Departments of 1Neuroscience and 2Neurosurgery, and 3Graduate Program in Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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Chen, G., C. L. Hanson, R. L. Dunbar, and T. J. Ebner. Novel form of spreading acidification and depression in the cerebellar cortex demonstrated by neutral red optical imaging. A novel form of spreading acidification and depression in the rat cerebellar cortex was imaged in vivo using the pH-sensitive dye, Neutral red. Surface stimulation evoked an initial beam of increased fluorescence (i.e., decreased pH) that spread rostrally and caudally across the folium and into neighboring folia. A transient but marked suppression in the excitability of the parallel fiber-Purkinje cell circuitry accompanied the spread. Characteristics differentiating this phenomenon from the spreading depression of Leao include: high speed of propagation on the surface (average of 450 µm/s), stable extracellular DC potential, no change in blood vessel diameter, and repeatability at short intervals. This propagating acidification constitutes a previously unknown class of neuronal processing in the cerebellar cortex.
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INTRODUCTION |
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Propagating waves of excitatory and inhibitory
activity across neuronal populations are of particular interest due to
their roles in neuronal signaling as well as pathophysiology. Calcium waves have been shown to occur in various neuronal-glial in vitro preparations and have been hypothesized to act as a signaling mechanism
(Cornell-Bell et al. 1990; Newman and Zahs
1997
). Spreading depression of Leao is a slowly propagating
wave that results in a dramatic extinction of all neuronal activity
(for review, see Lauritzen and Nicholson 1988
;
Leao 1944
; Ochs 1962
). This
"classical" form of spreading depression is characterized by a
large negative shift in the extracellular DC potential, relatively long
refractory period, and large ionic shifts including a transient
increase in extracellular K+ concentration. Classical
spreading depression may play a role in several pathophysiological
processes including migraine headaches and ischemia (Lauritzen
and Nicholson 1988
; Somjen et al. 1992
).
Extra- and intracellular pH are coupled closely to neuronal activity
(Chen et al. 1998; Chesler 1990
;
Chesler and Craig, 1989
; Grichtchenko and Chesler
1994a
,b
; Kraig et al. 1983
). Stimulation of the
cerebral or cerebellar cortex evokes an initial alkaline shift followed
by an acidic shift in the extracellular space. Intracellularly, this
sequence is reversed; neurons undergo an acidic shift while glial cells
become alkaline. The pH-sensitive dye, Neutral red, can be used to
monitor the spatial and temporal characteristics of pH shifts in vivo
and map neuronal activation (Chen et al. 1996
, 1998
).
This report demonstrates that electrical stimulation of the surface of
the cerebellar cortex stained with Neutral red evokes an optical signal
that propagates at high speed and is accompanied by a transient but
powerful depression of parallel fibers and their postsynaptic targets.
This novel form of spreading acidification and depression differs
fundamentally from the spreading depression of Leao and other
previously described wave-like phenomenon.
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METHODS |
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Details of the preparation are provided in previous publications
(Chen et al. 1996, 1998
). Briefly, adult Sprague-Dawley
rats of either sex (200-400 g) were anesthetized by an intramuscular injection of a solution of ketamine (60 mg/kg), xylazine (3 mg/kg), and
acepromazine (1.2 mg/kg). The animals were respirated artificially. After exposing Crus I and II of the cerebellar surface, Neutral red
(8-9 mM in rat Ringer solution) was applied topically for 2-3 h.
Surface folia were imaged using a CCD camera with modified Zeiss
epi-fluorescence optics. After washout of the dye, sequential epi-fluorescence images (excitation at 550 ± 10 nm, emission
>620 nm) of the stained cortex were acquired before (control), during, and after (stimulation image) a train of surface electrical stimulation with a tungsten microelectrode. Acquisition time for an individual frame was 100 ms with a slight delay between frames. Subtraction of a
single control image from a stimulation image was used as shown in Fig.
1. No averaging of the images was done.
Conventional electrophysiological techniques and glass micropipettes
were used to record extracellular field potentials evoked by surface
stimulation and to monitor extracellular DC potentials.
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In some experiments, the voltage-sensitive dye, RH-795, was used to
stain the cerebellar cortex and the optical response to surface
stimulation imaged. RH-795 was chosen because it yields a large optical
response to surface stimulation in vivo and has excitation-emission
characteristics similar to Neutral red (Ebner and Chen
1995). The exposed cortex was bathed in RH-795 (0.3 mg/ml) for
2 h. After washout of the dye, epi-fluorescence images (excitation at 546 ± 10 nm, emission >590 nm) were acquired. To detect the much weaker optical signals obtained from the cerebellum stained with
RH-795 (Ebner and Chen 1995
), frame averaging (30-100)
was required. Specifically, 30-100 pairs of images without and with surface stimulation were obtained (exposure time of 100 ms) and subtracted. The resultant set of "difference images" was averaged.
Classical spreading depression also was imaged in the
Neutral-red-stained cerebellar cortex. Because spreading depression in
the rat cerebellar cortex is difficult to evoke (Nicholson and
Kraig 1975; Nicholson et al. 1978
) and due to
the inhibitory effects of ketamine on the generation of spreading
depression (Gorelova et al. 1987
; Lauritzen et
al. 1988
), several changes to the experimental protocol were
required. The animals were anesthetized with pentobarbital (50 mg/kg
ip), and the cerebellar cortex was conditioned by bathing in a modified
Ringer solution that contained 123 mM sodium benzoate, completely
replacing the NaCl (Nicholson and Kraig 1975
;
Tobiasz and Nicholson 1982
). To evoke classical spreading depression, KCl was injected using a picospritzer pressure injection system (Medical Systems, Greenvale, NY). A glass
microelectrode with a 1- to 3 µ-diameter tip was filled with 0.5 M
KCl, and ~5 nl was injected over 1-3 s. A negative holding pressure
was applied to the electrode when not injecting. Optical images and the
extracellular DC potential were recorded in relation to the KCl injection.
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RESULTS |
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Electrical stimulation of the surface with a train of 100 pulses
initially evoked a narrow beam of increased fluorescence along the long
axis of the folium (Fig. 1A). An increase in fluorescence reflects a decrease in pH, and this beam-like signal is due to the
activation of parallel fibers and their postsynaptic targets (Chen et al. 1998). During the first 2.7 s of the
stimulus train, the fluorescence increase in the center of Crus Ia was
0.5% (
F/F) and remained confined to a
restricted beam. The increase in fluorescence began to rapidly spread
rostral-caudally across the surface of the folium ~2.85 s from the
onset of stimulation. At 3.45 s, the optical signal reached the
anterior edge (position b') and at 3.75 s the posterior edge
(position b) of Crus Ia (Fig. 1B). During the spread, the
intensity of the signal at the center of Crus Ia rapidly increased to
25% (
F/F) and remained elevated for the next
50 s (Fig. 1C). After a delay of 12.75 s, the increase
in fluorescence spread to the anterior edge (position c in Fig.
1B) of the neighboring folia, Crus Ib (16.05 s in Fig.
1A). The optical signal then spread rapidly across the
surface of Crus Ib and reached its posterior boundary within 16.95 s
(position d). The increase in fluorescence in Crus Ib persisted
throughout the remainder of the observation time (Fig. 1C).
The optical signal continued to spread and arrived at Crus IIa, which
was just visible in the lower right corner of the field of view, at
33.15 s (position e). The long delays in the arrival of the optical
signal in Crus Ib and II and the rapid but persistent increases in
fluorescence after reaching these folia are demonstrated in Fig.
1C.
The speed at which the acidification spread across the folium is in
sharp contrast to other forms of propagated activity in the CNS.
Calcium waves propagate at ~25 µm/s (Newman and Zahs 1997), whereas classical spreading depression in the cerebral cortex propagates at 25-125 µm/s with the highest speed of 150 µm/s reported in the rat cerebellar cortex (Leao 1944
;
Nicholson et al. 1978
; Somjen et al.
1992
). For the example shown in Fig. 1, the average speed was
470 µm/s with a peak speed of 1,100 µm/s on the surface of the
folia (a to b' and a to b on Crus Ia and from c to d on Crus Ib). In 14 animals in which the speed of the optical wavefront was calculated, the
average speed on the cerebellar surface was 450 ± 80 (SD)
µm/s with an average peak speed of 940 ± 220 µm/s. The speed
of propagation within the sulcus was 180 µm/s based on the assumption
that the spread followed the most direct path as estimated in Fig.
1D (from b to c and d to e). However, the actual path taken
by the spread is unknown and the speed in the sulcus may be
underestimated. In the experiment shown in Fig. 1, the optical signal
did not spread into lobulus simplex (SL), the most anterior folium in
the field of view. As shown in Fig. 1D, the sulcus between
Crus Ia and SL was relatively deep, suggesting that the distance over
which the spread occurs may be limited to a few centimeters. Spread of
the optical signal was observed in 40 of 73 animals. Stimulation
parameters were an important determinant of whether spread was evoked.
Increasing stimulation frequency (2-20 Hz), duration (2-30 s), and
intensity (75-300 µA) increased the likelihood of evoking the spread.
The gradients in the optical response both along the stimulation evoked parallel fiber beam and perpendicular to the beam are shown in Fig. 1, E-G, at four different times in the evolution of the response. At no time in the course of the spread was there an obvious gradient in the optical response along the beam (F). In contrast, a very large gradient in the optical response occurred perpendicular to the beam (Fig. 1G). The amplitude of the optical response was largest on beam, decreasing as a function of the distance from the beam. Furthermore, the optical response was symmetric relative to the beam throughout the course of the spread. The second peak of activity at 2,000 µm, shown in the amplitude profile at 27.90 s (Fig. 1G), reflects the spread of the optical response into Crus Ib.
The excitability of the cerebellar circuitry in relationship to the
spread of acidification was evaluated by recording both the optical
signals and the extracellular field potentials (Fig. 2) evoked by a second surface stimulating
electrode. Two stimulation electrodes were placed on the surface (Fig.
2A). The first electrode (S)
was used to evoke the spread of optical activity using a train of
stimulation (150-µA, 15-µs pulses at 10 Hz for 15 s). The
second, laterally placed electrode
(S
)
was used to stimulate the parallel fibers at 2-s intervals with a
single pulse (150 µA, 150 µs) and at the same time an optical image
was captured. The resultant parallel fiber volley
(positive-negative-positive deflection,
P1/N1/P2 components) and
postsynaptic response (longer latency negative deflection,
N2 component) were recorded "on beam" relative to the
second stimulation electrode (Fig. 2A). The amplitude of
P1 to N1 was used as a measure of parallel
fiber excitability and the amplitude of N2 as a measure of
the postsynaptic response. The stimulus train initially evoked a narrow
beam of increased fluorescence (b in Fig. 2A) that spread
across the entire folium (c-d). The increase in fluorescence peaked at
25.8 s after the onset of stimulation and decreased during the
next 50 s to a level ~5% above baseline (Fig. 2, A
and B).
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Examination of the extracellular field potentials (Fig. 2C)
revealed a transient but complete suppression of the parallel fiber
(P1/N1) and postsynaptic component
(N2) before and during the peak of the optical response.
Suppression of the parallel fiber volley developed rapidly and lasted
~20 s (Fig. 2 C and D). The postsynaptic
component (N2) also was suppressed completely and took
longer to recover. The postsynaptic component was abolished for 120 s
after the onset of stimulation, recovering to 95% of baseline at
420 s. In six of seven experiments in which the field potentials
were monitored, there was complete suppression of
P1/N1 and N2 components during the
peak of the spread of the optical signal.
In seven experiments, the cerebellar cortex was stained with the
voltage-sensitive dye, RH-795, and the epi-fluorescence optical response evoked by surface stimulation was imaged. Because the optical
signals obtained with voltage sensitive dyes are very small, frame
averaging was required. The optical response consisted of a
"beam" of decreased fluorescence (Fig.
3C) consistent with our
previously published findings (Elias et al. 1993) and
with the fact that depolarization leads to a decrease in
epifluorescence in this class of styryl dyes (Ebner and Chen
1995
). In no instance was a spread of the optical response
observed using RH-795.
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Is the spreading acidification and depression described in this report
the spreading depression of Leao? Both the high speed of propagation on
the cerebellar surface and the relatively brief suppression of the
parallel fiber volley suggest that the spread of acidification and
associated decrease in excitability is a unique phenomenon that has not
been described previously. Four additional experimental observations
confirm this conclusion. First, a hallmark of classical spreading
depression is an extracellular DC shift of 15 to
35 mV that lasts
up to several minutes (Kraig and Nicholson 1978
;
Lauritzen and Nicholson 1988
; Leao 1944
). In 12 experiments in which the extracellular DC potential was monitored, no shift in the extracellular potential occurred (Fig. 4A). Second, decreased
cerebral blood flow follows a period of increased blood flow and vessel
dilation in the spreading depression of Leao (Lauritzen and
Nicholson 1988
; Somjen et al. 1992
). Blood vessel dilatation did not occur at the stimulation parameters used in
this study. In seven experiments, 26 blood vessels with diameters
ranging from 50-180 µm were measured before and during the spread of
the optical signal (Fig. 4B). No significant change in blood
vessel diameter was observed (P = 0.71, Student's
paired t-test). It should be noted that increases in blood
vessel diameter on the cerebellar surface do occur with stimulus trains
of much higher frequency (30 Hz) and duration (40-60 s)
(Iadecola et al. 1997
). Third, classical spreading
depression is characterized by an absolute refractory period of
120-180 s during which it is not possible to evoke a second event
(Ochs 1962
). In this study, a second and third spread of
the optical signal could be evoked within 90 s (shortest interval
tested) of each other (Fig. 4C). Furthermore, parallel fiber
excitability returned to normal within 20 s of stimulation onset
(Fig. 2D), a much shorter recovery time than observed with
classical spreading depression (Ochs 1962
).
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To directly compare the spreading acidification described in this paper
with classical spreading depression, KCl was pressure injected into the
folium (Fig. 5). As described in
METHODS, ketamine had to be replaced with pentobarbital and
the Cl in the Ringer solution replaced with benzoate ion
(Nicholson and Kraig 1975
; Tobiasz and Nicholson
1982
). As shown in Fig. 5A, microinjection of KCl
evoked a strong optical signal that spread outward from the injection
site at a speed of 109 µm/s, ~5-10 times slower than the spreading
acidification of Fig. 1. The spread of the optical signal was
accompanied by an extracellular DC shift of
15 mV (Fig.
5B). Similar observations were made in five animals.
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DISCUSSION |
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What is the spreading phenomenon described in the study? The lack
of a DC shift, high speed, no change in blood vessel diameter, and a
short recovery period demonstrate that the spread of acidification evoked is not the spreading depression of Leao. Additional observations support this conclusion. First, it is difficult to generate classical spreading depression in the rat cerebellar cortex without extensive conditioning by replacing a large fraction of the extracellular Cl with proprionate or benzoate (Nicholson and
Kraig 1975
; Tobiasz and Nicholson 1982
). No
conditioning was required to evoke the high-speed propagation in this
study, but it was required to evoke spreading depression with
microinjection of KCl. Second, ketamine, at the concentrations used for
anesthesia in this study, is known to block classical spreading
depression (Gorelova et al. 1987
; Lauritzen et
al. 1988
). To evoke classical spreading depression with KCl
injection, pentobarbital anesthesia was required. Finally, spreading
depression and calcium waves propagate as a wavefront from a central
source outward (Newman and Zahs 1997
; Yoon et al. 1996
). In contrast, the optical signals at the point of
initiation increased and remained elevated as the signal spread into
the neighboring areas (Fig. 1, C and E-G). These
observations demonstrate that the propagating optical response
described in this study is not the spreading depression of Leao.
The role of Neutral red in the spread of the optical signal remains
unknown. Neutral red has been used as an intracellular vital dye and is
noted for its relatively benign characteristics (LaManna and
McCracken 1984). Neutral red does not appear to affect the
basic electrophysiological properties of the parallel fibers and their
postsynaptic targets (Chen et al. 1996
, 1998
). Still Neutral red may have an unidentified "conditioning" effect on the
cerebellar surface that contributed to the spread of acidification.
Previous work found that the pH shifts detected by Neutral red
are primarily intracellular (Chen et al. 1998). Several
mechanisms may contribute to intracellular acidification. These include
metabolic production of CO2 and lactic acid (Siesjo
1985
), GABA channel mediated HCO3
efflux (Kaila et al. 1990
), calcium influx resulting in
the release of H+ from internal storage sites
(Paalasmaa et al. 1994
), and glutamate-mediated H+ influx (Chen and Chesler 1992
). Ion
exchangers also may contribute including both the exchangers
Cl
/HCO3
(acidifying mechanism)
and Na+/H+ (alkalizing mechanism) known to
exist in the rat cerebellar Purkinje cells (Gaillard and Dupont
1990
). Surface stimulation and the resultant activation of
neuronal activity, including excitatory and inhibitory synaptic
connections, are likely to engage several of these mechanisms.
What is the relationship between the pH shift and the suppression
of the cerebellar field potentials? Shifts in pH are known to modulate
neuronal excitability (Chesler 1990). An increase in
neuronal acidity depresses voltage-gated sodium channels
(Tombaugh and Somjen 1996
), possibly accounting for the
suppression of parallel fiber excitability. Decreases in local pH also
augment GABAA channel Cl
conductance
(Robello et al. 1994
) but attenuate glutamate channel conductance (Traynelis and Cull-Candy 1991
),
probably contributing to the decreased postsynaptic responsiveness to
surface stimulation. Therefore the spread of acidification may
contribute directly to the depression in parallel fiber excitability
and their postsynaptic targets.
Suppression of synaptic activity outlasted the pH induced optical
responses suggesting that other mechanisms mediating cell excitability
may be involved (Fig. 2, B and D). One possible
mechanism involves an increase of intracellular calcium
[Ca2+]i, induced by activity and
acidification (Koch and Barish 1994). Intracellular
calcium activates a number of second-messenger cascades in cerebellar
neurons that have been associated with long-term depression (for
review, see Linden 1994
). In Purkinje cells, an increase
in [Ca2+]i leads to desensitization of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors
(Crepel and Krupa 1988
) and of
GABAA-receptor-mediated chloride current (Kano
1996
) and facilitates an excitatory amino-acid-dependent postsynaptic anion conductance (Kataoka et al. 1997
).
All of these mechanisms could contribute to the decreased excitability.
Last, nitric oxide produced in response to intracellular calcium
activation of a calmodulin-dependent nitric oxide synthase within
parallel fibers could diffuse to guanylyl cyclase in Purkinje cells and eventually lead to an inhibition of phosphatases responsible for dephosphorylating AMPA receptor channels in the Purkinje cell membrane
(Boxall and Garthwaite 1996
; Bredt and Snyder
1989
; Ito and Karachot 1992
; Lev-Ram et
al. 1995
; Shibuki and Okada 1991
).
The spread of acidification and associated depression in the excitability of the cerebellar circuits may be a neuronal signaling mechanism and/or play a role in pathophysiological processes. The high speed of the spread and relatively long distance traveled provides a mechanism by which neuronal activity in one folium can evoke profound changes in neighboring folia.
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ACKNOWLEDGMENTS |
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The authors thank L. King and J. Bailey for typing the manuscript and M. McPhee for graphics and illustrations.
This work was supported by National Institute of Neurological Disorders and Stroke Grants PO1-NS-31318 and RO1-NS-18338.
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FOOTNOTES |
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Address for reprint requests: T. J. Ebner, Dept. of Neuroscience, University of Minnesota, Lions Research Bldg., 2001 Sixth St. SE, #421, Minneapolis, MN 55455.
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 15 July 1998; accepted in final form 6 January 1999.
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REFERENCES |
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