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
Naama Rotem1,
Emanuel Sestieri1,
Dana Cohen2,
Mike Paulin3,
Hanoch Meiri4 and
Yosef Yarom1,4,*
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
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Fig. 1. The isolated brain stem of the shark Iago omanensis. (A) Careful
removal of the cartilage skull completely exposed the brain stem (white
circle), the cerebellum (green circle) and the cervical spinal cord (yellow
circle). (B) Top view of the shark's isolated brain stem. The black and yellow
lines outline the DON and the dorsal granular ridge (DGR), respectively. (C)
Side view of the shark's isolated brain stem where the DON is marked by black
line. (D) Schematic drawing of the isolated preparation, showing the relative
locations of the stimulating and the recording electrodes. PF, parallel
fibers; Aff, afferent nerve. Scale bars in A–C, 1 mm.
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Fig. 2. Characterizing the responses elicited by stimulation of either the parallel
fibers (PF) or the afferent nerve (Aff). (A,B) Extracellular recording of the
responses to Aff (A) and PF (B) stimulation. Each record represents the
average response to eight consecutive stimuli delivered at a rate of 0.1 Hz
and recorded at a depth of 500 µm below the surface of the DON. (A) The
response of the DON to Aff nerve stimulation is characterized by an early
positive component (arrowhead); the main triphasic component is marked by an
upward arrow and the second small negative component is marked by a double
arrowhead. (B) The response of the DON to PF stimulation is characterized by
significant longer delay, the pronounced second negativity and the overall
longer duration of the PF response in compare to Aff response. (C,D)
Intracellular recording of the responses to Aff (C) and PF (D) stimulation.
(C) Sub- and supra-threshold responses to Aff stimulation recorded
intracellularly. The subthreshold synaptic potential was occasionally composed
of two depolarizing phases separated by a short delay (arrow). (D) Sub- and
supra-threshold responses to PF stimulation recorded intracellularly. Note the
higher threshold of the PF evoked action potential.
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Fig. 3. Intracellular labeling of the ascending efferent neurons (AEN) of the DON.
(A–E) Unstained cross sections through the middle portion of the DON
developed for neurobiotin staining. (A) A low-power micrograph capturing the
entire DON. The molecular layer appeared as a homogenous tissue lightly
stained in brownish color. The principal cell layer appeared as a granular
tissue with occasionally spherical structures. This difference forms a
clear-cut border between the two compartments of the DON. (B) An enlarged view
of the area outlined in A, focusing on the labeled cell. (C–E) Several
examples of labeled neurons. The presume axon in C is marked with an arrow.
Note that the overall orientations of the cells are along the medio-lateral
axis. All of them emit a rather extensive dendritic tree that bifurcates
dorsally into the molecular layer and ventrally into the DONs principal cell
layer. Scale bar, 100 µm. D, dorsal; V, ventral; L, lateral; M, medial; R,
rostral; C, caudal.
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Fig. 4. The afferent nerve (Aff) has a lower threshold and a shorter dynamic range
of stimulation than the parallel fibers (PF). (A,B) The responses to Aff and
PF stimulation, respectively, following increase in stimulus intensity (from
top to bottom). Each record is an average response to eight consecutive
stimuli delivered at a rate of 0.1 Hz and recorded at a depth of 500 µm
below the surface of the DON. (C,D) Superposition of the responses shown in A
and B, respectively. Note the change in delay of the response to PF
stimulation. (E,F) Amplitude of the responses to Aff stimulation and PF
stimulation, respectively, as a function of the stimulus intensity. The
amplitude of the response in each experiment was normalized by the maximal
amplitude (N=4; each symbol represents one experiment). Note the
different scale of the x-axes; hence, the threshold of the PF is
higher than that of the Aff nerve.
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Fig. 5. Intracellular recordings showing the lower threshold and the shorter range
of stimulations of the Aff. (A,B) The responses to Aff (A) and PF (B)
stimulation following increased stimulus intensity. (C,D) Amplitude of the
synaptic potential as a function of stimulus intensity for Aff (C) and PF (D)
stimulation. The stimulus intensity in each experiment (N=6) was
normalized by the threshold of detectable response. Each symbol corresponds to
the behavior of one neuron for both inputs. Note that the range of Aff stimuli
is considerably shorter than that of PF stimulation. (E) Superimposed
normalized traces of the synaptic response to Aff and PF stimulation. Note the
fast rise time of the Aff synapse compared to the PF synapse. (F) Increase in
PF stimulus intensity evoked a prolonged response that can support the
generation of a short burst of action potentials.
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Fig. 6. The different thresholds for action potential generation. (A) Middle trace:
intracellular recordings of the responses to just threshold, afferent nerve
(Aff) and parallel fibers (PF) stimulation at resting membrane potential.
Lower trace: the two responses were superimposed on voltage hyperpolarization
evoked by 1 nA negative current injection. Upper trace: the response to
supra-threshold positive injection of 1 nA current. (B) The area marked in A
(broken line) is displayed at higher gain in B. Arrows point toward the
different thresholds (note the sharp transient deflections on the onset and
offset of the current pulse, reflecting bridge balancing artifacts).
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Fig. 7. The depth profile of the response to afferent nerve (Aff) and parallel
fibers (PF) stimulation. (A,B) The responses to Aff nerve stimulation (A) and
PF stimulation (B) at different locations along a single path of the
microelectrode through the dorsal ventral axis of the DON. Each record is an
average response to eight consecutive stimuli delivered at a rate of 0.1 Hz.
The depth from the DON surface is indicated. (C,D) The changes in the
responses to Aff (C) and PF (D) stimulation, as a function of the depth of
recording. The relative voltage was measured at three times along the response
as indicated on the corresponding insets. In C, the voltage was measured at 3,
6 and 8 ms from the time of stimulation and it is represented as diamonds,
squares and triangles, respectively. In D, the voltage was measured at 8, 12
and 16 ms from the time of stimulation and it is represented as diamonds,
squares and triangles, respectively. These specific times were selected to
denote the three phases of the triphasic response.
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Fig. 8. The response to afferent nerve (Aff) nerve stimulation propagates along the
medio-lateral axis. (A,B) The responses to Aff stimulation recorded at three
different locations and at 5–6 different depths along the rostro-caudal
axis of the lateral (A, red traces) and the medial (B, black traces) sides of
the DON. Each record is an average response to eight consecutive stimuli
delivered at a rate of 0.1 Hz. The location of each record is marked on a top
view of the DON as seen through a dissecting microscope. Arrows in B demark
the nerve terminal potential. (C) Superposition of the responses recoded at
the medial (black) and lateral (red) locations. Right to left, panels
correspond to the three different locations along the rostro-caudal axis. Note
the significant delay between the medial and lateral responses. L, lateral; M,
medial; R, rostral; C, caudal.
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Fig. 9. Bicuculline-sensitive inhibition induced by afferent nerve (Aff) nerve
stimulation. (A,B) Superimposed traces of the responses to paired pulse
stimulation of the Aff nerve. The paired stimuli were delivered at 20, 40, 60,
80, 100 and 120 ms intervals. Each record is an average response to eight
consecutive stimuli delivered at a rate of 0.1 Hz. Arrows denote the second
negative peak that increased as a function of the paired pulse interval. (A)
Before and (B) after the addition of bicuculline (100 µmol
l–1). (C) The amplitude of the responses to the second
stimulus, normalized by the amplitude of the first response, as a function of
the inter-stimuli interval. Diamonds indicate responses before, and squares,
responses after addition of bicuculline. N=9 (control),
N=7 (+bicuculline). (D,E) The inhibition induced by Aff stimulation
increases as a function of stimulus intensity. (D) The responses to a pair of
stimuli delivered to the Aff at an interval of 40 ms. Each trace shows the
response to different stimulus intensity, increasing from top to bottom. (E)
The amplitude (right axis) of the response to the first (circles) and second
(squares) stimulus as a function of stimulus intensity. Triangles indicate the
relative amplitude of the second response (left axis) as a function of
stimulus intensity.
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Fig. 10. Bicuculline-insensitive inhibition induced by parallel fibers (PF)
stimulation. (A,B) Superimposed traces of the responses to paired pulse
stimulation of the PF. The controlled paired stimuli in A were delivered at
30, 40, 60 and 80 ms intervals. The paired stimuli in the presence of
bicuculline (100 µmol l–1) (B) were delivered at 30, 50,
70, 90 and 110 ms intervals. Each record is an average response to eight
consecutive stimuli delivered at a rate of 0.1 Hz. (C) The amplitude of the
responses to the second stimulus, normalized by the amplitude of the first
response, as a function of the inter-stimuli interval. Diamonds indicate
responses before, and squares, responses after addition of bicuculline.
N=9 (control), N=3 (+bicuculline). (D) The responses
to a pair of stimuli delivered to the PF at an interval of 30 ms. Each trace
shows the response to different stimulus intensity, increasing from top to
bottom. (E) The amplitude (right axis) of the response to the first (circles)
and second (squares) stimulus as a function of stimulus intensity. Triangles
indicate the relative amplitude of the second response (left axis) as a
function of stimulus intensity.
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Fig. 11. Quasi-schematic representation of the electroresponsive structure of the
DON. Schematic representation of the postulated neuronal circuitry
superimposed on a cross-section of the DON. The molecular layer at the top of
the cross section (green dots) is the location of all the apical dendrites of
the principal neurons. The elongated cell bodies (in red) are located at the
middle of the DON and the incoming afferent nerve innervates the basal
dendrites in the deep layer (in yellow). The broken yellow line delineates a
hypothetic path of the recording. AEN, ascending efferent neurons; D, dorsal;
V, ventral; L, lateral; M, medial; R, rostral; C, caudal.
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© The Company of Biologists Ltd 2007
