They spread to varying degrees throughout
the neuropil so that it seldom presents a
homogeneous appearance. Each neuron,
however, tends to have its own domain of
arborizations. Synaptic junctions are not
easily recognized with light microscopy, but
are probably more heterogeneous than in
plexiform neuropil. There is no good anatomical information concerning the existence of unpolarized synapses or syncytial
interneuron bridges.
Diffuse neuropil is characteristic of the
more posterior central ganglia of most
higher invertebrates, and usually contributes to the cerebral ganglia. It is also found
in the brainstem of lower vertebrates and
in many peripheral invertebrate ganglia;
the crustacean stomatogastric ganglion is
an example.
tions. Areas of fiber interaction are usually
clearly distinguished from surrounding
fiber networks. The domain of a single neuron may be quite limited, but it often
makes contact with other elements whose
domain is extensive. The rigidity and preciseness of the fiber patterns is impressive.
They usually seem to represent a reduplicating system; a complex structure built up
of large numbers of relatively few kinds of
units which in turn are grouped into repeating sub-structures. Such precise but redundant structural fabric probably occurs
where stability, accuracy, and temporal or
spatial complexity are functional prerequisites. It seems unlikely that functional lability and the potentiality for "learning"
predominate in the more excessively structured neuropil.
Structured neuropil is most evident in
anterior ganglia of complex organisms. It
may be directly associated with complex
sensory organs. There are many geometrical patterns of structured neuropil, but
Structured neuropil
Structured neuropil is characterized by
regular, often repeating fiber configura-
ORGANIZATION OF NEUROPIL
riG. 6. Glomerular neuropil. Nucleus rolundus of
sea robin, l'rionolus evolans. Thin section, upper
part drawn as it appears after silver impregnation
according to Bodian. Lower part, reconstruction
from Golgi preparations. Ch., commissura horizontalis of Kritsch; Gl., glomeruli; Tr.r.-l., tractus rotundo-lohaiis. (I'rom Scharrer, 1945, J. Coinp. Neurol.)
most seem to fall into one or the other of
two groups, glomerular and stratified.
Glomerular neuropil is characterized by
tight, profuse ramifications of pre- and postsynaptic elements at the site of junction
(Fig. 6). These ramifications form the discrete, knot-like clusters of fibers that give
this kind of neuropil its name. The glomeruli themselves rarely occur in precise
relationship with each other; they may be
in layers or apparently unstructured clumps.
The size and form of the glomerulus and
the number and pattern of elements contributing to it may vary greatly. For example, an afferent fiber in the vertebrate
olfactory bulb or the insect antennal lobe
terminates in but one glomerulus, while
neurons of the insect corpora pedunculata
or mitral cells of bird olfactory lobes may
have branching processes which enter a
number of glomeruli. The minimal number of neurons necessary to form a glomerulus is not clear, but the olfactory glomeruli of the rabbit must represent a near
maximum. Each glomerulus is claimed to
receive as many as 26,000 incoming fibers
and has about 90 outgoing fibers (Allison,
1953). This is a particularly clear example
of the convergence that often seems to occur in glomeruli.
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The internal structure of glomeruli is
largely unknown (see Bodian, 1952). Silver
and methylene blue stains suggest that differences exist between the arborization patterns of pre- and post-units.
The neurons whose processes form glomeruli range widely in function and anatomy. They may be motor, but more commonly are sensory or internuncial elements.
Among the latter, the dendrites of small
globular neurons of the invertebrates (Hanstrom, 1928) and granule cells of the vertebrate cerebellum characteristically form glomerular junctions. It is evident that glomerular neuropil does not represent a homogeneous class, but further subdivision is
undesirable until more information on
structure and function is available.
Stratified neuropil (Fig. 7) is a regular,
three-dimensional fiber lattice formed from
precisely oriented neuron processes. The
anatomy presumably reflects the functional
connectivity. The basic pattern consists of
vertically or radially oriented fibers that
pass at right angles through horizontal or
tangential layers of ramifying processes. Synaptic junctions occur between the vertical
and horizontal fibers.
The arthropod optic ganglia represent a
typical, highly structured example of stratified neuropil (Fig. 7). The vertical elements are quite different in form from the
horizontal fibers. Some of the vertical elements have collaterals and junctions in only
one layer, others send collaterals into several layers. Often the collateral structure differs according to the layer or the particular
vertical neuron; presumably it reflects differences in the functional properties of the
junction. The lateral extent of the synaptic field of a vertical unit is usually limited. Consequently most vertical units probably synapse with relatively few horizontal
fibers in any one layer. In contrast, the
horizontal fibers branch diffusely. They
often travel in preferred directions, but apparently form junctions with a large number of vertical units in their level. Within
a layer they are probably the major link between vertical units, just as the vertical
units appear to be the major link between
layers.
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DONALD M.
MAYNARD
areas of cerebral ganglia. The cortex of the
vertebrate or possibly the central body of
the arthropod are examples. These regions,
however, often lend to merge in form with
diffuse, unstructured neuropil, and the
fiber domain of a single neuron appears less
rigidly defined than in the optic systems described above.
Hislochemistry and Sub-microscopic
Anatomy
The above discussion gives a general
view of fiber patterns in neuropil, but it is
necessarily superficial. In addition to the
obvious limitations resulting from this superficiality, there are at least two serious
drawbacks in the methods themselves which
reduce the functional significance of the
findings. On the one hand, the usual preparative techniques of classical micro-anatomy do not reveal chemical differences
among fibers of the neuropil, and on the
other, light microscopy does not have sufficient resolution to show the detailed intracellular structure of fibers and synapses.
Both of these aspects are particularly relevant in a consideration of the functional
anatomy of neuropil.
Histochemislry
FIG. 7. Stratified neuropil. Diagram of retina and
optic ganglia of honey bee, Apis melifica L. I, retina; II, lamina ganglionaris; III, medulla externa;
IV, medulla interna. (From Cajal and Sanchez,
1915, Investig. Biol.)
Both vertical and horizontal fiber groups
contain afferent and efferent elements. In
the case of optic ganglia, however, the primary input fibers from the visual receptors
are vertical rather than horizontal. In contrast, only horizontal fibers seem to carry
information from the optic ganglion complex into more central areas of the brain.
The retina and superior colliculus in the
vertebrate and the retina profundus of the
octopus are formed from stratified neuropil
essentially like that of the arthropod. Such
a configuration may be important in dealing with complex spatial patterning, and
will be considered in more detail below.
Less precise layering is evident in other
There is considerable evidence for several
neurotransmitters in the nervous system
(Florey, 1962). A detailed map showing
the localization of these, or substances involved in their production or break-down
could prove of aid in determining the functional properties of a given neuropil. Many
of the transmitters seem to be specific for
particular kinds of neurons.
We have been interested in the localization of cholinesterases in crustacean ganglia (Fig. 8, Maynard and Maynard, 1960a).
There are high concentrations of the enzyme in peripheral glial tissue (see also,
Wigglesworth, 1958), but it also occurs in
some neuron somata and particularly in
certain regions of the neuropil. Experiments with more peripheral structures in
the lobster (Maynard and Maynard, 1960b)
suggest that concentrations of the enzyme
may correlate with physiological character-
ORGANIZATION OF NEUROIML
dendrite and axon in the neuropil is lacking. The peripheral cell bodies are devoid
of terminal contacts, and fiber junctions
are confined to the neuropil. Three kinds
of junction are recognized: 1) cross contacts, 2) longitudinal contacts, and 3) endknobs (Fig. 9). In all, neuron membranes
come into direct contact with each other,
but there is no fusion of neuroplasm. Although membranes are often flattened or
indented at the point of contact, invaginations as found in the synapse between giant
fibers in the crayfish (Robertson, 1953) are
apparently absent. Only the end-knob junction contains the accumulations of microvesicles and mitochondria often considered
characteristic of synapses (De Robertis,
1958; Palay, 1958). Microvesicles, however,
also occur along fibers in non-junctional
areas.
ol
FIG. 8. Distribution of cholinestcrase in brain of
lobster, Homarus americmuis. Thin section, acelylthiocholine substrate. Al, accessory lobe, a glomerular neuropil; gc, globular cells, two different
groups; ol, olfactory lobe, another glomerular neuropil. Enzyme concentration to right of accessory
lobe represents a fiber tract; light areas surrounded
by a ring of enzyme are large neuron somata; other
areas of high enzyme concentration are in diffuse
neuropil. (Courtesy of E. A. Maynaid)
istics, but this remains to be established in
the central ganglia. Perhaps the final interpretation o£ such chemoarchitectonics
must await clarification of the role of acetylcholine esterase in neural activity.
Sub-microscopic anatomy
A number of scattered observations on
the fine structure of invertebrate nervous
systems exists, but a systematic, comprehensive investigation of neuropil has not been
published. One of the closer approximations to such a needed work is a preliminary
investigation of caterpillar ganglia (Trujillo-Cenoz, 1959).
Most fibers in the (diffuse?) neuropil of
a caterpillar ganglion are naked and lack
glial sheaths. In this they differ from the
sheathed fibers in interganglionic connectives. Fine structural distinction between
FIG. 9. End-knob in neuropil of caterpillar, Pholus
labruscoe L. Electron micrograph. E.K., end-knob;
M, mitochondria; S.M., synaptic membranes; T.F.,
thin fibers (post-synaptic?). Microvesicles accumulate to left of mitochondria. Width of field, about
2.3 microns. (From Trujillo-Cenoz, 1959, Zeit. f.
Zellforsch.)
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DONALD M. MAYNARD
Several important conclusions can be
drawn from these observations. 1) Neuron
interaction in caterpillar ganglia, if dependent upon close anatomical contact, seems to
be confined to the neuropil. 2) Some junctions in the neuropil are similar to the classical synapse; one may therefore anticipate
similar properties of synaptic transmission.
3) Several forms of fiber junction occur,
suggesting the possibility of more than one
mechanism of neuron interaction. It should
be clear, however, that full benefit of the
increased resolution of electron microscopy
will be apparent in neuropil analysis only
when correlations between fine structure
and specific microscopic structures are possible.
Glia and Capillaries
Thus far we have spoken of neuropil as
though it were composed solely of neuron
processes. This is misleading, for both glial
cells and vascular elements are also present.
As mentioned earlier, glial elements usually form an outer sheath enveloping the
entire nervous system. They may also surround cell bodies and individual fibers in
tracts and connectives (Wigglesworth, 1959).
Just how far glial processes interpenetrate
the denser fiber tangles of invertebrate neuropil is not known (Trujillo-Cenoz, 1959).
In the mammalian grey matter, of course,
glia and neuron processes intermingle, and
glia-neuron junctions have been described
(Scheibel and Scheibel, 1958).
The physiology of glia is of current interest. In the past glia have been considered
supporting or nutritive elements (Wigglesworth, 1960), but they also have been implicated in the control of extracellular environment in the neuropil (Hoyle, 1953;
Twarog and Roeder, 1956; Treherne, 1961).
In addition, there are reports of electrical
activity in vertebrate glia (Tasaki and
Chang, 1958), and current speculations suggest that they may be involved in integration in the nervous system (Galambos,
1961). This topic is beyond the scope of the
present paper, but obviously glia cannot be
completely dismissed from a final functional analysis of the nervous system.
Capillaries. Capillary beds are usually
dense in neuropil areas. In fish, for example, the glomeruli of the olfactory lobe
and nucleus rotundus are heavily vascularized. This often correlates with concentrations of mitochondria or oxidative enzymes
(Scharrer, 1945; Friede, I960) suggesting
that neuropil is a region of unusually high
physiological activity. Even in the lobster
and crayfish, which are generally considered to have an open circulatory system, the
central nervous system, particularly areas
of structured neuropil, is laced with a capillary network. In both vertebrates and lobsters, the higher correlative functions of the
nervous system are very sensitive to oxygen
deprivation.
FUNCTIONAL PROPERTIES OF NEUROPIL
Forms of Electrical Activity
Electrical changes provide one of the
most sensitive measures o£ neural activity.
For the past two years, in collaboration
with J. Clarridge, V. Shoemaker, R. Stephens, and M. J. Cohen, we have been
using intracellular microelectrodes to probe
into parts of neurons in the perfused spiny
lobster brain. One of our purposes was to
answer the simple question: What kind of
electrical activity normally occurs in complex central ganglia? Are there propagated
impulses? Are there slow potentials? Do
all parts of the neuron act alike?
General remarks
First, we find that in fibers in tracts only
full-sized action potentials occur. These are
indistinguishable from those found in any
peripheral nerve.
Second, electrodes placed in peripheral
cell bodies record only attenuated, slow depolarizations in our preparations. The soma membrane seems to be inactive, and the
recorded potentials presumably represent
passive, electrotonic spread from distant
sites of activity in the neuropil (Hagiwara
and Bullock, 1957; Preston and Kennedy,
1960). Such a finding perhaps is not surprising in the light of observations made by
Bethe (1897) and Hardy (1929) that reflex
activity can continue for short periods after
surgical removal of cell bodies.
87
ORCAN'IZATIO.N OF NF.UROPIL
FIG. 10. Synaptic noise recorded with intracellular
electrode from a nerve process in brain of spiny lobster. Read from right to left. One spike potential
rises from unusually large synaptic potential. Both
epsp and ipsp probably contribute to subthreshold
activity. Calibrations, 0.1 second; 4 millivolts.
Thirdly, ramifying processes in the unstructured neuropil are the site of much
complex electrical activity. This is gratifying, for neither the all-or-none spikes of the
tracts nor the small, relatively simple slow
potentials of the neuron soma are the kinds
of activity expected in a center of complex
integration. Some of the neuropil elements
are silent, but many are either spontaneously active or subject to continuous subthreshold, presynaptic bombardment (Fig.
10). There are at least four types of activity:
1. Excitatory post-synaptic potentials (epsp) are the most prominent. These are depolarizing potentials lasting several milliseconds. They are non-propagated, and consequently of greatest amplitude at the site
of the synaptic junction. Since those recorded in a single cell often vary considerably in amplitude, they must represent different junctions either at unequal distances
from the recording site, or possibly, of unequal effectiveness. Epsp initiated by different presynaptic elements may sum algebraically, and upon reaching critical depolarization, initiate propagated potentials
that travel down the axon (Fig. 11).
2. Action potentials are recorded from
most neuropil processes as small, all-ornone spikes lasting 1-2 milliseconds. These
may propagate distally, for they correlate
perfectly with impulses recorded in peripheral nerves. They usually rise from generator or synaptic potentials. In the latter
case, they tend to be smallest in those units
with the largest epsp. This suggests that
they may originate some distance from the
site of the synaptic junction and do not invade the junctional regions. Elements may
have more than one size spike potential, implying that one neuron may have several
different impulse sites, each capable of
maintaining an independent action potential (Bullock and Terzuolo, 1957; Preston
and Kennedy, 1960; Spencer and Kandel,
1961).
3. Generator potentials are slowly growing depolarizations that usually give rise to
propagated action potentials. (They are
not to be confused with the generator potentials of receptors.) They presumably
originate spontaneously in the normal unit,
but their genesis is obscure and they also
occur in injured axons. It is not clear how
much of the spike activity of neuropil elements results from generator potentials and
how much from synaptic noise.
4. Inhibitory post-synaptic potentials (ip-
*
•
I
«
•
*
*
t
•
FIC. 11. Epsp. Intracellular recording from unit
process in diffuse neuropil of spiny lobster brain
(tracing). Input via homolateral antennular nerve
with increasing stimulus strengths from bottom
trace up. Strongest stimulus evokes an action potential whose peak is not shown. Time mark, 2
millisecond intervals; voltage calibration, 10 millivolts.
88
DONALD M. MAYNARD
n
FIG. 12. Ipsp. Iniracellular recording as in Fig. 11.
Read from right to left. Input at 50 stimuli per
second via medial bundle of heterolateral antennular nerve. Calibrations, 0.1 second; 10 milivolts.
Upper trace of this and following figures taken from
monitoring electrodes on antennular nerve; lower
trace from microelectrode within ganglion.
sp) are similar to the epsp but often hyperpolarize rather than depolarize (see Furshpan and Potter, 1959b). On occasion these
reduce the excitability of the unit and
either block spike initiation or reduce spontaneity (Fig. 12). Like the epsp, the ipsp
originate from activity in presynaptic elements, and like them, help comprise the
continuous subthreshold synaptic noise
found in most units of the neuropil.
T h e activity described above is well
known from other preparations, but it is
important that it be demonstrated in neuropil itself. Other kinds of electrical activity are probably to be found in the ganglia
(see below), but the above are the most obvious, and from them we may begin to
build a tentative picture of functional neuropil as a coding center of the nervous system.
1. Integrative activity is largely confined
to the neuropil. Tracts only conduct impulses, and cell bodies seem to be electrically inactive.
2. Although both input and output from
the ganglion, and communication between
neuropils within, involves propagated action potentials and a pulsed code, the electrical events concerned with actual integration may be more similar to a non-pulsed,
analog system.
3. Portions of the neuron concerned with
reception and integration of information
from other neural elements are distinct
from, and have much more variable activity than portions concerned solely with impulse propagation. T h e entire neuron,
therefore, does not a d as an all-or-none
unit, and the eventual propagated impulse
leaving the neuropil depends upon the preceding summed, integrated, non-propagated
activity of the dendritic arborizations.
4. At least some neuropil integration involves typical synaptic transmission, both
excitatory and inhibitory.
5. Many, if not most, of the neuropil association or motor elements receive continuous, subthreshold presynaptic bombardment. This is not immediately apparent in the output of the system, but is probably the basis of a variable central excitatory state. It seems unlikely that normal
neuropil can ever be considered truly at
rest or in a static state.
Specific remarks on single units
One of the remarkable things about many
neuropil elements is the extent of their arborizations. We have suggested that functional differences exist between arborizations and axons, but one may also ask: Are
all excitatory synapses alike? How does one
branch of a neuron affect another if it is
several millimeters distant? Although we
cannot fully answer these questions, certain
observations on units in the lobster brain
are suggestive.
First: Many of the synaptic potentials
caused by primary afferent fibers from the
antennular nerve in the spiny lobster do
not show temporal facilitation. In fact,
they begin to diminish in amplitude at low
frequencies of repetitive stimulation and
soon fall below the spike-initiating threshold. In contrast, other epsp with longer latencies do not fatigue and usually require
temporal summation to reach maximum
effectiveness in spike initiation. There are,
therefore, at least two kinds of epsp in central as well as peripheral crustacean ganglia
(see Terzuolo and Bullock, 1958; Furshpan
and Potter, 1959b). These give the integrating neuron the potential of responding
in very different manners to similar temporal patterns from different inputs (Fig.
13).
Second: Several observations suggest that
a neuron can maintain independent spike
potentials in more than one of its processes.
Although evidence is not conclusive, it is
possible that these potentials represent
propagated impulses rather than synaptic
89
ORGANIZATION OF NEUROPIL
RMB
I I I 1 I M 1 11
T
T
I
r
LMB
0.2 sec
FIG. 13. Patterned responses from different inputs.
Intracellular recording from one unit as in Fig. 11.
Read from right to left. Input at 10 per second via
medial bundle of homolateral antennular nerve
(upper pair of traces) and via medial bundle of het-
erolateral antennular nerve (lower pair of traces).
Note different patterns of response to first stimulus
of train and differences in adaptation or fatigue.
Synaptic potentials were not present in this record.
potentials (Furshpan and Potter, 1959a).
Jf so, they provide a mechanism of intracellular interaction in extensively branched
neurons (Spencer and Kandel, 1961; Preston and Kennedy, 1960). The propagated
impulse would occur in stretches of the dendrite connecting separate arborizations, and
not appear as the final output of the neuron unless capable of exciting the efferent
axon itself. Our records indicate that this
does not always occur (Fig. 14), and, if our
hypothesis is correct, that synaptic potentials initiating a propagated impulse in one
portion of a single neuron's arborizing processes may ultimately fail because of failure
of the impulse at some dendrite-axon junction. This implies that the single neuron
may be analogous to a two-neuron chain,
and the point of potential spike failure or
lowest safety factor is analogous to a synaptic junction. Possibly the extensively
branched neurons often found in the arthropod neuropil (see Fig. 5) should not be
regarded as functional units. They may
embody in one morphological structure
two or more functional elements and may
be, in fact, similar to a synaptic chain of
several less ramifying neurons. One may recall that many invertebrates have remarkably few neurons in their nervous system
and yet are capable of much complex, adaptive behavior.
FIG. 14. Various spike potentials in one unit. Recording as in Fig. 11. Unit responds in different
manner to each of four different inputs: LLB, lateral bundle of left antennular nerve: LMB, medial
bundle of left antennular nerve; RLB, lateral bundle of right anlciinular nerve; RMB, medial bundle
of right antennular nerve. Note synaplic potential
following RMB stimulation and smaller spikes following RLB. Under other conditions, these smaller
spikes were capable of initialing the larger spike.
Small spikes never appeared upon stimulation of
bundles of the left antennular nerve.
AsyitapUc in term: I ion
The observations reported above indicate
that at least some of the integrative activity
of the neuropil involves synaptic transmis-
90
DONALD M. MAYNARD
sion. There is evidence from other preparations, however, that synapses may not be
the only means of significant interaction between neurons; non-synaptic, electronic influences may exist (Watanabe, 1958; Bennett, I960). Unlike synaptic potentials, the
electrotonic influence does not occur as discrete events, but appears in the second unit
as an attenuated and distorted reflection of
prolonged potential changes originating in
the first unit. This kind of interaction
should tend to insure that a group of neighboring neurons remain at the same excitability state. In two preparations where
they have been analyzed, electrotonic interactions are associated with synchronized
discharge of elements of the system. Synchronized discharges of neighboring neurons as found in the cockroach corpora pedunculata (Maynard, 1956) may also result
from such mechanisms of interaction.
Summary
T o return to our first question, we may
say that the electrical activity in neuropil
is complex, and that a large portion of the
complexity appears as a property of individual units rather than as the result of complex connectivity patterns. The relatively
simple model of synaptic action as we know
it from the neuro-muscular junction, the
vertebrate motor horn cell, or the squid
giant axon synapse therefore seems less applicable to an analysis of central integration than the more complex patterns found
in such peripheral autonomic ganglia as
the crustacean cardiac ganglion. Tt may be
misleading, when analyzing central integration, to concentrate too exclusively on wiring diagrams, functional or anatomical, and
to ignore the properties of the switches and
oscillators of the system.
Functional
Connectivity
Functional connectivity patterns are important, however, and much of our structural analysis of neuropil is based on the
anatomical substrate of such patterns. I
should like to turn about and review several observations which seem to demonstrate this importance in the determination
of physiological characteristic* of total neu-
ropil. They also suggest correlations between the morphology and the physiology
of specific neuropils.
Connectivity in diffuse neuropil
From anatomy one might presume that
in diffuse neuropil each element connects
with almost every other, in an unorganized
fashion. Preliminary electrical recordings
from single elements in the lobster brain do
not entirely support such a conclusion in so
far as functional connections are concerned.
It is true that many neurons form junctions
with a variety of presynaptic elements, but
these are not disorganized.
Central connections of the lobster antennular nerve can serve as an illustration.
Massive stimulation produces the responses
shown in Figure 15. Variation of stimulus
parameters leads to the conclusion that the
beginning of the initial response is produced by direct action of afferent fibers.
The prolongation of the initial response
seems to be the result of activity in interposed excitatory internuncials, and the following drop, the result of inhibitory internuncials. The terminal rise in excitation
shows different properties and must result
either from a second population of internuncials or much more slowly conducting
afferent fibers. Such a patterned sequence
seems less likely to result from random central connections than from some specific
pattern like that illustrated. Further evidence for regular connectivity patterns in
diffuse neuropil in lobsters comes from
stimulating identical nerves on each side of
the body while recording from one central
element. Responses to the two inputs may
be qualitatively different. On the other
hand, stimulation of normal and heteromorphic appendages may cause similar responses, again suggesting specific connectivity within the diffuse neuropil. Although it
has been implied (Horridge, 1961) that
functional connectivity patterns in unstructured neuropil can be explained solely by
physiological mechanisms such as neurotransmittor and neuroreceptor specificity,
it seems more likely that functional patterns reflect appropriate anatomical wiring
diagrams.
ORGANIZATION OF NEUROPIL
A.
B.
PHh
l-'IG. 15. Connectivity in dill use neuropil. A. Intracellular recording of response to antennular
stimulation from unit in spiny lobster brain (tracing). Calibrations, 10 milliseconds, 10 millivolts. B.
Diagram of possible connections from antennular
nerve with schematic post-synaptic potential changes
which, when summed, could produce the above response. Antennular nerve fibers enter from left;
dot and vertical line represent excitatory and inhibitory synapses respectively. See text for further
details.
Connectivity in plexiform neuropil
In the above discussion we were concerned primarily with formal connectivity
between units, and not with geometrical
configurations. In many neuropils, however,
geometrical considerations seem fully as
necessary as formal connectivity diagrams
for an understanding of function. This may
be illustrated by two examples from a system in which spatial or geometrical patterning is unusually important, the visual system.
The first example is the subretinal plexus
of Liniulus as described by Hartline and
his colleagues (see Ratliff, 1961). This is a
plexiform neuropil interposed between the
receptors of the compound eye and the
91
fibers of the optic nerve. Each ommatidium
acts as a unit and serves as a node in the
network of nerve branches forming the
plexus. The discharge from each ommatidium depends upon both the incident light,
which stimulates, and the activity in the
plexus, which depresses. The plexus, therefore, is the site of the first stage of integration of the visual pattern received by the
receptors. Our concern is to show how geometrical parameters play a large role in the
function of this rather simple system.
Non-geometrical properties of the junctions between ommatidia in the Limulus
eye are as follows: 1) All interaction between elements is inhibitory. 2) The degree of inhibition is proportional to the frequency of discharge of the fiber from the
presynaptic ommatidium (when above a
threshold frequency). 3) The effect of one
presynaptic unit may sum with the effect of
others. 4) Interaction is reciprocal, but
need not be symmetrical with respect to effectiveness. 5) With the possible exception
of ommatidia around the borders of the
eye, all seem to have similar junctions with
the neuropil. 6) Under normal circumstances, some ommatidia pairs do not interact, suggesting that not all ommatidia are
directly connected with each other. These
properties permit fairly accurate prediction
of the result of simultaneous visual stimulation of any two receptor elements, but they
do not allow one to say how the message in
the optic nerve, resulting from patterned illumination of the entire eye, differs from
the one that would obtain if interaction in
the plexus were absent. Some additional
statement about geometrical connectivity is
necessary.
An anatomical wiring diagram of the
subretinal plexus is not available, but its
apparent homogeneity suggests either of the
following possibilities: 1) Connectivity in
the eye is truly random, and the probability of interaction between elements is completely unrelated to the position of or distance between elements. 2) Connectivity in
the eye is biased so that the probability of
interaction between elements is a function
of the direction and distance between elements. If the first were true, one might ex-
92
DONALD M. MAYNARD
0.5 mm. at the eye
FIG. 16. Connectivity in f.itnuhis eye. Discharge
of impulses from a single receptor unit in response
to a "step" pattern of illumination in various positions on the retinal mosaic (see inset). The upper
(rectilinear) graph shows the frequency of discharge
of the test receptor, when the illumination was occluded from the rest of the eye by a mask with a
small aperture, minus the frequency of discharge
elicited by a small control spot of light of constant
intensity also confined to the facet of the test receptor. Scale of ordinate on the right. The lower
(curvilinear) graph is the frequency of discharge
from the same test receptor when the mask was removed and the entire pattern of illumination was
projected on the eye in various positions, minus the
frequency of discharge elicited by a small control
spot of constant intensity confined to the facet of
the receptor. Scale of ordinate on the left. (From
Ratliff and Harlline, 1959, J. Gen. Physiol.)
pect visual patterns to be transmitted with
relatively little distortion, but with an increase in the uncertainty or noisiness of the
message. If the second were true, and if interaction increased as distance decreased,
then one might expect intensity discontinuities in the spatial or temporal visual
pattern to be emphasized in the afferent
message. The latter, in fact, appears to be
the case in Limulus (Fig. 16). It seems of
particular interest that insertion of a single,
rather simple geometrical bias in an apparently random system transforms it from one
which increases noise in the afferent message to one which, among other things, reduces redundancy in the message without
great loss of information about the spatial
pattern (Barlow, 1961).
Connectivity in stratified neurofril
The second example is the retina-collicular system in the frog (Maturana, et al.,
I960; Lettvin, et al., 1961). The pertinent
anatomy is summarized first.
Ganglion cells in the retina collect from
rods and cones via interposed bipolars. According to our terminology the bipolars are
radial or vertical elements in the structured
lattice and have relatively small terminal
fields, while the ramifying dendrites of the
ganglion cells in the inner plexiform layer
are horizontal elements. There are five
kinds of ganglion cells. They differ in size,
lateral extent of the dendritic tree, and the
stratum or strata of the plexiform layer in
which the dendrites ramify. Ganglion cell
axons pass from the retina directly to the
colliculus or optic tectum where they presumably separate and terminate in four
horizontal strata according to ganglion cell
type. There is a point-to-point representation of the retina in each layer of the colliculus, and these points are in vertical
alignment. Input to the colliculus, therefore, may be likened to a stack of superimposed geographical maps, each showing a
different feature of the same countryside.
Output originates in dendrites of collicular
cells that pass vertically through the horizontal strata.
Recordings from the terminal arborizations of ganglion cells in the optic tectum
show that the four horizontal layers correspond to five functional classes, one each in
layers 1, 2, and 4, and two (Classes 3 and 5)
in layer 3. Each class responds optimally to
a different aspect of a single complex stimulus. For example, convex edge detectors
(Class 2) do not respond to changes in general light intensity, but do discharge when
a small object (1-3°) with a sharp edge is
moved across a lighter background. All
parameters are important, and if, say, the
background moved with the object or were
darker than the object, the response would
be greatly diminished or absent. These
units have also been called "fly detectors."
The other classes are: Class 1, sustained
edge detectors; Class 3, changing edge detectors; Class 4, dimming detectors; and
Class 5, dark detectors. The functional
properties of the five classes correlate rather
well with the five anatomical ganglion cell
types. If the suggested correlation proves
correct, then the dendrites of edge detectors
and of dimming detectors ramify in differ-
ORGANIZATION OF NEUROPIL
ent strata of the inner plexiform layer, each
collecting from a different set of bipolar terminals.
Only two functional classes of tectal cells
have been described, "newness" detectors
and "sameness" detectors. It is not necessary to go into their detailed properties at
this time, but only to indicate that their receptive fields are larger than that of any
ganglion cell, and that their optimal stimulus, if not more complex, at least involves
new combinations of qualities not found in
the single ganglion cells.
Let us review: Complex integration occurs in the layered neuropil of the retina so
that by the second internuncial neuron,
units recognize and respond optimally to
specific temporal-spatial configurations of
the complex visual pattern within their receptive field. These configurations, or effective attributes of the stimulus, are limited in number and apparently correspond
to the kinds of neurons involved at the pertinent level of integration. The effective attributes become potentially more complex
at each stage of transmission; from receptor
to bipolar, from bipolar to ganglion cell,
from ganglion cell to collicular cell, and so
on. The layered neuropil in this system,
therefore, permits successive separation and
recombination of stimulus attributes while
retaining the original geometrical relations
of the primary sensory layer. Physical as
well as functional separation seems necessary, for each layer maps a separate attribute. Vertical elements select from these
layers and apparently recombine in new
configurations at later stages in the system.
An analogy between the retina or colliculus
and a punch card system seems rather apt.
Both employ a geometric code and both are
concerned with cross-reference and recombination of a limited number of attributes.
.Since the attributes themselves can involve
geometrical configuration of the stimulus,
this may be a system which permits recognition of universals such as "triangle" or
"circle." Further information about the
mechanisms of attribute recognition or sorting is necessary, but if the above interpretations are correct, proper geometry as well as
connectivity and electrical phenomena are
93
required for neural integration in visual
systems.
Complex Patterning in Neuropil
The last section pointed out that many
units in afferent neuropil may act as event
detectors. Such neurons tell whether or not
a pre-determined complex sensory input is
present, but do not describe that input in
the patterns of their own activity. One may
ask whether the reverse occurs: Are there
significant, complex, and relatively autonomous patterns of activity that may be
elicited by simple stimuli in the appropriate neurons? To use Wiersma's phrase
(1952), is there a "push-button" to initiate
appropriate events as well as a signal light
to indicate when events have occurred?
Several analyses of animal behavior suggest that many relatively complex acts are
elicited by specific stimuli and may be
treated as units with all-or-nothing properties. This implies that complex, preset
activity patterns exist in certain areas of the
neuropil and that once initiated, these patterns are like the regenerative action potential in a single nerve fiber and are independent of the initiating stimulus. Direct
recordings of responses meeting these requirements have been described in several
preparations (Wiersma, 1952; Hagiwara
and Watanabe, 1956; Horridge, 1961).
The response of an isolated lobster stomatogastric ganglion to repeated trains of
presynaptic stimuli emphasizes another aspect of such "push-button reflexes" (Fig.
17). The first train elicits no post-synaptic
response; with repetition, facilitation occurs and increasing numbers of post-synaptic units become active. Eventually the pattern of Uie response alters altogether, shifting from an irregular continuous barrage to
periodic bursts that continue for considerable time after the termination of the
stimulus. One is reminded in some ways of
the initiation of seizure activity in the vertebrate brain. Although the mechanism is
not yet clear, it does appear that the ganglion shifts in a stepwise manner from one
functional state into another, and that in
one of these states it produces complex,
maintained responses to rather brief, un-
94
DONALD M. MAYNARD
1'IG. 17. Complex response of isolaletl stomatogastric ganglion of lobster, Homarus americanus, to one
second trains of presynaptic stimuli at 10 per second, repealed at five second intervals. Extracellular
recording electrodes placed distal to the ganglion.
Numbers indicate the number of the stimulus train;
dots on records indicate individual stimuli. There
is no post-synaptic response in the first record, the
spikes shown come from fibers that pass without in-
Structured stimuli. The potentialities for
such activity must lie in the organization of
the ganglion neuropil itself, for extra-ganglionic neural feed-back is absent in this
preparation.
If the above observations can be generalized to apply to central ganglia, then we
may answer our first question as follows:
Yes, there are "push-button reflexes," but
activation requires two operations; first the
switch must be turned on and then the button pushed. It is not sufficient for a given
neural system to have the structural organization requisite for complex, semi-autonomous activity; the system must also be
in the proper physiological state in order to
respond to the specific, triggering stimulus.
If much of an organism's behavior is organized around push-button responses, then
such a priming or activating mechanism
gives an added degree of freedom, and
should facilitate adaptive behavior. Several
functional states with corresponding differ-
terruption through the ganglion, and consequently
represent direct responses to the electrical stimuli.
Two post-synaptic impulses occur with the 6th
train of stimuli, three with the 8th, 15 with the
10th, and bursts with the 12th. Bursts continued
for several seconds beyond the records shown, and
the changed state of the ganglion continued still
longer, as shown by test trains of stimuli. Calibration, 0.5 second.
ent responses would extend still further the
functional potentialities of a limited number of neurons with rigid, preset activity
patterns. The anatomical and physiological
properties of the hypothetical priming
mechanism remain to be specified in invertebrates.
Braiyi Waves
A discussion of neuropil organization
should not be concluded without reference
to the rhythmic, often sinusoidal potential
oscillations called "brain waves." These are
usually recorded by surface electrodes from
specific masses of nerve tissue. Although
there is no complete agreement on their
genesis or significance, most would concede
that the waves are not envelopes of action
potentials, that they reflect potential oscillations in neurons, that they appear in typical form only in masses of nerve tissue, and
that they represent some synchronized activity in large numbers of units. The alpha
95
ORGANIZATION OF NEUROPIL
rhythm of the vertebrate cortex is perhaps
the best known example, but analogous sinusoidal oscillations have been recorded
from olfactory bulb, cerebellum, optic ganglia of insects, and recently, the ocelli of
some insects (see Gerard and Young, 1937;
Crescitelli and Jahn, 1942; Bullock, 1945b;
Bum and Catton, 1956; Ruck, 1961). The
structures giving rise to the above oscillations differ rather markedly in general appearance, but they do have one factor in
common. In all cases the tissue appears to
be structured neuropil. Smooth oscillations
apparently have not been recorded from unstructured neuropil. If the synchronization
characteristic of brain waves is mediated by
some mechanism not involving action potentials and synapses, possibly electrotonic
potentials or chemical agents, then such
geometric organization may have greater
significance than originally postulated.
SUMMARY
Much of the factual information and
many of the ideas presented in the above
discussion are neither new nor original.
Nevertheless, they are worth considering
for they stress several points of some importance:
1. Although acceptable for general conversation, neuropil is unsatisfactory when unmodified as a technical term in that it does
not refer to a commonly accepted, well-defined structure. Its original anatomical connotations have broadened to include functional aspects, and thus it logically encompasses the entire portion of the nervous system devoted to neural integration, cell
bodies, processes, and all.
2. Neuropil structure is complex, but not
haphazard. Similar patterns reappear in
widely divergent organisms. Where adequate analyses are available, the structural
pattern proves to have functional significance. A preliminary classification on the
basis of fiber configuration divides all neuropil into four categories: unstructured
plexiform, unstructured diffuse, structured
glomerular, and structured stratified. Geometrical configurations as well as connectivity diagrams are important.
3. Neuropil is the site of most complex in-
tegrative neural activity. The mechanisms
of such interaction, however, are not limited to those we know from peripheral junctions and nerve processes. They also involve
other properties, many as yet inadequately
studied. Indeed, it seems likely that specific
forms of neuropil may have their own
unique functional characteristics.
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