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INTRODUCTION |
Brain rhythms occurring at various frequencies appear to represent
a basic mode of operation of large neural networks, allowing for the
selective processing of information related to behavioral and
perceptual states. Theta-band oscillation and synchrony in the
hippocampal formation (HPC) and related limbic structures is recorded
as an extracellular field potential consisting of a sinusoidal-like
waveform with an amplitude 
2 mV and a narrowband frequency range of
3-12 Hz in mammals. The asynchronous activity termed large-amplitude
irregular activity (LIA) is an irregular waveform with a broadband
frequency range of 0.5-25 Hz (Leung et al. 1982
). Many
populations of cells in the HPC and related structures exhibit
discharge properties that are precisely related to hippocampal theta
field activity. Such theta-related cells comprised two distinct
populations termed theta-ON and theta-OFF, first described in acute preparations using extracellular recordings by
Colom et al. (1987), followed by a detailed cell
classification paper by Colom and Bland (1987) and
subsequently used to classify theta-related cells in the HPC in a
number of studies (Bland and Colom 1988, 1989;
Bland et al. 1996; Colom et al. 1991;
Konopacki et al. 1992; Smythe et al.
1991). Theta-ON and -OFF cells have also been recorded in the medial septal nucleus and nucleus of the
diagonal band of Broca (MS/vDBB) (Bland et al. 1990,
1994; Colom and Bland 1991; Ford et al.
1989), the entorhinal cortex (Dickson et al. 1994,
1995), cingulate cortex (Colom et al. 1988), caudal diencephalon (Bland et al. 1995; Kirk et
al. 1996), rostral pontine region (Hanada et al.
1999), the superior colliculus (Natsume et al.
1999), the basal ganglia (Hallworth and Bland
1999), the red nucleus (A. Dypvik and B. H. Bland,
unpublished data), and the neocortex (Lukatch and MacIver
1996; but see review by Buzsaki 2002 that does
not acknowledge the literature on theta-ON and theta-OFF cells). The preceding data suggest, because many
regions of the brain from the lower brain stem to the cerebral cortex display "theta-related" neuronal activity, that
theta-ON and -OFF cells may represent a general
organization of the cellular mechanisms underlying "theta band"
oscillation and synchrony (Bland 2000; Bland and
Oddie 1998). The morphological identity of
theta-ON and -OFF cells is therefore crucial to
the understanding of the cellular interactions involved in the
generation of theta field activity. Earlier studies by Fox and
Ranck (1975, 1981) provided indirect evidence that theta cells
(thetaon cells in our scheme) in the HPC were
interneurons, a view supported by some recent work on identified cells
(see review by Buzsaki 2002). On the other hand, Bland
and Colom (reviewed in Bland and Colom 1993) have
proposed, also based on indirect evidence, that a subpopulation of HPC
projection cells (pyramidal and granule cells) were
theta-ON cells and a subpopulation of HPC interneurons were
theta-OFF cells.
Theta-band oscillations may also be recorded intracellularly in some
populations of cells in the HPC, during the simultaneous occurrence of
the extracellular theta field oscillations. In agreement with previous
work (Leung and Yim 1991), we have adopted the term membrane potential oscillations (MPOs) to designate the slow
intracellular oscillations that occur at theta frequencies in subsets
of hippocampal cells. The occurrence of MPOs in HPC pyramidal cells,
dentate granule cells, and interneurons has been well documented
(Bland et al. 1988; Chapman and Lacaille
1999; Fox 1989; Fox et al. 1983; Fujita and Sato 1964; Konopacki et al.
1992 Leung and Yim 1986, 1988, 1991;
Leung and Yu 1998; MacVicar and Tse 1989;
Munoz et al. 1990; Nunez et al. 1987,
1990a-c; Ylinen et al. 1995). At present, there
is little agreement on how intracellular theta (MPOs) is generated,
some arguing that inhibitory postsynaptic potentials (IPSPs) make the
major contribution, whereas others argue the case for excitatory
postsynaptic potentials (EPSPs) or intrinsic mechanisms (Bland
and Colom 1993). As well, there is a lack of understanding of
the relationship between MPOs and the rhythmic discharge properties of
HPC cells. Do the mechanisms underlying the rhythmic discharge
properties of the cell produce the MPOs or do the mechanisms underlying
MPOs control the rhythmical cell discharges? The objectives of the
present study were to determine the contributions made by MPOs in the
control of the rhythmic discharge properties of HPC cells meeting the
criteria for being classified as theta-ON and
-OFF cells and to establish their morphological identity by
intracellular labeling. To do this, we investigated the effect of four
levels of depolarizing and hyperpolarizing constant current injections
in HPC cells in vivo, applied during the spontaneous occurrence of both
theta and LIA HPC field activities, on cellular MPOs and the rate and
pattern of cell discharges. Whenever possible, after completion of the
experimental protocol, an attempt was made to label cells through the
intracellular injection of Neurobiotin.
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METHODS |
Subjects and surgical procedures
The data were obtained from 38 male black-hooded rats
(125-130g) supplied by the Life and Environmental Sciences Animal Care Facility at the University of Calgary. The rats were initially anesthetized with Halothane while tracheal and jugular cannulae were
inserted. Halothane was then discontinued, and urethan was administered
via the jugular cannula to maintain an appropriate level of anesthesia
during the remaining surgical and experimental procedures. The rats
were placed in the stereotaxic instrument with the plane between Bregma
and lambda leveled to horizontal. Body temperature was maintained at
37°C, and heart rate was monitored constantly throughout the
experiment. An uninsulated tungsten wire placed in the cortex, anterior
to Bregma, served as the indifferent electrode and the stereotaxic
frame was connected to ground. A tungsten microelectrode (0.2-0.5
M
) for recording hippocampal (HPC) field activity was placed in the
right dorsal hippocampal formation in the dentate molecular layer
(3.0-3.3 mm posterior to Bregma, 2.0-2.5 mm lateral to the midline
and 2.4-2.8 mm ventral to the dural surface). Intracellular recordings
in the left HPC were made at the same posterior and lateral
coordinates, starting at the alvear surface and continuing ventral to
the lower (endal) blade of the dentate granule cells. Procedures for
preparing the site for intracellular recordings described previously
(Bland and Konopacki 2000) are briefly the following: a
dental acrylic circle was made on the skull encompassing the whole
recording area on the left side of the skull. This served to form the
pool for holding either glycerine or, later when recording, distilled water. At this point a bone window was drilled out using a 1/4 carbide drill bit, the dimensions ~3.0 mm long by 2.0 mm wide. The
well was filled with glycerine to keep the brain moist. Using the same carbide drill bit and holding the bone fragment down with a pair of
forceps, a hole was drilled in the center of the bone and rough edges
removed from the bone fragment by polishing with the drill bit. Dura
was removed using a No. 26 syringe with a small hook in the end and a
pair of fine forceps, and the position of the large blood vessels
observed because this determined the exact location of the bone
fragment. With the layer of glycerine in the pool and using fine
forceps, the bone fragment was placed upside down in the hole in the
skull with the long side oriented to the midline and gently pushed
under the skull at the midline enough to allow it to be pulled back
under the lateral edge. The location of the bone fragment was finessed
with respect to blood vessels, as discussed in the preceding text.
Electrophysiological recordings
Intracellular recordings in HPC cells were made with glass
microelectrodes (80-120 M) filled with 2 M potassium acetate and 2% Neurobiotin (Vector Laboratories). Details of these procedures have
been published previously (Bland and Konopacki 2000;
Konopacki et al. 1992). Cells meeting the criteria for
classification as phasic theta-ON or phasic
theta-OFF were selected for subsequent analysis.
Theta-ON cells were defined as cells that increased their
activity during theta field activity as reflected by an overall mean
increase in discharge rate or as a linear positive increase in
discharge rate in relation to increasing frequencies of
simultaneously-recorded theta field activity. Theta-OFF
cells were defined as cells that decreased their activity during theta field activity as reflected by an overall mean decrease in discharge rate (to 0 in many cases) or as a linear negative increase in discharge
rate as theta frequency declines. A further criteria relates to the
pattern of cell discharges. A given theta-related cell discharges in
one of two characteristic patterns during theta field activity. A
rhythmic discharge pattern was defined as two or more cell discharges
occurring per extracellular theta wave and was termed phasic because
the discharges occurred with a consistent phase relation to each cycle
of theta field activity. The second pattern is either regular or
irregular discharges termed tonic because they consisted of a
nonrhythmical discharge pattern with no observable phase relation to
theta field activity. Further criteria were developed on the basis of
intracellular recordings of theta-related cells in the hippocampal
formation. At theta field onset, phasic theta-ON cells
underwent a depolarizing shift of the membrane potential along with
prominent theta frequency MPOs that were highly coherent with the field
oscillations. Rhythmic cell discharges occurred on the positive peaks
of the MPOs. At theta field onset, phasic theta-OFF cells
underwent a hyperpolarizing shift of the membrane potential, prominent
MPOs appeared that were highly coherent with the field oscillations,
and cell discharges ceased. As theta frequency slowed, the cell would
discharge single spikes, gradually making a brief transition to
rhythmic cell discharges (Bland et al. 1988;
Konopacki et al. 1992). Signals were lead through an
active bridge circuit (Axon Instruments, Axoclamp 2A) allowing
simultaneous injection of current and measurement of membrane potential
(Vm). The bridge balance was monitored
and adjusted as necessary throughout the recording procedures. These and all other signals were displayed on a digital oscilloscope (Tektronix TDS 420) and stored on an FM tape recorder (Teac XR 
30)
for subsequent off-line data analysis. Once a recording was considered
stabilized the experimental protocol was initiated. First, recordings
were taken during the spontaneously occurring HPC field activities of
synchrony (theta) and asynchrony or large-amplitude irregular activity
(LIA), ensuring that 
1 min in duration of each type was accumulated.
Next a series of hyperpolarizing (100, 200, 300, and 400 pA)
and depolarizing (100, 200, 300, and 400 pA) constant current pulses
were administered during 30 s of each of the spontaneously occurring
HPC field activities of theta and LIA. In our first such series of
experiments, the current pulses were administered in random order. In
later experiments, the protocol consisted of administering the
hyperpolarizing pulses in ascending magnitude, followed by the
depolarizing pulses in ascending magnitude. Analysis revealed no
differences between the two stimulation protocols in terms of their
effects on MPOs and cell discharge rate and pattern. After completion
of this part of the protocol, a series of short-duration
hyperpolarizing current pulses (100-ms duration, 100, 200, 300,
and 400 pA) and depolarizing current pulses (100, 200, 300, and 400 pA) were administered in ascending magnitude, respectively, for
purposes of carrying out standard electrophysiological measurements.
Procedures for intracellular staining were modified slightly from the
procedures previously described (Kita and Armstrong
1991). Positive pulses were applied (5 nA, 2 Hz, 100-ms
duration) for 1-10 min and the electrode was left inside the cell for
2-3 min before it was withdrawn.
Data analysis
Data were analyzed using the Axon Instruments Axoscope 7, Microcal Origin 4.1, and Data Waves 6.1 software. Spike height and duration was determined from the first action potential evoked at
threshold levels of depolarization. Resting membrane potentials were
defined as an average of a series of a minimum of 30 membrane potentials measured between the spike discharges occurring during the
LIA field conditions. The membrane potential values for the spontaneous
theta control (no current) condition and the spontaneous theta
conditions plus intracellular current injections were defined as
average membrane potentials measured at the midpoint of the MPOs when
present. Input resistance was provided as the slope of the linear
regression line fitted through the linear portion of the
current-voltage plots derived from the family of hyperpolarizing and
depolarizing current pulse injections. MPOs, defined as the slow
intracellular membrane potential oscillations occurring at extracellular theta frequencies, were measured at the positive and
negative peaks (see Fig. 2A) using the cursor facility in the Axoscope 7 program, and calculated as the statistical average of a
minimum of 50-60 individual MPOs measured in each experimental condition. Additional data analyses included: fast Fourier transforms of the field activities of theta and LIA, autocorrelation histograms and first-order interval spike histograms of the cell discharges during
theta and LIA, cross-correlation's between MPOs and theta field
activity, and quantification and statistical analysis using t-tests of the spike discharges occurring during each
experimental condition.
Imaging techniques and cellular identification
At the termination of a recording session, all animals were
administered an overdose of pentobarbital sodium and perfused transcardially with 50 ml phosphate-buffered saline (PBS, 0.1 M, pH
7.4) followed by 300 ml of 4% paraformaldehyde in PBS. The brain was
removed and immersed in the fixative for an additional 4 h and
then cryoprotected, overnight, in a solution of 30% sucrose in PBS.
The brains were frozen, and coronal sections were cut at 50 µm on a
sliding microtome. Every section through the hippocampal formation was
collected in PBS and then incubated for 2 h in a 1:500 solution of
avidin-HRP in PBS. After three washes in PBS, the
Neurobiotin-avidin-HRP complex was visualized by incubating the
sections in a chromogen solution consisting of 10 ml 0.1 M Tris-buffered saline containing 5 mg diaminobenzidine, 40 µg nickel ammonium sulfate, and 10 µl 30% hydrogen peroxide. When the reaction was complete, the sections were washed three times in PBS and then
mounted onto gelatin-coated glass slides and allowed to dry. The
sections were dehydrated in an ascending series of ethanol, cleared in
xylene and cover slipped using Permount. The labeled cells were
identified using a Zeiss Axioplan 2 microscope at ×20, and digital
images were captured and serially reconstructed using Open lab (v. 3.0, Improvision) and Adobe Photoshop (v. 6.0, Adobe) running on an Apple G4.
| |
RESULTS |
Cells classified as phasic theta-ON cells
CA1 PYRAMIDAL CELLS.
We describe here the results of intracellular recordings of cells
(n = 18) in the HPC of urethan-anesthetized rats,
classified as phasic theta-ON cells (comparison of group
mean discharge rates for theta vs. LIA significant at P < 0.05, paired t-test). These cells discharged in a
rhythmic pattern during theta but not during LIA, and MPOs were
recorded only during the theta field condition, their onset signaled by
a 5- to 10-mV depolarizing shift in the membrane potential. Each
rhythmic discharge occurred on the depolarizing phase of the MPO and
was phase locked to the simultaneously occurring extracellular theta
waves. The average resting membrane potential was 62.6 ± 0.91 (SE) mV, average spike height 62.5 ± 1.8 mV, and average
input resistance 31.3 ± 1.6 M. Of the 18 cells classified as
phasic theta-ON cells, 7 were successfully labeled and
morphologically identified as CA1 pyramidal cells. Unlabeled cells were
inferred to be pyramidal cells as well based on their anatomical
location in the CA1 pyramidal layer and their electrophysiological
characteristics, which did not differ from those of the labeled cells.
Figure 1A shows an example of
a CA1 pyramidal cell (102) recorded during the simultaneous
occurrence of either HPC theta (left half) or LIA
(right half). MPOs were recorded during the theta field
condition only and their onset was signaled by a 5- to 10-mV depolarizing shift in membrane potential. Gamma oscillations were recorded in all cells during both theta and LIA (average frequency = 70 Hz). During theta field activity, the cell discharged in a
rhythmic pattern, had a higher discharge rate (12.4 ± 0.7 Hz) compared with the LIA condition (10.3 ± 0.5 Hz), and cell
discharges occurred on the depolarizing phase of MPOs with an average
amplitude of 9.7 ± 0.3 mV. The MPOs occurred at the same
frequency (2.9 ± 0.04 Hz) and phase of the extracellular theta.
The positive peak of the MPO corresponded to the positive peak of the
theta recorded from the dentate. This would correspond to the negative peak of theta recorded from the CA1 cell layer since the theta field
activity recorded from these two regions is ~180° out of phase
(Bland and Whishaw 1976). During LIA, the cell
discharges were irregular and MPOs were absent. Average spike amplitude
during both the LIA and theta conditions was 60 mV. Figure
1B shows the fast Fourier transform (FFT) of the theta field
seen in A, top left, which revealed a peak in power at 2.9 Hz. Figure 1C shows the autocorrelation histogram of the
rhythmical cell discharges associated with the theta field seen in
A, top left. The intervals of regular peaks in the histogram
(340 ms) coincided with the theta field frequency. The bimodal
distribution of the interspike interval histogram of cell discharges
during theta field activity (Fig. 1D) shows the within
rhythmic discharge and between rhythmic discharge intervals
respectively, indicating a rhythmical discharge pattern. Figure
1E shows the FFT of the LIA field activity seen in A,
top right, indicating the absence of a peak in power at any
narrowband frequency. The autocorrelation histogram (Fig. 1F) of the cell discharges associated with the LIA field
seen in A, top right, shows the absence of peaks, thus
indicating an irregular cell discharge pattern. This is also
illustrated by the interspike interval histogram (Fig. 1G)
of cell discharges during LIA field activity showing the absence of a
bimodal distribution.
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Fig. 1.
A: relationships between spontaneously occurring
hippocampal (HPC) field activity and CA1 pyramidal cell discharges in
the no current control condition. Top: the HPC field
activity recorded from the molecular layer of the dentate region,
bottom: the discharge pattern of a CA1 pyramidal cell
(102) classified as a phasic theta-ON cell
(positivity up in all traces). The 1st half of the panel
shows the rhythmic cell discharge pattern and membrane potential
oscillations (MPOs) occurring during HPC theta and the 2nd
half of the panel shows the irregular cell discharge pattern
occurring during HPC large-amplitude irregular activity (LIA). Note the
absence of MPOs. Gamma oscillations (mean frequency = 70 Hz) were
visible in all cell traces during the occurrence of both theta and LIA.
B: fast Fourier transform of the theta field in
A, top left. C: autocorrelation histogram of the
rhythmic cell discharges associated with the theta field in A,
top left. The intervals of regular peaks in the histogram (340 ms) coincided with the theta field frequency. D:
interspike interval histogram of cell discharges during theta field
activity. The bimodal distribution shows the intraburst and interburst
intervals respectively, indicating a rhythmic discharge pattern.
E: fast Fourier transform of the LIA field activity in
A, top right, showing the absence of a peak in power at
any narrowband frequency. F: autocorrelation histogram
of the irregular cell discharges associated with the LIA field in
A, top right, showing the absence of peaks.
G: interspike interval histogram of cell discharges
during LIA field activity showing the absence of a bimodal
distribution, indicating the irregular cell discharge pattern.
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Figure 2A shows a segment of
spontaneous theta with the cell trace amplified to illustrate the MPOs
and the cursor method used to measure MPO amplitude, whereas Fig.
2B is a segment of LIA illustrating the absence of MPOs in
the cell trace. Figure 2C shows the response of this cell to
a 200-pA depolarizing intracellular current pulse, applied during the
occurrence of spontaneous theta field activity. All cells responded in
this simple spike pattern of stimulus-graded trains of independent
action potentials (Jensen et al. 1996). Figure
2D presents the current-voltage plot along with the linear
regression curve (input resistance = 27.2 M).
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Fig. 2.
A: segment of spontaneous theta from left
half of Fig. 1A with cell trace amplified to
show the slow MPO and method of measurement using cursors (MPO = 8.6 mV; positivity up in all traces). B: segment of
spontaneous LIA from Fig. 1A, right half, showing
absence of MPOs. C: intracellular depolarizing current
pulse (200-pA, 100-ms duration) applied to cell shown in Fig.
1A during spontaneously occurring theta. Note the simple
spike pattern. D: current- voltage plot of the cell
shown in Fig. 1A, input resistance = 22.5 M.
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Figure 3 shows a CA1 pyramidal cell
(154; spikes truncated) during the control (no current)
condition and the cell's responses to hyperpolarizing
(left) and depolarizing (right) intracellular constant current injections administered during spontaneously occurring
HPC theta field activity, highlighting the effects on cell discharge
pattern and MPO amplitude. Phase measurements (data not shown) made
comparing the simultaneously occurring extracellular theta field and
MPOs revealed maximal phase shifts of <5° occurred during any level
of the hyperpolarizing or depolarizing constant current injections. The
HPC theta field frequency remained stationary throughout the
administration of all current levels (3.9 ± 0.04 Hz). Figure 3,
top middle, shows that during the no current control condition the cell exhibited rhythmical discharges with average MPO
amplitudes of 6.5 ± 0.4 mV occurring at an average membrane potential of 61 mV. Figure 3 (left) shows that during the
injection of 100 pA the cell retained a rhythmical discharge pattern
and average MPO amplitudes were reduced (5.7 ± 0.6 mV) at an
average membrane potential of 64.7 mV. Figure 3 (left)
shows that during the injection of 200 pA, the rhythmical discharge
pattern was abolished (although single spikes were still phase-locked
to the extracellular theta field) and average MPO amplitudes were
reduced (3.4 ± 0.4 mV) at an average membrane potential of 67.3
mV. Figure 3 (left) shows that during the injection of 300
pA all cell discharges were abolished and average MPO amplitudes were
reduced (2.4 ± 0.4 mV) at an average membrane potential of 73.3
mV. Figure 3 (left) shows that during the injection of 400
pA all cell discharges were abolished and average MPO amplitudes were
reduced to zero with an average membrane potential of 80.9 mV. Figure
3 (right) shows that during the injection of 100 pA, the
cell exhibited rhythmical discharges. The MPOs decreased in amplitude
(compared with the no current condition; 4.9 ± 0.7 mV) at an
average membrane potential of 58.4 mV. Figure 3 (right)
shows that during the injection of 200 pA the cell exhibited rhythmical
discharges and the MPOs decreased in amplitude (4.9 ± 0.5 mV) at
an average membrane potential of 55.4 mV. Figure 3 (right)
shows that during the injection of 300-pA rhythmical discharges were
abolished and the MPOs decreased in amplitude (3.4 ± 0.3 mV) at
an average membrane potential of 54 mV. Figure 3 (right)
shows that during the injection of 400-pA cell rhythmicity was
abolished at an average membrane potential of 51 mV, and there were
no longer any measurable MPOs.
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Fig. 3.
The control (no current) condition (top middle) and
hyperpolarizing (left) and depolarizing
(right) intracellular constant current injections during
the occurrence of spontaneous theta field activity, highlighting the
effects on the average value of the membrane potential, the average MPO
amplitudes and cell discharge patterns of a CA1 pyramidal phasic
theta-ON cell (154). Spikes truncated
(positivity up in all traces).
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Data in both Fig. 4, A and
B, is plotted against the average membrane potential values
produced by the current injections and the average membrane potential
value occurring during the control (no applied current) condition for
cell 154. Figure 4A graphically summarizes the
effects of hyperpolarizing and depolarizing constant current injections
and the control (no applied current) condition on the spike discharge
pattern and rate during the theta and LIA conditions for the cell shown
in Fig. 3. In the control (no current) condition, the rhythmic cell
discharge pattern occurred at a mean rate of 6.5 ± 0.4 Hz,
whereas during LIA cell discharge pattern was irregular at a mean rate
of 5.0 ± 0.7 Hz. As the membrane potential was more depolarized
from the value of the membrane potential during the spontaneous (no
current) control condition, the number of cell discharges increased
during both the theta and LIA conditions. The discharge rate remained
significantly higher during theta compared with LIA (P < 0.005) and rhythmic cell discharges occurred only during the theta
condition. Within the theta condition, rhythmical discharges were
abolished at membrane potential values depolarized above 55.4 mV.
Cell amplitude during the 200- and 300-pA conditions decreased to 55 mV
and decreased further to 42 mV during the 400-pA condition.
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Fig. 4.
A: graph summarizing the effects of hyperpolarizing and
depolarizing constant current injections and the control (no applied
current) condition on the cell discharge pattern and rate during the
theta and LIA conditions for cell 154 shown in Fig. 3.
B: graph summarizing the effects of hyperpolarizing and
depolarizing constant current injections and the control (no applied
current) condition, on the MPO amplitudes during the theta condition
for cell 154. Data in both A and
B are plotted against the average membrane potential
values produced by the current injections and the average membrane
potential value occurring during the control (no applied current)
condition. Standard error bars showing positive half only in
A.
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As the membrane potential was hyperpolarized from the value of the
membrane potential during the spontaneous (no current) control
condition, the number of cell discharges decreased during both the
theta and LIA conditions. Rhythmical cell discharges occurring during
the theta condition only were completely abolished at membrane
potentials hyperpolarized below 64.7 mV. In the 200-pA current
condition, rhythmic discharges were abolished during theta; however,
when single discharges did occur, they remained phase-locked to HPC
theta waves. In the 300-pA current condition, rhythmic discharges
were also abolished during theta; but again, when single discharges
occurred they remained phase-locked to HPC theta.
Figure 4B graphically summarizes the effects of
hyperpolarizing and depolarizing constant current injections and the
control (no applied current) condition on the MPO amplitudes during the theta condition for the cell (154) shown in Fig. 3. The
effects of current injections during the theta field condition on MPO amplitudes revealed an inverted U-shaped curve. The curve was asymmetrically distributed around the average value of the membrane potential occurring during the spontaneous theta (no current) control
condition. A comparison of the graph in Fig. 4A with the graph in B revealed that rhythmical cell discharges during
the theta condition occurred in a critical range of membrane
potential values from 55.4 to 64.7 mV. This corresponded to a range
of MPO amplitudes of ~4-7 mV. The 4- to 7-mV amplitude range was skewed such that more of the larger MPO amplitudes occurred at membrane
potentials depolarized above the average value in the control condition
compared with the MPO amplitudes that occurred when the membrane
potential was hyperpolarized.
The group data for all 18 CA1 phasic theta-ON cells
are summarized in Fig. 5, presented as
means and standard errors of the mean. In this figure, each level of
constant current injection has again been converted along the abscissa
to the average value of the membrane potential produced by the current
injection for each cell, and then averaged across all 18 cells (SEs
across all levels of current injections ranged from ±1.0 to ±1.4 mV,
whereas the range for +400 pA was 45 to 55 mV and the range for
400 pA was 73 to 90 mV). The effects of constant current
injections during the theta field condition on MPO amplitudes, averaged
across all cells, again revealed an inverted U-shaped curve. The curve was asymmetrically distributed around the maximal amplitude (6.9 ± 0.3 mV, range: 5.4-9.9 mV) of the MPOs occurring during the average
membrane potential (64 mV) associated with the spontaneous theta no
current condition. Phase measurements (data not shown) revealed no
significant phase shifts occurring in any of the 18 CA1 phasic
theta-ON cells.
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Fig. 5.
Graph summarizing the group data for all CA1 pyramids classified as
phasic theta-ON cells (n = 18), plotted
against the average membrane potential value for the control (no
applied current) spontaneous theta condition and the average membrane
potential values produced by the constant current injections. The
left axis shows the mean cell discharge rate and the
right axis shows the average MPO amplitude. The plot of
MPO amplitudes revealed an inverted U-shaped curve asymmetrically
distributed around the value of the membrane potential during the
spontaneous theta condition. Rhythmic cell discharges only occurred
during theta and only when MPO amplitudes were in the range of
~4.0-7.0 mV. All data plotted as means ± SE.
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The effects of current injections during the theta field
condition on the rate of spike discharges averaged across all cells revealed an increase from near 0 Hz (12 of the 18 cells had cell discharge rates reduced to 0) at the maximum hyperpolarized membrane potential of - 81 mV to 18.1 Hz at the maximally depolarized membrane potential of 49 mV. The effects of current injections during the LIA
field condition on the rate of cell discharges revealed an increase
from 0 to 13.7 ± 0.5 Hz over the range of membrane potentials
from 81 to 49 mV, respectively. With the exception of the two most
hyperpolarized values of 77 and 81 mV where cell discharges were
reduced to zero in 12 of the 18 cells, the discharge rates during LIA
were significantly lower than those occurring during the theta
condition (P < 0.005). Also, unlike the theta
condition, rhythmical discharges did not occur at any value of the
membrane potential during the LIA condition.
A comparison of the graphs revealed that rhythmical cell discharges
during the theta condition occurred in a critical range of membrane
potential values from approximately 57 to 68 mV. This corresponded
to a range of MPO amplitudes of ~4-7 mV. The 4- to 7-mV
amplitude range was skewed such that more of the critical range of MPO
amplitudes occurred at membrane potentials depolarized above the
average value in the control (no current) condition compared with the
MPO amplitudes that occurred when the membrane potential was
hyperpolarized. Thus the group data for all 18 cells did not differ
from the single cell data shown in Fig. 4.
Figure 6, left, shows an
example of a CA1 pyramidal cell (226) recorded during the
simultaneous spontaneous (no current) occurrence of either HPC theta
(left half of the panel) or LIA (right half of
the panel). MPOs were recorded during the theta field condition only.
The Fig. 6, right, shows that injecting Neurobiotin into cell 226 resulted in the labeling of 2 CA1 pyramidal cells.
Of 7 labeled cells, 57% (n = 4) resulted in double
labels (2 pyramidal cells) and 3 were single labels.
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Fig. 6.
Relationships between spontaneously occurring HPC field activity and
CA1 pyramidal cell (226) discharges in the no current control
condition. Left, top: is the HPC field activity recorded
from the molecular layer of the dentate region; bottom:
the discharge pattern of a CA1 pyramidal cell classified as a phasic
theta-ON cell. (positivity up in all traces). The
1st half of the panel shows the rhythmic cell discharge
pattern and MPOs occurring during HPC theta, and the 2nd
half of the panel shows the irregular cell discharge pattern
occurring during HPC LIA. Note the absence of MPOs. Gamma oscillations
(mean frequency = 70 Hz) were visible in all cell traces during
the occurrence of both theta and LIA. Right: the
intracellular injection of Neurobiotin into this CA1 pyramidal cell
resulted in the labeling of 2 CA1 pyramids. These results were observed
when injecting 4 of 7 (57%) successfully labeled cells.
Top: a low-power magnification showing the location of
the cells in the CA1 cell pyramidal layer. Bottom: a
higher power magnification showing the details of the cell morphology
Calibration bar = 50 µ.
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BISTRATIFIED INTERNEURONS.
The left side panel in Fig.
7 shows an example of one of two
bistratified cells (216) recorded during the simultaneous
occurrence of either HPC theta (left half of the panel) or
LIA (right half of the panel). We were unable to measure
MPOs of these cells during the occurrence of either theta or LIA field
activity. Both cells had a pronounced afterhyperpolarization (see Fig.
7) and showed the highest number of rhythmic cell discharges per theta
wave (mean = 6.5 ± 1.2 Hz) compared with all other cells in
the study. The mean discharge rate during theta field activity was
24 ± 3.1 Hz and during LIA was 20 ± 2.7 Hz. The right side
panel shows that cell 216 was located in the CA1 pyramidal
layer (as was the other cell) and was identified as a bistratified
interneuron.
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Fig. 7.
Relationships between spontaneously occurring HPC field activity and
cell discharges of a bistratified cell (216) classified
as a phasic theta-ON cell, in the no current control
condition. Top panel, top trace: the HPC field activity
recorded from the molecular layer of the dentate region, bottom
trace: the discharge pattern of the cell (positivity up in all
traces). The 1st half of the panel shows the rhythmic
cell discharge pattern occurring during HPC theta and the 2nd
half of the panel shows the irregular cell discharge pattern
occurring during HPC LIA. Note the absence of MPOs. Bottom
left: a single spike at a fast sweep to illustrate the
prominent afterhyperpolarizing potential. Bottom right:
the intracellular injection of Neurobiotin resulted in the labeling of
a bistratified interneuron; top: low-power magnification
showing the location of the cell in the CA1 cell pyramidal layer;
bottom: is a higher-power magnification showing the
details of the cell morphology. Calibration bar = 50 µ.
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Figure 8 shows cell 216 (spikes truncated) during the control (no current) condition (top
middle) and the cell's responses to hyperpolarizing
(left) and depolarizing (right) intracellular constant current injections administered during spontaneously occurring
HPC theta field activity, highlighting the effects on cell discharge
pattern and MPO amplitude. The effects of these manipulations on cell
discharge pattern and frequency of the two bistratified cells were
different in some respects from those reported in the preceding text
for CA1 pyramidal phasic theta-ON cells (see Fig.
9).
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Fig. 8.
The control (no current) condition (top middle) and
hyperpolarizing (left) and depolarizing
(right) intracellular constant current injections during
the occurrence of spontaneous theta field activity, highlighting the
effects on the average value of the membrane potential, the average MPO
amplitudes and cell discharge patterns of bistratified phasic
theta-ON cell 216. Spikes truncated
(positivity up in all traces).
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Fig. 9.
Graph summarizing the effects of hyperpolarizing and depolarizing
constant current injections and the control (no applied current)
condition, on the cell discharge pattern and rate during the theta and
LIA conditions for cell 216 shown in Figs. 7 and 8. Data
are plotted against the average membrane potential values produced by
the current injections and the average membrane potential value
occurring during the control (no applied current) condition. SE bars
showing positive half only.
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Figure 9 reveals that the bistratified cell 216 responded
overall to depolarizing current injections with more cell discharges during theta field activity compared with LIA field activity similar to
CA1 pyramidal phasic theta-ON cells. However, during theta and LIA, increasing levels of depolarizing current injections resulted
in a steady decline in the number of cell discharges, the opposite to
the response of CA1 pyramidal phasic theta-ON cells. The
response of the bistratified cells to increasing levels of
hyperpolarizing current injections was similar to that of CA1 pyramidal
theta-ON cells in that increasing levels of hyperpolarizing current resulted in decreasing cell discharge rates, with complete cessation of discharges at 300 and 400 pA.
Cells classified as phasic theta-OFF cells
We describe here the results of intracellular recordings
of cells (n = 22) in the HPC of urethan-anesthetized
rats classified as phasic theta-OFF cells. These cells were
mostly silent during theta and discharged during LIA while MPOs were
recorded only during the theta field condition, their onset signaled by
a 5- to 10-mV hyperpolarizing shift in membrane potential. The cell discharges that did occur during low-frequency theta discharged on the
depolarizing phase of the MPOs and were phase locked to the
simultaneously occurring extracellular theta waves. Nine of the phasic
theta-OFF cells were recorded from the stratum pyramidale, 11 were recorded from the dentate granule cell layer, and 2 were recorded in the hilus. The average resting membrane potential of the
CA1 layer basket cells was 66.6 ± 0.91 mV, average spike height
60.5 ± 1.8 mV, and average input resistance 29.4 ± 6.1 M. Of the nine cells recorded in the CA1 pyramidal cell layer and
classified as phasic theta-OFF cells, one was successfully labeled and morphologically identified as a CA1 pyramidal layer basket
cell. Unlabeled cells were inferred to be CA1 pyramidal layer basket
cells as well based on their anatomical location in the CA1 pyramidal
layer, electrophysiological characteristics, and responses to current
manipulations, which did not differ from that of the labeled cell. The
average resting membrane potential of the dentate layer cells was
65 ± 4.91 mV, average spike height 58 ± 1.8 mV, and
average input resistance 32 ± 3.1 M. Of the 11 cells recorded
in the dentate layer, 3 were labeled and identified as dentate granule
cells. Unlabeled cells were inferred to be granule cells as well, based
on their anatomical location in the denate granule layer and their
electrophysiological characteristics, which did not differ from that of
the labeled cells. Of the two cells recorded in the hilus, one was
labeled and identified as a mossy cell interneuron.
CA1 PYRAMIDAL LAYER BASKET CELLS.
Figure 10, A and
B, shows examples of a CA1 layer basket cell
(229) recorded during the simultaneous occurrence of either
LIA (left half of the panel) or HPC theta (right
half of the panel). MPOs were recorded during the theta field
condition only. In Fig. 10A, theta frequency was slightly
higher (4.3 Hz) and the cell failed to discharge. In Fig.
10B, theta frequency was lower (3.4 Hz) and the cell began
to discharge, first single discharges and then a doublet. Figure
10C shows the response of this cell to a 200-pA depolarizing
intracellular current pulse, applied during the occurrence of
spontaneous theta field activity. Figure 10D reveals that
the intracellular injection of Neurobiotin into cell (229)
resulted in the labeling of a cell identified as a basket cell
interneuron. The top panel is a low-power magnification
showing the location of the cell in the CA1 cell pyramidal layer. The bottom panel is a higher-power magnification showing the
details of cell morphology (axons not drawn). The axons of the basket cell were visually confirmed to form a plexus restricted to the CA1
pyramidal layer. Figure 10E presents the current-voltage
plot along with the linear regression curve (input resistance = 29.4 M).
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Fig. 10.
A and B: relationships between
spontaneously occurring HPC field activity and the cell discharges of a
CA1 layer basket cell (229) classified as a phasic
theta-OFF cell, in the no current control condition.
Top trace in each panel is the HPC field activity
recorded from the molecular layer of the dentate region; bottom
trace is the discharge pattern of the cell (positivity up in
all traces). The 1st half of the panels in
A and B show the irregular cell discharge
pattern occurring during HPC LIA. Note the absence of MPOs. The
2nd half of the panel in A shows the
complete cessation of cell discharges during theta field activity with
at a higher frequency (4.3 Hz) and the occurrence of MPOs. The 2nd half
of the panel in B shows that as theta field frequency
slowed to 3.4-Hz, phase-locked cell discharges began to occur. Again,
MPOs were recorded during theta field activity. C:
intracellular depolarizing current pulse (200-pA, 100-ms duration)
applied to the cell during spontaneously occurring theta.
D: the intracellular injection of Neurobiotin into
cell 229 resulted in the labeling of a cell identified
as a CA1 pyramidal layer basket cell. Top: a low-power
magnification showing the location of the cell in the CA1 cell
pyramidal layer. Bottom: a higher-power magnification
showing the details of cell morphology. Calibration bar = 50 µ.
E: current-voltage plot of the cell shown in
Fig. 1A, input resistance = 29.4 M.
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Figure 11 shows CA1 layer basket cell
(229; spikes truncated) during the control (no current)
condition and the cell's responses to hyperpolarizing
(left) and depolarizing (right) intracellular constant current injections administered during spontaneously occurring
HPC theta field activity, highlighting the effects on cell discharge
pattern and MPO amplitude. Phase measurements (data not shown) made
comparing the simultaneously occurring extracellular theta field and
MPOs revealed that during the control (no current) condition, the
positive peak of the MPOs corresponded with the negative peak of the
extracellular theta field activity recorded from the dentate region.
This was exactly opposite for the phase relations described in the
preceding text for the CA1 pyramidal phasic theta-ON cells.
As discussed in the preceding text, the positive peak of the MPO would
correspond to the positive peak of theta recorded from the CA1 cell
layer because the theta field activity recorded from these two regions
is ~180° out of phase (Bland and Whishaw 1976). A
maximal phase shift of ~90° began to occur during the injection of
a 100-pA depolarizing constant current injection and shifted ~180°
to become in phase with the extracellular theta field activity during
the injection of 200, 300, and 400 pA (Fig. 11, right). The
MPOs remained ~180° out of phase with the extracellular theta field
activity during the injection of 100, 200, 300, and 400 pA
(Fig. 11, left). The HPC theta field frequency remained
stationary throughout the administration of all current levels
(4.1 ± 0.06 Hz). Figure 11, top middle, shows the cell
failed to discharge during the no current control condition with
average MPO amplitudes of 3.9 ± 0.5 mV occurring at an average membrane potential of 66.4 mV. Figure 11 (left) shows that
during the injection of 100, 200, 300, and 400 pA the cell
failed to discharge. Figure 11 (right) shows that during the
injection of 100 pA, the cell exhibited single cell discharges
occurring on average just prior to extracellular theta peak positivity. During the injection of 200 pA, the cell exhibited cell discharges now
occurring at peak positivity. Figure 11 (right) shows that during the injection of 300-pA rhythmical discharges phase-locked to
the extracellular theta field began to occur. During the injection of
400 pA, cell phase-locked rhythmical discharges continued with an
increase in the number of cells per burst. Note this is exactly opposite for the results of these levels of depolarizing current injections in CA1 pyramidal phasic theta-ON cells (i.e.,
rhythmicity was abolished in these cells).
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Fig. 11.
The control (no current) condition (top middle) and
hyperpolarizing (left) and depolarizing
(right) intracellular constant current injections during
the occurrence of spontaneous theta field activity, highlighting the
effects on the average value of the membrane potential, the average MPO
amplitudes and cell discharge patterns of CA1 pyramidal layer basket
phasic theta-OFF cell (229). Spikes
truncated (positivity up in all traces).
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Data in Fig. 12, A and
B, are plotted against the average membrane potential values
produced by the current injections and the average membrane potential
value occurring during the control no applied current condition for
cell 229. Figure 12A graphically summarizes the
effects of hyperpolarizing and depolarizing constant current injections
and the control (no applied current) condition on the cell discharge
pattern and rate during the theta and LIA conditions for the cell shown
in Figs. 10 and 11. In the control (no current) condition, the cell
failed to discharge while during LIA cell discharge pattern was
irregular at a mean rate of 0.8 ± 0.1 Hz. As the membrane
potential was more depolarized from the value of the membrane potential
during the spontaneous (no current) control condition, the number of
cell discharges increased during both the theta and LIA conditions.
Interestingly, cell discharges increased more during the LIA condition
compared with the theta condition, the exact opposite of the data for
CA1 pyramidal phasic theta-ON cells. Also, in contrast
to the data for CA1 pyramidal phasic theta-ON cells,
rhythmic cell discharges occurred only during the theta condition and
then only at the most depolarized membrane potential values (300 and
400 pA). In the 100- and 200-pA current conditions, the single-cell
discharges that occurred were phase-locked to HPC theta. At all
hyperpolarized membrane potential values and during the spontaneous (no
current) control condition, there was a total failure of cell
discharges.
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Fig. 12.
A: graph summarizing the effects of hyperpolarizing and
depolarizing constant current injections and the control (no applied
current) condition on the cell discharge pattern and rate during the
theta and LIA conditions for cell 229 shown in Figs. 11
and 12. B: graph summarizing the effects of
hyperpolarizing and depolarizing constant current injections and the
control (no applied current) condition on the MPO amplitudes during the
theta condition for cell 154. Data in both
A and B are plotted against the average
membrane potential values produced by the current injections and the
average membrane potential value occurring during the control (no
applied current) condition. SE bars showing positive half only in
A.
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Figure 12B graphically summarizes the effects of
hyperpolarizing and depolarizing constant current injections and the
control (no applied current) condition on the MPO amplitudes during the theta condition for cell 229. The effects of current
injections during the theta field condition on MPO amplitudes revealed
a curve asymmetrically distributed around the average value of the membrane potential occurring during the spontaneous theta (no current)
control condition. There was a trend for MPO amplitudes to reduce with
increasing membrane hyperpolarization that was rendered nonsignificant
due to variability but there was a significant increase in MPO
amplitudes with increasing levels of depolarization (P < 0.001).
The data presented in the preceding text for basket cell 229 was representative of all the data collected for the remaining eight
basket cells.
MOSSY CELL INTERNEURONS.
Figure 13 (left side panel)
shows a transition from LIA to theta field activity (top
trace) and the accompanying cell discharges of a labeled mossy
cell (203) located in the hilus (right side panel). The cell discharged at a mean rate of 7.0 ± 1.1 Hz
during LIA and no MPOs were recorded. The beginning of theta was marked by a hyperpolarizing shift in the membrane potential along with the
presence of clear MPOs and the cessation of cell discharges. As the
membrane potential became more depolarized, a few cell discharges
occurred. In the interest of identifying the cell, the stimulation
protocol was not carried out. Figure 13, right, shows that
injecting Neurobiotin into cell 203 resulted in the labeling
of a mossy cell interneuron in the hilus of the dentate gyrus. Similar
relations between cell discharges and spontaneous theta and LIA were
observed for one other unlabeled cell recorded from the hilar region.
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Fig. 13.
Relationships between spontaneously occurring HPC field activity and
the cell discharges of a mossy hilar cell (203)
classified as a phasic theta-OFF cell in the no current
control condition. Top trace: the HPC field activity
recorded from the molecular layer of the dentate region; bottom
trace: the discharge pattern of the cell (positivity up in all
traces). The 1st half of the panel shows the irregular
cell discharge pattern occurring during HPC LIA. Note the absence of
MPOs. The 2nd half of the panel shows the initial
cessation of cell discharges during theta field activity followed by a
few phase-locked discharges as theta frequency reduced. MPOs occurred
along with theta field activity. Right: the
intracellular injection of Neurobiotin into the cell. Right,
top: a low-power magnification showing the location of the
mossy cell in the hilar reion of the dentate gyrus.
Bottom: a higher-power magnification showing the details
of the cell morphology. Calibration bar = 50 µ.
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GRANULE CELLS.
Figure 14 (left side panel)
shows a transition from LIA to theta field activity (top
trace) and the accompanying cell discharges of a labeled granule
cell located in the lower blade of the denate granule layer
(right side panel). The cell discharged at a mean rate of
3.0 ± 4.1 Hz during LIA and no MPOs were recorded. The beginning
of theta was marked by a hyperpolarizing shift of 5.2 mV in the
membrane potential along with the presence of clear MPOs and the
cessation of cell discharges. These data are representative of all 11 granule cells recorded. We found these cells very difficult to hold for
long periods so in the interest of identifying the cell, the
stimulation protocol was not carried out. Figure 14, right,
shows that injecting Neurobiotin into cell 190 resulted in
the labeling of three granule cells in the lower blade of the granule
cell layer. The injection of Neurobiotin into granule cells
218 and 220 both resulted in the labeling of two
granule cells (data not shown).
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Fig. 14.
Relationships between spontaneously occurring HPC field activity and
cell discharges of a denate granule cell (190)
classified as a phasic theta-OFF cell in the no current
control condition. Top trace: the HPC field activity
recorded from the molecular layer of the dentate region; bottom
trace: the discharge pattern of the cell (positivity up in all
traces). The 1st half of the panel shows the irregular
cell discharge pattern occurring during HPC LIA. Note the absence of
MPOs. The 2nd half of the panel shows the complete
cessation of cell discharges during theta field activity. MPOs occurred
along with theta field activity. Right: the
intracellular injection of Neurobiotin into the cell resulted in the
labeling of 3 dentate granule cells. Right, top: a
low-power magnification showing the location of the cells in the lower
blade of the dentate gyrus. Bottom: a higher-power
magnification showing the details of the cells morphology.
Calibration bar = 50 µ.
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DISCUSSION |
Cells classified as phasic theta-ON cells
In the present study, all 18 CA1 pyramidal cells responded with a
simple cell discharge pattern to depolarizing current pulses. Jensen et al. (1996) demonstrated that CA1 pyramidal
cells fell into two categories according to their response to
depolarizing current pulses: nonbursters (simple spikes) and bursters
(complex spikes). Nonbursters generated stimulus-graded trains of
independent action potentials with nondecrementing amplitudes, whereas
bursters generated clusters of three or more closely spaced spikes of
descending amplitude riding on a distinct depolarizing envelope.
The present study has provided evidence supporting the following
conclusions concerning cells classified as phasic theta-ON cells: 1) morphologically identified hippocampal CA1
pyramidal cells formed a subset of cells meeting the criteria for
classification as phasic theta-ON cells. Previous studies
(Fujita and Sato 1964; Leung and Yim 1988,
1991; Nunez et al. 1987, 1990a) have clearly demonstrated the rhythmical discharge properties of CA1 pyramidal cells; 2) MPOs occurred only during theta field activity,
their onset signaled by a 5- to 10-mV depolarizing shift in membrane potential; 3) the amplitude of membrane potential
oscillations in CA1 pyramidal phasic theta-ON cells was
voltage dependent and frequency was voltage independent; 4)
there were no phase changes observed during current injections;
however, amplitude analysis of MPOs revealed an inverted U-shaped curve
asymmetrically distributed around the average value of the membrane
potential occurring during the spontaneous theta (no current) control
condition; 5) the rate of rhythmic cell discharges in the
CA1 pyramidal phasic theta-ON cells during the theta
condition was precisely controlled within a critical range of membrane
potential values from approximately 57 to 68 mV, corresponding to a
range of MPO amplitudes of ~4-7 mV. Outside the critical range,
rhythmical discharges were abolished. And 6) there appear to
be some interneurons that meet our criteria for being phasic
theta-ON cells. These are the bistratified cells first
described by Buhl et al.(1994). However, MPOs were not
recorded in these cells during theta field activity and they responded in a different manner to depolarizing constant current injection.
Mechanisms underlying the generation of MPOs in identified CA1
pyramidal cells
The present study demonstrated that MPOs occurred only during
spontaneous theta field activity and not during the spontaneous occurrence of LIA field activity. Previous data on the mechanisms underlying the generation of MPOs may be summarized as supporting three
main conclusions: they are generated by inhibitory postsynaptic potentials (IPSPs); they are generated by excitatory postsynaptic potentials (EPSPs); and they are generated by intrinsic properties of
the cell membrane. Evidence supporting the IPSP hypothesis came from
observations that the laminar profile of antidromically evoked IPSPs in
the CA1 region was similar to that of the theta rhythm of anesthetized
animals (Artemenko 1973; Fox et al.
1983). Leung and Yim (1986) provided stronger
support by demonstrating that MPOs recorded in CA1 pyramidal cells
reversed in phase with respect to extracellularly recorded theta at a
reversal potential corresponding to GABA-mediated IPSPs. Further
support for the importance of IPSPs has come from studies by Fox
(1989), Soltesz and Deschenes (1993), and
Ylinen et al. (1995). The main support for the EPSP
hypothesis has derived from two main observations: MPOs do not reverse
with membrane voltage or the intracellular injection of
Cl
and the MPO was larger with
hyperpolarization. (Fujita and Sato 1964; Nunez
et al. 1987, 1990a). The main support for the intrinsic generation hypothesis has come from studies using in vitro hippocampal preparations. Leung and Yim (1988, 1991) demonstrated
theta frequency MPOs could be induced in hippocampal cells solely by
depolarization and even when synaptic transmission was blocked by low
Ca2+, low Na+, and
tetrodotoxin. In the present study, the demonstration of an inverted
U-shaped function following the injection of depolarizing and
hyperpolarizing constant current pulses also argued against the
dependence of MPOs on synaptic activity. There were no phase reversals
observed between the MPOs and the extracellularly recorded theta field
activity and hyperpolarization of the membrane potential did not
increase the size of the MPOs. Nunez et al. (1990a) also reported that the injection of hyperpolarizing current pulses into CA1
pyramidal cells recorded in vivo failed to produce any phase reversals,
although they did report that the amplitude of the intracellular theta
(MPOs) increased. In the present study, we did not observe an increase
in amplitude during hyperpolarizing current injections; in fact,
increasing hyperpolarization eventually totally abolished MPOs (as did
increasing levels of depolarization). On the other hand, Soltesz
and Deschenes (1993) did observe an ~180° phase shift in
CA1 cells that were hyperpolarized from 65 to 85 mV, and similar
phase shifts were reported by Ylinen et al. (1995). In
the present study, we failed to see a phase reversal in three cells
hyperpolarized in the range from 85 to 90 mV. A possible
explanation for the difference between these two studies and the
present study may lie in the way the CA1 pyramidal cells were selected
for data analysis. Indeed, in both the Solstez and Deschenes and Ylinen
et al. studies only low-firing (<1 and 2 Hz, respectively) CA1
pyramidal cell were selected for analysis compared with discharge rates
of 10-12 Hz during theta in the present study. Soltesz and
Deschenes (1993) and Ylinen et al. (1995) also
reported that the frequency of intracellular theta rhythm in CA1
pyramidal cells was independent of membrane potential, a result
supported by the present study. The absence of any measurable MPOs
during the 300- and 400-pA applied current conditions could be due to
the fact that the membrane potential would be largely determined by the
spike conductances, which may short-circuit those responsible for the
generation of MPOs. If this was the case, then rhythmic spike
discharges would be abolished due to the large current-induced
depolarization and not the abolishment of the MPOs. In the current
study, we had no direct experimental evidence to determine which
explanation was correct. It is also of interest to note that in the
present study the four levels of depolarizing constant current failed
to produce MPOs during spontaneously occurring LIA field activity, in
agreement with the results of Ylinen et al. (1995). On
the other hand, in the in vitro studies of Leung and Yim
(1998), they were able to manipulate MPO frequency with
intracellular current injections. We have no explanation for the
differences except to note that our experiments were carried out in
vivo. In addition, it's possible that our current steps were too large
and thus missed producing the appropriate membrane potential for
activating the membrane currents underlying the generation of MPOs.
Relationship between MPOs and rhythmic cell discharges at theta
frequencies in identified CA1 pyramidal cells
The present study demonstrated that the occurrence of MPOs in a
critical range of amplitudes and the rhythmical cell discharges at
theta frequencies was highly correlated. These data provide evidence
that once extrinsic synaptic inputs enable the theta state,
voltage-dependent intrinsic MPOs functioned to control the rhythmic
discharge properties of CA1 hippocampal pyramidal cells classified as
phasic theta-ON cells. Thus intrinsically generated MPOs
allow CA1 phasic theta-ON cells to be tuned selectively to
theta-band frequencies. The question remains as to whether MPOs control
the rhythmical discharge properties or vice versa. W
TOP
ABSTRACT
INTRODUCTION
