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First published online July 20, 2007
Journal of Experimental Biology 210, 2730-2742 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001784
The functional architecture of the shark's dorsal-octavolateral nucleus: an in vitro study
1 The Otto Loewi Center, the Inter University Institute, Eilat,
Israel
2 The Gonda Interdisciplinary Brain Research Center, Bar Ilan University,
Ramat Gan, Israel
3 Department of Zoology and Centre for Neuroscience, University of Otago,
Dunedin, New Zealand
4 Department of Neurobiology, the Institute of Life Sciences, Hebrew
University, Jerusalem, 91904, Israel
* Author for correspondence (e-mail: Yarom{at}vms.huji.ac.il)
Accepted 16 May 2007
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Using extracellular and intracellular recordings we compared the dynamics and spatiotemporal organization of the electrosensory afferent nerve and parallel fiber inputs to the DON. The afferent nerve has a low threshold and high conduction velocity; a stimulus that recruits a small number of fibers is sufficient to activate the principal neurons. The excitatory postsynaptic potential in the principal cells evoked by afferent nerve fibers has fast kinetics that efficiently reach the threshold for action potential. In contrast, the parallel fibers have low conduction velocity, high threshold and extensive convergence on the principal neurons of the DON. The excitatory postsynaptic response has slow kinetics that provides a wide time window for integration of inputs.
The highly efficient connection between the afferent nerve and the principal neurons in the DON indicates that filtration occurring in the DON cannot be mediated simply by summation of the parallel fibers' signals with the afferent sensory signals. Hence we propose that the filtering may be mediated via secondary neurons that adjust the principal neurons' sensitivity to afferent inputs.
Key words: dorsal-octavolateral, nucleus, electrophysiology, electroreception, shark, synaptic interactions
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Introduction |
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). However, the
suggestion that cerebellar-like hindbrain circuitry performs this function in
the electrosensory system of elasmobranches has been supported by experimental
evidence for more than two decades
(Bodznick et al., 2003;
Montgomery, 1984;
Montgomery and Bodznick,
1994).
In the electrosensory system of elasmobranches the dorsal-octavolateral
nucleus (DON) is the site where unexpected sensory input is retrieved from the
overall sensory information. The DON is a structure in the medulla that serves
as the first stage that processes sensory information. It receives direct
inputs from the peripheral electroreceptors – the ampullae of Lorenzini
– via the afferent nerve (Aff) known as the superficial
opthalmic ramus of the anterior lateral line nerve. The information conveyed
by the afferent nerve comprises the electrical field generated by the
elasmobranch's own movements (reafferent signals) and electrical fields
generated by external sources. In the output of the DON, transmitted by the
axon of the ascending efferent neurons (AENs), the reafferent signals are
significantly attenuated, and information about electrical fields generated by
the external sources is enhanced. Hence, it has been hypothesized that the DON
predicts self-generated electrosensory information and subtracts it from the
incoming sensory information, thus leaving essential sensory information about
possible prey intact (Montgomery and
Bodznick, 1999; Bodznick et
al., 2003).
The DON is a cone-shaped structure divided into three layers organized
along the dorso-ventral axis. The principal cell layer, located at the center
of the DON, is composed of the AENs. The AENs have extensive dendritic
arborizations, spreading ventrally and dorsally to form the ventral and dorsal
dendrites, respectively. The ventral dendrites of the AEN receive monosynaptic
input from the terminals of the Aff, thus forming the afferent fiber layer.
The dorsal dendrites spread dorsally into the molecular layer where they are
innervated by the parallel fibers (PF)
(Paul and Roberts, 1977).
Inhibitory interneurons have been described in both the Aff layer and the
molecular layer (Montgomery and Bodznick,
1994; Paul et al.,
1977). The PF of the DON, which arise from a mass of granular
cells forming the dorsal granular ridge (DGR), carry three categories of
information: proprioceptive information, reflecting body movements; descending
electrosensory information and corollary discharge signals associated with the
motor commands (Bell,
2002).
The term `adaptive filter' has been used to denote the ability of the DON to filter out the irrelevant, namely the expected, sensory information. It has been suggested that plasticity in the PF synapses underlies the adaptive capabilities of the filtration process. Specifically, when PF input precedes or coincides with AEN action potential, the strength of the PF synapse is supposedly decreased, allowing adaptive filtration of the electrosensory information. This adaptive filtering, which is anti-Hebbian in nature, decreases AEN output in correlation with the activity in the PF.
Knowledge about functional signal processing in the DON has been derived from in vivo preparations. In order to gain insight into the cellular and network mechanisms underlying the adaptive filtering properties of the DON, we developed a unique in vitro preparation of a shark (Iago omanensis) brainstem, consisting of the DON and the afferent nerve originating at the Ampullae of Lorenzini. This preparation allows us to examine anatomical and electrophysiological properties of the intact DON and its afferents. Specifically, we characterize the temporal and spatial propagation of local field potential (LFP) reflecting inhibitory and excitatory responses to stimulation of the Aff and the PF.
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Surgical procedure
Sharks were immobilized by cooling to 8–10°C. Following
decapitation, at the level of the first gill, the cartilaginous skull
containing the brain was removed and immersed in a 7–10°C shark
Ringer solution. As shown in Fig.
1A, the cartilaginous bone was carefully removed to expose the
cerebellum (green), the brain stem (white), the spinal cord (yellow) and the
cranial nerves. Subsequently the brainstem and the Aff were isolated from the
level of the dorsal granular ridge (DGR,
Fig. 1B,C) to the level of
cervical vertebrate (c3). The isolated brainstem was incubated in the
experimental chamber, which was continuously superfused with aerated cold
(15°C) Ringer solution for at least 1 h prior to recording. During
incubation, the temperature was gradually increased to 20°C, the
temperature of the Iago omanensis natural habitat.
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),
contained (in mmol l–1) 280 NaCl, 6 KCl, 5
CaCl2-2H2O, 3 MgCL2-6H2O, 0.5
Na2SO4, 1
NaH2PO4-12H2O, 8 NaHCO3, 350 urea,
72 trimethylamine N-oxide dehydrate (TMAO, Sigma, Rehovot, Israel), 5
glucose, 0.75 polyvinylpyrrolidone (PVP-40T, Sigma). The pH of this modified
Ringer solution was 7.4–7.6.
The isolated preparation is shown in Fig. 1B,C. Continuous lines outline the DON (black) and the DGR (yellow). A schematic illustration of the isolated preparation (Fig. 1D) was reconstructed from images such as those shown in Fig. 1B,C. The locations of the stimulating and the recording electrodes were marked. To the best of our knowledge this is the first isolated intact preparation of the DON. This unique preparation maintains the integrity of the network, which is extremely advantageous over the commonly used in-vitro slices when studying network connectivity and integration in the DON.
Electrophysiological recording
4–7 M
electrodes filled with 2 mol l–1 NaCl
were used to measure local field potentials (LFP) via an amplifier
(Axoclamp 2A, Foster City, CA, USA). Homemade bipolar electrodes were prepared
from Teflon coated, 75 µm diameter silver wires. The wires were wrapped
together so that the two exposed tips were separated by 100 µm. These
stimulating electrodes were placed either on the ipsilateral afferent nerve or
on the dorsal surface of the DON. Since the entire preparation is continuously
immersed in the physiological solution the tips of both electrodes were also
immersed. The stimulating electrodes were driven by 0.1 ms pulses, of various
intensities. In experiments where bicuculline was used to block
GABAA receptors, it was added to the external solution reaching a
final concentration of 50–100 µmol l–1, and
recording started 30 min after initial bicuculline application. Sharp glass
pipettes filled with 2 mol l–1 potassium acetate (30–60
M) were used for intracellular recordings. For intracellular labeling,
the electrodes were filled with 5% neurobiotin (Sigma) diluted in 1 mol
l–1 potassium acetate. These electrodes had a d.c. resistance
of 60–80 M. Positive current pulses of 0.5–1.2 nA in
amplitude, 50 ms duration repeated at 1 Hz for 10–60 min were used to
deliver the neurobiotin into the cells.
Histological procedures
Following neurobiotin injection, the isolated brain stem was fixed for 24
h, imbedded in a gelatin block (made of 7% gelatin solution) and incubated for
48 h in the fixative solution. The fixative solution consisted of 4%
formaldehyde diluted in 730 mmol l–1 phosphate buffer
solution (PBS). 200 µm cross-section slices were cut by a vibratome and
immersed in PBS for 30 min. The free-floating sections were `prebleached' by
soaking for 20 min in 0.5% H2O2 in PBS. The sections
were then washed 3x and incubated in ABC solution for 4–6 h [2
drops A+2 drops B in 5 ml PBS (ABC Standard Elite Kit, Vector Labs,
Burlingame, CA, USA)]. After thorough washing in PBS, the sections were
incubated for 10–15 min in a solution containing 5 mg diaminobenzidine
(DAB; Sigma), 20 ml PBS, 1 ml 0.3% NiCl and 5 µl
H2O2. The reaction was stopped by washing in PBS.
Sections were mounted onto chrome alum gelatin-coated slides and left to dry
overnight. Slides were then immersed in a series of alcohol and xylene for
dehydration and clearness.
Data analysis
Data acquisition board (PCI-MIO-16XE-10, National Instruments, Austin, TX,
USA), controlled by software written in LabView (National Instruments), was
used to sample the data at a rate of 10 000 samples s–1 and
stored for offline analysis. The LFP recorded traces were averaged 5–10
times before storing and the subthreshold synaptic potentials were
occasionally averaged five times. The amplitude of the LFP response was
measured from the positive peak to its following negative peak. The durations
of the negative and positive components were measured between the points at
which the potential reversed its polarity. The delay was measured from
stimulus onset to the negative peak in the response. The amplitudes of both
the action potentials and the synaptic potentials were measured from the
resting potential to the peak of the response and their duration was measured
at half amplitude. The rise time of the synaptic potentials was measured from
10% to 90% of the amplitude.
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Response shapes
The field potential evoked in response to the Aff stimulation
(Fig. 2A) is characterized by a
fast positive wave (arrowhead), which most likely reflects the propagation of
the action potential volley in terminals of the Aff. This positive wave is
followed by a triphasic slower response that reaches maximum negativity after
4.2 ms; an average delay of 4.36±0.81 ms (mean ± s.d.) was
measured in 13 preparations. The triphasic response probably reflects somatic
action potentials (maximum negativity marked with an arrow) superimposed on
the synaptic currents (positive slow wave). Occasionally, the characteristic
triphasic response was followed by another small negative component (double
arrowhead) (see also Fig. 9D),
suggesting that a single peripheral stimulation can elicit a short burst of
postsynaptic action potentials. On average, the amplitude of the triphasic
response was 2.76±1.44 mV (N=15). The average duration of the
negative and the second positive components were 2.91±0.7 ms and
5.45±1.07 ms, respectively (N=13).
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The longer delay of the response to PF stimulation and the shorter distance to the stimulating electrode compared to Aff stimulation suggests that the PF have slower conduction velocities. The conduction velocity was estimated by measuring the difference in the delay of the response after relocating the stimulating electrode along the activated axons. The average conduction velocities of the PF and the Aff were 0.13±0.04 m s–1 and 10.3±4.4 m s–1, respectively. In addition, the response duration to PF stimulation was an order of magnitude longer than that of the Aff stimulation, suggesting that PF synapses have a slower kinetics than that of Aff synapses.
In contrast to the differences in response delays and response durations, the negative wave in the two responses had a similar shape, suggesting that both stimuli activated the same population of postsynaptic elements. The different amplitude of the two negative waves suggests that the two inputs either activate the DON to a different extent or to a different level of synchronization.
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The identity of the postsynaptic neurons was established by intracellular labeling (see Materials and methods). Fig. 3 shows five neurons that were filled with neurobiotin. Indeed, these neurons share common features. First, as demonstrated in a lower power micrograph (Fig. 3A), they are located at the border between the molecular layer and the principal cell layer. Second, they are oriented at a similar plane as evident in Fig. 3C showing two labeled neurons. Third, all of them have a rather extensive dendritic tree that ramifies dorsally into the molecular layer and ventrally into the DON's principal cell layer. The dorsal dendritic tree is more elaborate in terms of number of primary dendrites and their secondary and tertiary bifurcations. Usually there are only two ventral dendrites that rarely bifurcate (Fig. 3B,D,E) compared with 3–5 dorsal dendrites that form a complex planar dendritic tree reminiscent of the Purkinje cell dendrite. Fourth, dendritic spines could not be identified. Fifth, presumed axons were occasionally identified as a fine homogeneous process that travels ventrolaterally without bifurcations into the output track of the DON (arrow in Fig. 3D). Since all the labeled neurons responded to both inputs we assume that most of our intracellular recordings were made from the AEN, the DON principal neurons.
The response amplitude
As expected, the amplitude of the PF and the Aff responses increased with
stimulus intensity (Fig. 4A,B).
A more careful analysis of these two responses revealed that they differed in
four distinctive features. (1) The response to Aff stimulation was detected at
lower stimulus intensities compared to PF stimulation (compare
Fig. 4E and F; note the
different scale). This can be attributed to the low threshold of the Aff, as
expected in myelinated fibers. Alternatively, the low threshold of the
response can be explained by a small number of Aff fibers needed to elicit a
detectable response in the DON. (2) The delay following PF stimulation
decreased as stimulus intensity increased
(Fig. 4D), whereas the delay
following Aff stimulation was less dependent on stimulus intensity
(Fig. 4C). This difference is
likely to reflect a slow rise time of the PF synaptic response. (3) The second
negative wave in the response to PF input appeared at a lower stimulus
intensity than that of the Aff input. This rather low activation threshold of
the second response to PF input suggests a slow decay time of PF synapses.
Based on the slow rise time and slow decay time of the PF synapse we suggest
that this input has slow kinetics. (4) The response to Aff stimulation
saturated within a short range of stimulus intensities, whereas the PF
response rarely saturated and seemed to increase linearly within a wide range
of stimulus intensities. This difference in saturation levels also supports
the possibility that activation of a smaller number of Aff fibers is
sufficient to activate the DON whereas more PF need to be activated in order
to generate a detectable response in the DON.
These four features that distinguished the PF and the Aff inputs were further examined by analyzing the responses recorded intracellularly. The results are summarized in Fig. 5. The postsynaptic response to both PF and Aff activation increased in amplitude with stimulus intensity, eventually reaching the threshold and eliciting an action potential (Fig. 5A,B). Six experiments are summarized in the plots shown in Fig. 5C,D. The range of stimulus intensities of the Aff that evoked sub-threshold synaptic potentials was much smaller than that of PF stimulation. As a result there is a narrow distribution of the amplitude of the synaptic potential. This limited range of Aff sub-threshold activity, further supports the possibility that only a small number of Aff fibers converges on a single AEN neuron. Furthermore, these plots show that the depolarization required to evoke an action potential by Aff input is smaller than that of PF input (see Fig. 6). This difference can be due either to the existence of two distinct spike generating mechanisms or the site of action potential generation being closer to the Aff input and distal to the recording site. The intracellular recording also confirmed the lower threshold for the second response and led to the conclusion that the second negativity detected in the LFP does indeed represent prolonged input into the AEN which, with high intensity, can elicit a compound response at the single cell level. This prolonged response, shown in Fig. 5F, suggests that the PF input activates an intrinsic conductance that can support the generation of a burst of action potentials. Activation of such an intrinsic conductance was validated by blocking the delayed response and the prolonged depolarization by hyperpolarizing the cell's membrane (not shown). Finally the possibility that the Aff input has a faster kinetics than the PF was directly confirmed by comparing these two inputs (Fig. 5E). The two responses were normalized and superimposed to show, beyond doubt, that the Aff input has a faster rise time. Summarizing the results from ten neurons revealed that the average Aff rise time was 3.9±1.8 ms and that of the PF was two times slower, averaging at 8.8±3.5 ms.
The action potential threshold
The intracellular recordings strongly suggest that the threshold to elicit
action potential by Aff input is much lower than the threshold to elicit
action potential by the PF input. This observation, in addition to being
interesting in its own merit, touches a fundamental issue; the interaction
between these two inputs is the suggested mechanism for cancellation of
self-generated responses. Therefore, we directly compared the threshold for
spike initiation by activating a neuron with three different inputs. The
procedure is shown in Fig. 6.
We stimulated the Aff nerve at just threshold intensity; this was followed by
just threshold stimulation of the PF input. The two responses were then
superimposed on a hyperpolarizing pulse that prevented the cell from firing,
thereby revealing the threshold synaptic potentials. Then we used the
intracellular current injection to activate the cell directly and measured the
voltage threshold for spike initiation. The area marked by the broken line is
displayed at higher gain in Fig.
6B, where the arrows point the different thresholds. Indeed in
this example the threshold for direct current injection was 10 mV, that of the
PF was 4.5 mV and the Aff threshold was only 2 mV. Summarizing the results
from 11 cells shows that the average spike threshold for Aff input is
2.9±1.74 mV where as that of PF input is 12±5.58 mV and the
direct current is 14.7±5.65 mV. Whereas the threshold for Aff is
significantly lower than that of the PF
(P
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The electroresponsive structure of the DON was further analyzed by measuring the responses to Aff stimulation along the rostro-caudal and medio-lateral axes. As shown in Fig. 8, the shape of the field depends on the site of recording as well as the depth of the electrode within the tissue. Specifically, it is apparent that: (1) the nerve terminal potentials were larger at medial locations (arrows in Fig. 8B), suggesting that the Aff input arrives via the medial side of the DON; (2) there are very small variations in the response delay recorded along the rostro-caudal axis of the DON (Fig. 8A,B), and these delay variations, which are much smaller than those observed along the medio-lateral axis, suggest that peripheral information arrives simultaneously to all areas along the rostro-caudal plan of the DON; (3) the responses in the lateral DON exhibited a longer delay and had a wider negative peak compared to those recorded in the medial DON (Fig. 8C). The calculated propagation velocity of the response along the mediolateral axis was 0.26 m s–1, which is rather slow compared to the conduction velocity in the Aff fibers (10.3 m s–1; see above). This suggests that the delay of the response was due to post-synaptic propagation of the signal. Finally (4), the fast negative response was observed in all recorded locations, suggesting that the sites of action potential initiation (likely the soma of the AEN or the ventral dendritic tree) are within the middle portion of the DON at a depth of about 500 µm. It is tempting to speculate that this medio-lateral propagation of the post-synaptic response represents `back propagation' of action potential along the dorsal dendrite of the AENs.
Short-term interactions between the PF and the Aff inputs
Interactions between the PF and the Aff inputs have been hypothesized to
underlie the ability of the DON to extract relevant information from the
sensory input (Nelson and Paulin,
1995; Montgomery and Bodznick,
1994). According to these `adaptive filter' models, feed-forward
inhibition in both the PF and the Aff circuits plays a major role in signal
adaptation. Therefore, to further understand how the DON processes information
we characterized the short-term interactions between these inputs. We measured
the responses at the middle portion of the DON where the AEN cell bodies are
located (Fig. 3). We
implemented paired pulse protocols in which either the Aff nerve or the PF
were stimulated. The responses were measured at several time intervals between
the two stimuli.
Paired pulse stimulation of the Aff
Paired pulse protocol applied to the Aff nerve revealed significant
depression. It reached maximum at about 20 ms and lasted almost 100 ms
(Fig. 9A). The amplitude of the
response to the second stimulus delivered at 20 ms after the first stimulus,
reached 30% of the initial amplitude. The average time course of the
depression clearly shows that at 20 ms it is larger than at 10 ms
(Fig. 6C; N=9). The
maximum depression averaged over nine preparations was 68.6±15.2%. The
amplitude of the second response gradually increased with the interval between
the two stimuli while maintaining the shape of the initial response,
suggesting that the depression Affects all stimulated components equally.
Moreover, a second negative peak (Fig.
9A, arrows) appeared in the response to the second stimulus. This
second peak indicates the presence of a facilitatory process occurring in
addition to inhibition of the AENs.
The delayed peak of the depression indicates that it is mediated via chemical synapses. We therefore examined the effect of bicuculline, a commonly used GABAergic blocker, on the inhibition measured by paired pulse protocol. Bicuculline completely abolished the inhibition (Fig. 9B,C). Nonetheless, at an interval of 10 ms, the amplitude of the response remained smaller than the initial response despite the presence of bicuculline. This reduction in amplitude is probably due to refractory period. Furthermore, the amplitude of the response to the first stimulus increased (compare Fig. 9A with Fig. 9B), suggesting that the feed-forward inhibition is fast enough to decrease significantly the initial excitatory response or that some of the GABAergic receptors on AENs are tonically active. On average, after blocking the GABAA receptors the amplitude of the response to Aff stimulation increased by 22.2±12.2% (N=5; P<0.002). The fast onset of the inhibitory response suggests that the inhibitory neurons have a low threshold and a short time constant. To further elucidate the source of inhibition, we examined the dependence of inhibition on Aff stimulus intensity. In the example shown in Fig. 9D, the inhibition evoked by Aff stimulation was measured at 40 ms intervals. The amplitudes of the first response (circles), the second response (rectangles) and their ratio (triangles) are shown in Fig. 9E. The inhibitory effect started at very low stimulus intensities. The response to the first stimulus increased monotonically with stimulus intensity while the response to the second stimulus was rather small, even at high stimulus intensities, and was intensity independent. Thus, we suggest that the inhibition evoked by Aff nerve stimulation is highly efficient and is widely distributed throughout the DON.
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The electroresponsive architecture of the DON
We have demonstrated that the PF input is located at the dorsal surface of
the DON whereas the Aff input is probably located deeper in the ventral side
of the nucleus. We have also shown that the response to Aff nerve stimulation
propagates along the medio-lateral axis of the DON. These observations are
summarized in the quasi-schematic representation in
Fig. 11, superimposed on a
micrograph of the DON cross-section. The cell bodies of its principal neurons,
the AEN (Fig. 11, thick red
line), are located in the middle portion of the DON. Based on our histology
(Fig. 3) and electrophysiology
data (Fig. 8), the dorsal
dendrites of the AEN extend up into the molecular layer located at the top of
the cross section (marked by green dots). The information arriving
via the Aff reaches the ventral dendrites of the AEN at the bottom
layer of the DON (marked in yellow). Assuming that the trajectory of recording
electrode was as shown in Fig.
11 (broken line), the synaptic response within the molecular layer
is expected to be negative (sink) during PF stimulation and positive (source)
during Aff nerve stimulation, as shown in
Fig. 7. It is also expected
that the generation of action potentials at the AEN's soma will cause a
negative wave in the middle portion of the DON. Indeed, both PF and Aff
stimulation induced a negative response in this part of the DON. Nonetheless,
due to location of the electrode that stimulates only a beam of PF, the
response to PF was markedly attenuated once the recording electrode crossed
the border between the molecular layer and the cell body layer. At this point
the deeper AEN dendrites are not activated by the PF-stimulating electrode. In
contrast, when the Aff is stimulated, it activates most of the AEN; therefore,
the response to Aff nerve stimulation could be detected deep inside the DON
area, which was not activated by the surface electrodes that stimulated only a
narrow beam of PF. The possibility that the action potential back-propagates
along the dorsal dendrite could also contribute to the fact that the Aff
response can be detected throughout the dorso-ventral axis of the DON. It is
expected that the current flow of the back-propagating action potentials,
activated by the Aff input, will be different from the forward-propagating
action potentials activated by the PF. Non-symmetric arrangement of the inputs
and several spike initiation sites will cause the relationship between the
different components of the field potential evoked by Aff to be different from
those evoked by PF.
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The PF input to the DON
There are four properties of the PF input that should be discussed: the
conduction velocity, the dependence on stimulus intensity, the kinetics of the
response, and the activation of feed-forward inhibition. Since the DON is
regarded as a component of the elasmobranch cerebellum
(Bell, 2002;
Paulin, 1993), the properties
of the PF are expected to resemble those of cerebellar PF. Indeed, the
measured conduction velocity of 0.13 m s–1 is similar to 0.2
m s–1 measured in dogfish Scyliorhinus canicula
(Paul, 1969), 0.2 m
s–1 in frogs (Llinas et
al., 1969) and 0.3 m s–1 in cats
(Eccles et al., 1966). As in
the cerebellum, the number of PF is quite large while each fiber seems to form
low efficacy synapses. This can explain the high stimulus intensity required
to evoke a response when the PF were stimulated (see Figs
4 and
5); many fibers should be
activated in order to evoke a detectable postsynaptic response. It also
explains the almost linear relationship between stimulus intensity and
response amplitude. Unlike cerebellar Purkinje cells, the AEN's response to PF
stimulation is characterized by multiple peaks, suggesting that the dorsal
dendrites may produce complex regenerative activity. Indeed the intracellular
recordings (Fig. 5F) show that
the burst of action potentials that was evoked at high stimulus intensity was
elicited by a prolonged plateau-like depolarization that lasted much longer
than the underlying synaptic potential. The significant decrease in the delay
of the response that occurred when stimulus intensity was increased
(Fig. 4D) suggests a slow
kinetics of the synaptic input. A slow rise time of synaptic potential will
generate a significant decrease in the delay as the time to reach the
threshold decreases with the increase in amplitude. This interpretation of the
LFP response was confirmed by intracellular recordings showing that the rise
time of the PF synaptic potential is twice as long as the Aff synaptic
potential (Fig. 5E). Finally,
paired pulse protocol delivered to the PF system revealed two types of
interactions: an inhibitory interaction at high stimulus intensities and
facilitatory interaction at low intensities. The facilitation may represent an
increase in synaptic transmission and or the summation of prolonged synaptic
potential. The latter possibility is supported by the exceptionally prolonged
field potential (see Fig. 2B),
which can last for over 100 ms, and the plateau-like depolarization
(Fig. 5E). The inhibitory
interactions occurred at high stimulus intensities, peaked immediately after
the stimulus and decayed exponentially with a time constant of 10–20 ms.
The inhibitory effect of the PF on themselves and on the response to Aff nerve
stimulation had a similar time course and was insensitive to bicuculline.
These observations suggest that the inhibitory effect evoked by PF stimulation
represents inactivation of the regenerative responses in the AEN
(Poulter et al., 1993), rather
than activation of inhibitory synapses. However, the possible involvement of
glycine receptors should be examined.
The Aff input to the DON
The Aff nerve that transmits the peripheral information to the DON has a
low threshold and a high conduction velocity. A conduction velocity of
10.3±4.4 m s–1 measured in our isolated preparation is
within the range of conduction velocities measured in peripheral nerves of
other species such as frogs (10 m s–1)
(Poulter et al., 1993) and
mammals (30–80 m s–1)
(Nicholls et al., 2001). This
high conduction velocity is also in accordance with the high rate of activity
observed in vivo in this preparation
(Tricas and New, 1998). The
rather low threshold and rapid saturation of the response
(Fig. 4E and
Fig. 5C) can be explained by
assuming that the number of afferent terminals that innervate a given area of
AEN neurons is rather small compared to the number of PF that innervate the
same area. A low number of Aff nerve terminals will cause the field potential
to saturate at low stimulus intensities, as observed (see
Fig. 4). The low threshold
causes the AENs to be easily activated by Aff input and suggests that a small
number of fibers is required to activate the AENs. This possibility was
further supported by the intracellular recordings, which show that action
potentials in AEN are triggered by a rather low amplitude synaptic potential
(see Figs 2 and
5) that attained a limited
number of possible amplitudes. The latter strongly suggests that each AEN is
innervated by a small number of Aff terminals. This conclusion argues against
one of the mechanisms postulated in the adaptive filter model of the DON.
According to this mechanism, the output of the DON occurs only when the Aff
input integrates with the PF input. A very low threshold of the Aff input will
leave no range for integration with PF input. On the other hand, the low
threshold of the Aff input supports the inhibition role in this model. If the
threshold is low and there are few inputs, an inhibitory input located at a
strategic site can act as an efficient gatekeeper, enabling only unpredicted
information to pass through the gate.
The sharp negative wave in the field potential, which characterizes the response to Aff stimulation, suggests that synaptic input is relatively fast and synchronized. This possibility is further supported by the insensitivity of the delay of the response to stimulus intensity, and it is clearly demonstrated in the intracellular recordings. Hence, we can conclude that the Aff input has a low threshold for activation of the AEN neurons and relatively fast kinetics. It is located at the ventral dendrites of the AENs and each neuron is innervated by a small number of afferent axons.
Threshold differences are one of the most remarkable observations. In order to activate the neuron by PF input, the membrane had to be depolarized by 12 mV, as measured at the recording site. However, only 3 mV of membrane depolarization was needed to activate the neurons when Aff was stimulated. Such a fourfold difference in threshold is a puzzling result. The most straightforward possibility is that the spike initiation site is located closer to the location of the Aff input. This simple explanation is supported by the fact that presumed axons seem to emerge from a ventrally oriented dendrite (Fig. 3) and agrees with the rather superficial recording site. On average, the recording electrode was located 200 µm below the surface. We assume that at this depth the electrode is still within the molecular layer. Therefore we have to conclude that most of the recordings were performed from dorsal dendrites. This possibility is in agreement with the recording of relative large PF synaptic potentials. From the site of recording, this synaptic potential will passively propagate toward the cell body and down the ventral dendrite to the presumed site of spike initiation. Such a passive propagation over a rather long distance will lead to voltage attenuation and thus, at the site of spike initiation, the voltage threshold is similar for all inputs. This explanation can also account for the similarity between the threshold for direct current injection and the PF threshold. From the site of recording these two signals have to propagate along the same path to reach the site of spike initiation and therefore will have to be of similar amplitude. Small variations between these two signals can be reconciled by assuming that the PF input is distributed all over the dorsal dendrites whereas the current injection is limited to one point only. However, the rise time of the Aff synaptic potential is shorter than that of the PF synapse. The cable theory demonstrated that the rise time of synaptic events is the most sensitive parameter that is affected by the electrotonic distance. If two inputs that have the same kinetics are distributed in space, the proximal input will have a faster rise time. Accordingly the faster rise time of the Aff synaptic potential implies that the Aff input is located closer to the recording site.
This conclusion is in sharp contrast to the observations summarized above.
For this reason we are force to conclude either the Aff input has faster
kinetics or that there are two different spiking mechanisms, one with high
threshold located close to the PF input and the other with low threshold
located closer to the Aff input. It is interesting to note that in a very
recent publication, Bell and his colleagues
(Zhang et al., 2007) show that
the afferent input in an analogous electrosensory structure in teleosts fishes
has an electrotonic component. Such a mechanism can account for a rather fast
rise time of the Aff synaptic potential.
The Aff nerve activates inhibitory pathways that exert significant inhibition on the Aff input. This feed-forward inhibition has several unique properties. First, it peaks after 20 ms from the stimulus onset. Such delay onsets are usually associated with metabotropic type of receptors; however, the complete blockade of this inhibition by bicuculline argues against this possibility. An alternative posibility to account for the prolongued delay is that the inhibition is activated via feedback mechanisms from the AEN to the midbrain through the DGR back to the DON. Second, although this inhibition is highly effective and can eliminate up to 90% of the Aff response, it does not affect the PF response. This is a puzzling result, which argues against the possibility of inhibition through the DGR, and can be explained by activation of presynaptic inhibition that limits the effect to the Aff terminals, and/or by activation of feed-forward inhibition that is located at distal compartments of the ventral dendrites (i.e. close to the site of Aff input and remote from the site of PF input). The latter possibility implies that the spike initiation zone of the PF input differs from that of the Aff input. The absence of inhibitory synaptic potentials in the intracellular recordings supports both possibilities.
Further study is required to understand the synaptic mechanisms of the
inhibitory and excitatory inputs in this unique preparation. However, it is
clear that our data do not support the cancellation mechanism suggested in the
adaptive filter model of the DON
(Montgomery and Bodznick,
1994; Nelson and Paulin,
1995). Alternative models, according to which parallel fibers
adjust the response properties of the Purkinje-like AENs, have been suggested
(Paulin, 2005;
Paulin and Nelson, 1993;
Paulin et al., 1998). These
alternative models are consistent with experimental evidence
(Bastian, 1986;
Bower, 2002;
Santamaria and Bower, 2005)
about neuronal interactions in cerebellum and cerebellar-like sensory
filtering structures.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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