1Neuroscience Program and 2Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136; and 3Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
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ABSTRACT |
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Baccus, Stephen A., Brian D. Burrell, Christie L. Sahley, and Kenneth J. Muller. Action Potential Reflection and Failure at Axon Branch Points Cause Stepwise Changes in EPSPs in a Neuron Essential for Learning. J. Neurophysiol. 83: 1693-1700, 2000. In leech mechanosensory neurons, action potentials reverse direction, or reflect, at central branch points. This process enhances synaptic transmission from individual axon branches by rapidly activating synapses twice, thereby producing facilitation. At the same branch points action potentials may fail to propagate, which can reduce transmission. It is now shown that presynaptic action potential reflection and failure under physiological conditions influence transmission to the same postsynaptic neuron, the S cell. The S cell is an interneuron essential for a form of nonassociative learning, sensitization of the whole body shortening reflex. The P to S synapse has components that appear monosynaptic (termed "direct") and polysynaptic, both with glutamatergic pharmacology. Reflection at P cell branch points on average doubled transmission to the S cell, whereas action potential failure, or conduction block, at the same branch points decreased it by one-half. Each of two different branch points affected transmission, indicating that the P to S connection is spatially distributed around these branch points. This was confirmed by examining the locations of individual contacts made by the P cell with the S cell and its electrically coupled partner C cells. These results show that presynaptic neuronal morphology produces a range of transmission states at a set of synapses onto a neuron necessary for a form of learning. Reflection and conduction block are activity-dependent and are basic properties of action potential propagation that have been seen in other systems, including axons and dendrites in the mammalian brain. Individual branch points and the distribution of synapses around those branch points can substantially influence neuronal transmission and plasticity.
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INTRODUCTION |
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Identifying the changes responsible for learning
in a given system is a complex problem. Although a number of mechanisms
of synaptic plasticity have been identified (Bear and Malenka
1994), it is less clear how these mechanisms relate to learning
and operate in systems with thousands or millions of neurons. Even in
invertebrates, with relatively fewer cells, hundreds of neurons can
participate in multiple behaviors and scores of cells can modify their
activity during learning (Wu et al. 1994
; Zecevic
et al. 1989
).
In contrast, it has been found in the leech that a single interneuron,
the S cell, is required for a form of nonassociative learning
(Modney et al. 1997; Sahley et al. 1994
).
The leech can modify its reflexes including reflex shortening
(Debski and Friesen 1985
; Lockery and Kristan
1991
). The leech defensively shortens in response to stimuli
that activate mechanosensory neurons including P cells. A prior noxious
stimulus will sensitize this behavior, increasing the contraction
produced by the weaker shortening stimulus (Boulis and Sahley
1988
). The S cell is necessary for sensitization of the
shortening reflex. Killing one S cell in the electrically coupled chain
of S cells, or cutting its axon, abolishes this form of learning while
leaving the basic reflex intact. The S cell is also required for
complete dishabituation, another form of nonassociative learning
(Sahley et al. 1994
). The single S cell in each ganglion
is excited by sensory neurons, is electrically coupled through
nonrectifying junctions to a pair of coupling interneurons, the C cells
(Muller and Scott 1981
), and in turn excites motoneurons
active throughout the entire length of the leech (Gardner-Medwin
et al. 1973
). Therefore, understanding changes in sensory
transmission to the S cell is important for understanding the cellular
mechanisms of learning.
The structure of neurons, including leech sensory cells, can influence
synaptic transmission in a dynamic manner. One mechanism that depends
on axonal branching and influences transmission is conduction block,
which enables an impulse to activate some synapses within a neuron but
not others, reducing transmission in a stepwise manner (Gu
1991; Macagno et al. 1987
; Muller and
Scott 1981
). Recently it was found that reflection of action
potentials, a related mechanism, allows single branch points of an axon
to increase transmission (Baccus 1998
). Reflection
occurs when impulses are sufficiently delayed as they travel through
branch points. Thus when the delay exceeds the refractory period of the
conducting axon, the impulse propagates backward as well as forward
from the branch point, creating a reflection. In leech mechanosensory neurons, the reflected impulse activates presynaptic terminals on one
side of a branch point a second time, facilitating transmission from
those synapses and not others (Fig. 1). Repetitive activity can cause
these branch points to change between the states of reflection,
conduction block, and the state where impulses pass through the branch
point without reflection, known as full conduction (Baccus
1998
), but the separate effects of these three states on
synaptic transmission has not been reported.
The P to S synaptic connection, which has been cited as an unpublished
observation (Shaw and Kristan 1995), is characterized here. To understand the extent that a neuron's morphology can dynamically influence synaptic transmission, the effect of presynaptic reflection and conduction block on transmission at this synapse is examined.
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METHODS |
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Preparations and electrophysiology
Leeches Hirudo medicinalis were obtained from a supplier
(Leeches USA, Westbury, NY) or raised in the laboratory. Preparations were dissected as described (Baccus 1998;
Nicholls and Baylor 1968
). The bath contained leech
saline composed of (in mM) 115 NaCl, 4 KCl, 1.8 CaCl2, and 10 Tris maleate (pH 7.4 adjusted with NaOH) (Kuffler and Potter 1964
). For experiments using
elevated [Ca2+]-[Mg2+] saline, the bath
contained (in mM) 15 Ca2+ and 18 Mg2+, each replacing Na+
mole for mole. Experiments were conducted at room temperature (20-22°C). Preparations consisted of a chain of two segmental ganglia with one ganglion attached to the skin by the dorsal peripheral nerve root. Intracellular recordings were made in the adjacent ganglion, which was either anterior or posterior to the ganglion attached to the skin, using sharp microelectrodes (20-25 M
) filled with 4 M potassium acetate. In some experiments the recording electrode
in the P cell contained 1 M tetraethylammonium chloride (TEA+), which diffused into the cell and
broadened the action potential by blocking potassium channels. The
broader action potentials increased transmitter release in a graded
fashion for the purpose of testing whether a connection was
physiologically monosynaptic (see RESULTS). Medial P cells
were identified by their size, shape, and position within the ganglion.
The S cell was identified by its position and action potential because
it is the only small cell in the central glial packet that produces a
large, fast overshooting action potential in response to weak
stimulation of the segmental nerve cord or stimulation of the soma.
Resting membrane potentials for P cells and S cells ranged between
41
and
55 mV. P cells were peripherally stimulated using a suction
electrode applied to the anterior or posterior minor receptive fields
in the dorsal skin, which are contiguous with the central, major
receptive field as shown in Fig.
1A. In some experiments, to
prevent the S cell from firing in response to large synaptic
potentials, continuous hyperpolarizing current was injected into the
soma. Values of n indicate number of preparations, not
measurements.
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Laser axotomy
When measuring the S cell synaptic potential produced by
peripherally stimulating the P cell, it was necessary to eliminate the
contribution from P, touch (T), and nociceptive (N) mechanosensory neurons in the ganglion attached to the skin. This is because these
sensory neurons also innervated the area of skin being stimulated and
synapsed on the S cell in the adjacent ganglion where the recording was
made. Therefore the peripheral axons of these neurons were cut with a
laser microbeam as described (Baccus 1998; Gu et
al. 1989
). In brief, the cells were pressure injected with 0.17 M 6-carboxyfluorescein, neutralized to pH 7.4 with KOH, and their
peripheral axons cut by irradiation for a few seconds with 488 nm light
from a 20-mW argon laser attenuated two- to fourfold with neutral
density filters.
Histology
In one series, S cells were injected with Lucifer yellow dye by
either pressure or iontophoresis and P cells were pressure injected
with either 2% horseradish peroxidase (HRP) (Muller et al.
1981) or 5% (wt/vol) biocytin (Horikawa and Armstrong
1988
). Cells injected with HRP were processed as described by
incubation in leech saline saturated with diaminobenzidine (DAB)
(Gu 1991
; Macagno et al. 1987
). Biocytin
injected cells were processed as described (Peinado et al.
1993
) by incubation with rhodamine-conjugated streptavidin at a
concentration of 0.2% wt/vol for 3 h. To count apparent synaptic
contacts, ganglia were viewed at ×400 with a ×40 oil-immersion
objective having a 1.3 numerical aperture. For ganglia stained using
HRP, by balancing fluorescence epi-illumination and transmitted light
it was possible simultaneously to view the Lucifer yellow and
HRP-stained cells. To ascertain whether cells appeared to be in direct
contact, the focal plane was adjusted during viewing. Photomicrographs
were not used to identify synaptic contacts. Preparations stained with
rhodamine required alternate viewing through filters optimized for
fluorescein, which allowed simultaneous viewing of Lucifer yellow and
rhodamine and filters optimized for rhodamine. For this reason,
counting synaptic contacts using HRP was preferable. In a few cases,
fibers crossed each other in close proximity without an apparent
presynaptic varicosity; these were not counted as a synaptic contact.
In a complementary series, 5% lysinated tetramethylrhodamine dextran (104 Da, Molecular Probes) dissolved in 0.2% Fast Green FCF in 0.1 M KCl was injected into the S cell and 5% Lucifer yellow into the P cell. The dextran did not cross gap junctions and was excluded from C interneurons, permitting the identification of apparently direct contacts between the P and S cells. Preparations were fixed in paraformaldehyde, mounted in Fluormount (Gurr), and viewed with a laser scanning confocal microscope (Fluoview, Olympus) with fluorescein and rhodamine optics using a ×20 objective.
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RESULTS |
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Synaptic transmission from P cell to S cell
Activation of the P cell produced a synaptic potential in
the S cell of 1.1 ± 0.1 (SE) mV (n = 17) (Fig. 2). The synaptic potential had
an excitatory early component and later more variable components that
were excitatory, inhibitory, or both. To determine whether the synaptic
potential was physiologically monosynaptic, or direct, the excitatory
postsynaptic potential (EPSP) was measured with the ganglion bathed in
physiological saline containing 1.8 mM Ca2+
and the bath was switched to saline containing 15 mM Ca2+
and 18 mM Mg2+ (Nicholls and Purves 1970).
This solution reduced postsynaptic excitability without reducing
transmission at chemical synapses thereby eliminating or reducing
polysynaptic transmission involving spiking interneurons. The early
component of the synaptic potential had a constant latency and
persisted in high [Ca2+]-[Mg2+] saline,
indicating it was direct (Fig. 2, A and
B). The later, variable components were eliminated in
high [Ca2+]-[Mg2+] saline, indicating they
were polysynaptic. P cells also produced a synaptic potential of ~0.5
mV in S cells in adjacent ganglia (n = 2, data not
shown). In contrast to the touch mechanosensory cell (T cell) to S cell
connection, there was no electrical coupling detectable between the P
and the S cells because strong hyperpolarizing current (2 nA) injected
into either cell did not pass to the other (n = 4, data not shown).
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As an additional test to confirm that the early component was effectively monosynaptic, 1 M TEA+ was included in the presynaptic microelectrode and allowed to diffuse into the cell. TEA+ prolongs the action potential in a graded fashion by blocking presynaptic K+ channels in a concentration dependent manner, thereby increasing transmitter release. Monosynaptic potentials increase gradually as the concentration of TEA+ increases, whereas polysynaptic potentials do not increase or increase in a stepwise manner as an interneuron fires multiple times. During repeated trials, the presynaptic action potential recorded at the soma broadened (Fig. 2, C-E). As TEA+ diffused to presynaptic terminals, the early component of the synaptic potential increased steadily, confirming that it was monosynaptic (n = 6).
These criteria to verify that a connection is monosynaptic cannot rule
out the presence of an intervening electrically coupled interneuron
(Deschênes and Bennett 1974; Muller and
Scott 1981
). In fact, many contacts between the P and S cells
are mediated by two coupling interneurons (C cells) that are strongly
electrically coupled to the S cell (see Fig. 6) as described
previously for T cells. Such a connection is functionally monosynaptic,
is not distinguished physiologically from a true monosynaptic
connection, and is termed here a direct connection (Muller and
Scott 1981
) included with the monosynaptic connection.
The pharmacology of the P to S synapse was determined by applying
CNQX, which blocks glutamate receptors of the AMPA/Kainate type. CNQX
blocks input from the P cell to the swim interneuron TR1
(Thorogood and Brodfuehrer 1995) and blocks the chemical
monosynaptic connection between the P cell and the Anterior Pagoda (AP)
cell (Wessel et al. 1999
). The S cell synaptic potential produced by the P cell was eliminated by 25 µM CNQX (Fig.
3). CNQX blocked both the monosynaptic
component isolated using high
[Ca2+]-[Mg2+] saline (Fig. 3,
A-C; n = 4) and the polysynaptic
component recorded in physiological saline (Fig. 3,
D-F; n = 4).
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Branch point conduction states
The conduction state of P cell central branch points was
determined by stimulating the minor receptive field and recording the
impulses that arrived at the P cell soma. Reflection was distinguished from full conduction by stimulating the periphery twice at levels above
threshold to generate a pair of impulses in the periphery (Baccus 1998). When the branch point was fully
conducting, the second impulse reached the soma (Fig.
4A). Otherwise, if the first impulse reflected, the reflection traveled back toward the periphery, colliding with the second impulse and preventing it from reaching the
soma (Fig. 4B). The absence of the second impulse thus
indicated that the branch point was reflecting. The second impulse
could always be recovered by depolarizing the soma to produce full
conduction (Fig. 4D) or by hyperpolarizing the soma to
produce conduction block (data not shown). This confirmed that the
absence of the second impulse was not because the peripheral stimulus
was below threshold (Baccus 1998
). Additionally, action
potentials that reflected had a longer initial rising phase, or
"foot", indicating a delay in traveling through the branch point
(Baccus 1998
) (Fig. 4B, arrow). Reflection,
full conduction, and conduction block (Fig. 4C) occurred at
rest in different preparations as previously described and all states
occurred in all cells examined (n = 12) (Baccus
1998
). The conduction states were changed by injecting the soma
with steady depolarizing or hyperpolarizing current, or in some cases
depolarizing current pulses, to change membrane potential by <10 mV.
Pulses were used to produce full conduction in some cells that
exhibited reflection at rest (Fig. 4D), including those
cells that with steady depolarization fired repetitively in response to
a skin stimulus (data not shown).
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Effect of reflection and conduction block on synaptic transmission
The effect of presynaptic reflection and conduction block on
transmission to the S cell was measured by stimulating the anterior or
posterior P cell minor fields and injecting current into the P cell
soma to change the branch point conduction state. This level of current
injection (<0.5 nA) does not directly affect transmission from the P
cell to the AP cell (Baccus 1998). Indeed, there was no
change in transmission with increased current injection except when the
conduction state of the P cell branch point changed. Figure
5A shows the synaptic
potential recorded in the S cell when the P cell anterior branch point
was in the state of full conduction, activating all presynaptic
terminals, but only once. Reflection increased transmission by firing a
subset of synapses rapidly a second time, causing facilitation (Fig.
5B). Conduction block decreased transmission by activating
only a subset of synapses (Fig. 5C). Other sensory cells
that innervated the stimulated region of skin had been axotomized with
a laser (see METHODS). However, to confirm that the
synaptic potential in the S cell was produced by the P cell and not by
another presynaptic cell excited by the skin stimulus, an impulse was
generated in the P cell soma just before the peripheral stimulus. This
outgoing impulse produced an early synaptic potential and collided with the incoming peripheral impulse, eliminating the synaptic potential at
the time it would otherwise have been seen, indicated by an arrow in
Fig. 5D.
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Table 1 shows the effect of anterior and posterior reflection and conduction block in the P cell on the magnitude of the S cell synaptic potential. On average, anterior reflection increased the synaptic potential 1.8 times, whereas anterior conduction block reduced the synaptic potential to 0.55 times the value during full conduction (n = 4). Posterior reflection increased transmission 2.31 times and posterior block decreased transmission to 0.51 times that during full conduction (n = 5). The conduction state of two different branch points can thus change synaptic transmission between different levels.
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It has been previously reported that in ~12% of medial P cells, the
anterior and posterior thin axons join directly together before forming
a single central branch point with the thick axon (Gu
1991). Impulses can thus pass directly from anterior to
posterior thin axon and conduction block at the single branch point
between thin and thick axons does not reduce transmission. Two such P cells were encountered (Table 1, asterisks) and were excluded from
analysis. Reflection did however increase transmission from these cells
as would be expected.
During conduction block, by measuring the size of the synaptic
potential relative to its size when all terminals are activated, the
functional distribution of the synapse on both sides of the central
branch points can be measured (Gu 1991; Gu et al.
1991
; Macagno et al. 1987
). These values
indicate that on average the P to S synapse was divided functionally so
that one-half of the synaptic potential was produced anterior of the P
cell branch points and one-half was produced posterior of the branch
points (49 ± 7% anterior, n = 10).
P cell to S cell synaptic contacts are spatially distributed
To examine directly the spatial distribution of the synapse,
apparent synaptic contacts of the P cell with the S cell were seen by
injecting separate intracellular tracers into the presynaptic and
postsynaptic cells. Previous studies using HRP as a marker have
confirmed that contacts of the P cell identified in the light microscope are indeed synapses when examined in the electron microscope (Macagno et al. 1987; Muller and McMahan
1976
).
When Lucifer yellow was injected in the S cell soma, the small molecular tracer filled the S cell and the two lateral C cells (Fig. 6A). HRP was injected into the P cell to label all processes of the cell, including presynaptic terminals. Apparent synaptic contacts could be seen in the light microscope (Fig. 6, B and C). The P cell made 56 ± 6 (n = 4) contacts within a ganglion; 52 ± 0.5 (SD) % of the contacts were anterior of the P cell branch points,which agreed with the physiological results that the synapse was approximately equally distributed on both sides of the branch points. Because the synapse was spatially distributed (see Fig. 1A), reflection and conduction block at both branch points influenced transmission to the S cell.
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Because Lucifer yellow easily passes between the S cell and C cells, processes of these cells could not clearly be distinguished in all regions of the ganglion. The S cell was alternatively filled with lysinated tetramethylrhodamine dextran, which does not cross electrical synapses; the P cell was filled with Lucifer yellow (Fig. 7A). When the P and S cells were filled in this manner, apparent synaptic contacts were observed between them (Fig. 7B), indicating that P cells make monosynaptic contact with S cells.
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DISCUSSION |
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These results demonstrate that branch points can have multiple
dynamic effects on transmission from a single neuron, extending the
known influence that neuronal morphology has over signaling and
plasticity. P cell branch points control transmission produced by
impulses arising in a single branch while not affecting transmission by
impulses arising in other branches. Reflection and conduction block
only affect impulses arising in the minor receptive fields, as impulses
from the major field or the soma cross the central branch points in the
direction of safe propagation from the thick to the thin axons
(Baccus 1998; Gu 1991
; Macagno et
al. 1987
). Anterior and posterior branch points can also have
different conduction states at the same time (Mar and Drapeau
1996
).
During conduction block, synaptic transmission decreases in a number of
systems, including cat 1A afferents (Henneman et al. 1984), the crustacean neuromuscular junction (Grossman
et al. 1973
; Parnas 1972
), and hippocampal CA1
pyramidal neurons (Debanne et al. 1997
). Although
reflection has been observed in other systems (Ramón et
al. 1975
; Rinzel 1990
; Tauc 1962
;
Velte and Masland 1999
), its effect on synaptic
transmission has only recently been reported (Baccus
1998
). In mitral cells of the mammalian olfactory bulb, both
conduction block (Chen et al. 1997
) and reflection (Chen
and Shepherd, personal communication) have been observed for impulses
that initiate in dendrites. Dendrites of these cells are both pre- and
postsynaptic, making dendrodendritic synapses. It is therefore expected
that reflection will increase dendritic synaptic transmission.
In order for changes in branch point conduction state to act as a
useful mechanism for synaptic plasticity, the occurrence of reflection
and conduction block must be controlled. Conduction states at a P cell
branch point can be controlled by activity. Repetitive firing
hyperpolarizes the membrane by activating a Ca2+-dependent
K+ conductance and the
Na+-K+ ATPase. This
hyperpolarization can produce reflection and conduction block. After
activity, the cell will recover and return to its original conduction
state (Baccus 1998; Jansen and Nicholls
1973
).
Activity can also control conduction state in other systems. Activity
produces conduction block both in lobster motoneuron axons and
hippocampal CA1 dendrites (Grossman et al. 1979;
Spruston et al. 1995
). In contrast, in
Aplysia C2 sensory neurons, activity can relieve conduction
block by electrotonic summation of failed action potentials
(Weiss et al. 1986
). In addition to activity, other
mechanisms can also influence conduction state. For example, conduction
block in hippocampal CA1 axons is gated by a K+
conductance controlled by changes in membrane potential (Debanne et al. 1997
). Conduction state can also be changed by
neuromodulatory processes. In the leech, 5-HT changes the conduction
state of mechanosensory cells, including P cells (Mar and
Drapeau 1996
).
As opposed to other mechanisms such as activity-dependent facilitation
and short-term synaptic depression, which can change transmission in a
graded manner, the mechanisms described here produce discrete changes
in transmission. These discrete changes are large enough to be
distinguished from the background variability in synaptic transmission.
In a sensory neuron, discrete levels of transmission can encode
threshold changes in sensory input. A change between discrete levels is
more easily detected by a postsynaptic cell and this change is less
sensitive to fluctuations in synaptic transmission. In this respect,
change between discrete levels of transmission is analogueous to
digital encoding of signals. Stepwise changes in synaptic transmission
have also been observed during long-term plasticity at mammalian
central synapses (Murthy 1998).
The mechanism of reflection operates when an impulse in one region of a
cell produces a delayed flow of current through a second region of
membrane, and the resulting depolarization of the first region of
membrane outlasts its refractory period. Although this delay can be
produced by an abrupt increase in membrane area as may occur at a
branch point or at the soma, delays in membrane currents can also be
produced by spatial nonuniformities in membrane conductances.
Compartmental models indicate that in P cells, such variation in
membrane conductances is not necessary to account for the presence of
reflection and conduction block at rest, and that morphology is
sufficient to produce reflection (Baccus 1998). However,
models based on experimental results further indicate that when
reflection is produced after repetitive firing, spatially nonuniform
Ca2+-dependent K+
conductances and Na+/K+
ATPase currents develop as a result of differential accumulation of
Ca2+ and Na+ in different
diameter axons and that these spatial properties tend to maintain the
cell in a reflecting state (Baccus 1998
). In neocortical
pyramidal cells, impulses arising in the region of the soma can lead to
slower Ca2+ action potentials in apical dendrites
that reexcite the soma and axon. These Ca2+
action potentials and the resultant reflections can be gated by
synaptic inputs (Larkum et al. 1999
).
In this study, single brief stimuli were used to measure transmission
during full conduction, reflection, and conduction block. Single brief
stimuli have also been used during behavioral experiments to measure
sensitization and dishabituation (Modney et al. 1997; Sahley et al. 1994
). During trains of impulses however,
the effects of reflection on transmission will be more complex. If the
interval between peripheral impulses decreases to less than twice the
conduction time, reflected impulses will begin to collide with
peripheral impulses, thus reducing central transmission. Additionally,
at increased firing frequencies, synaptic transmission from P cells facilitates (Muller and Nicholls 1974
) so that at higher
frequencies, each fully conducting impulse will produce greater
transmission. This may reduce the difference in transmission produced
by reflection as compared with full conduction. Therefore the greatest
enhancement by reflection will be produced when reflection occurs at
low firing frequencies.
Although the effects of conduction state examined here involve transmission in the same ganglion that contains the soma, P cells, like other mechanosensory neurons, form synaptic connections in several ganglia (Fig. 1A). Because reflection and conduction block change the way impulses propagate throughout the presynaptic cell, these mechanisms will affect transmission to multiple S cells.
Depletion of 5-HT throughout the leech eliminates sensitization
and disrupts dishabituation, which are behavioral effects very similar
to killing the S cell, although the S cell is not itself serotonergic
(Ehrlich et al. 1992). Noxious stimuli of the type that
produce sensitization also activate cells that release 5-HT
(Sahley 1988
). Because one effect of 5-HT is to relieve
conduction block in mechanosensory neurons, 5-HT may increase sensory
transmission to the S cell during nonassociative learning.
Changes in transmission are thought to underlie various types of learning. Neuronal branching pattern can dynamically change transmission from subsets of synapses in multiple ways under cellular control; these properties are sufficient to influence synaptic and behavioral plasticity.
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ACKNOWLEDGMENTS |
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We thank Drs. J. Barrett, R. Bookman, A. Chen, D. Johnston, W. Nonner, and R. Rotundo for helpful discussions, and Dr. A. Caicedo for help in use of Dr. S. Roper's confocal microscope.
This work was supported by National Institute of Neurological Disorders and Stroke Grant RO1-NS-34927 to K. J. Muller and C. L. Sahley. S. A. Baccus was supported by a Howard Hughes Medical Institute Predoctoral Fellowship and B. D. Burrell by National Research Service Award F32-NS-10065.
Present address of S. A. Baccus: Dept. of Molecular and Cell Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138.
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FOOTNOTES |
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Address for reprint requests: K. J. Muller, Dept. of Physiology and Biophysics, R-430, RMSB 5092, University of Miami School of Medicine, 1600 NW 10th Ave., Miami, FL 33136.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 June 1999; accepted in final form 10 November 1999.
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REFERENCES |
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