1Department of Psychology, 2Interdepartmental Neuroscience Program, and 3Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beggs, John M., James R. Moyer Jr., John P. McGann, and Thomas H. Brown. Prolonged Synaptic Integration in Perirhinal Cortical Neurons. J. Neurophysiol. 83: 3294-3298, 2000. Layer II/III of rat perirhinal cortex (PR) contains numerous late-spiking (LS) pyramidal neurons. When injected with a depolarizing current step, these LS cells typically delay spiking for one or more seconds from the onset of the current step and then sustain firing for the duration of the step. This pattern of delayed and sustained firing suggested a specific computational role for LS cells in temporal learning. This hypothesis predicts and requires that some layer II/III neurons should also exhibit delayed and sustained spiking in response to a train of excitatory synaptic inputs. Here we tested this prediction using visually guided, whole cell recordings from rat PR brain slices. Most LS cells (19 of 26) exhibited delayed spiking to synaptic stimulation (>1 s latency from the train onset), and the majority of these cells (13 of 19) also showed sustained firing that persisted for the duration of the synaptic train (5-10 s duration). Delayed and sustained firing in response to long synaptic trains has not been previously reported in vertebrate neurons. The data are consistent with our model that a circuit containing late spiking neurons can be used for encoding long time intervals during associative learning.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work on perirhinal cortex (PR) identified
pyramidal neurons in layer II/III that generated long delays
to initiate spiking when injected with depolarizing current steps
(Faulkner and Brown 1999). Following the initial delay,
these late spiking (LS) neurons also exhibit sustained
firing during the current step, in contrast to the strong
accommodation more commonly seen in regular spiking (RS) cortical cells
(McCormick et al. 1985
). The intrinsic firing properties
of LS neurons in PR, combined with their apparent circuit-level
organization, gave rise to a model of how these cells might be used in
cortical circuits to process temporal information (Tieu et al.
1999
). In particular, the cellular anatomy and neurophysiology
suggested that LS neurons might be organized into delay lines that are
capable of encoding intervals of seconds to tens of seconds.
An untested prediction of our model is that these LS neurons can also
exhibit delayed and sustained firing in response to synaptic inputs. Several factors could preclude delayed and
sustained firing of LS neurons in response to a train of synaptic
inputs. For example, conventional feed-forward or feedback synaptic
inhibition, activity-dependent synaptic depression, and the presence of
certain voltage- or calcium-dependent conductances on the postsynaptic dendrites (cf. Magee 1998) could act to prevent both
delayed and sustained firing to synaptic inputs. Here we used visually
guided, whole cell recordings from rat PR layer II/III, which is known to contain numerous LS pyramidal neurons (Faulkner and Brown
1999
), while trains of synaptic stimuli were delivered to layer
I afferents. The experimental question was whether layer II/III
pyramidal neurons can exhibit delayed and sustained firing to trains of
synaptic inputs produced by repetitive electrical stimulation of layer I. Preliminary results have been presented in abstract form
(Beggs et al. 1997
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brain slices from 12- to 32-day-old Sprague-Dawley rats were
prepared and maintained as previously described (Moyer and Brown 1998). Whole cell recordings were made from layer II/III
pyramidal neurons in horizontal slices (300-400 µm) containing
perirhinal cortex (corresponding to plates 98 to 100 of Paxinos
and Watson 1998
) limited by the rostral and caudal extent of
the lateral amygdala (approximately
2.4 to
4.8 mm posterior to
bregma). Layer II/III PR pyramidal neurons were visualized and
identified with infrared-filtered, video-enhanced DIC optics
(Moyer and Brown 1998
; Xiang and Brown
1998
).
Recordings were done at room temperature (~24°C) in physiological
saline containing (in mM) 124 NaCl, 2 KCl, 2 CaCl2, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose, pH 7.4, 290 mosmol. The electrophysiological methods are described in detail
elsewhere (Moyer and Brown 1998). Patch pipettes (~4
M
), were filled with (in mM) 120 K-gluconate, 10 HEPES, 1.0 EGTA, 20 KCl, 2.0 MgCl2, 2.0 Na2·ATP, 0.25 Na3·GTP·2H2O, pH 7.3, 280-290 mosmol. Electrical signals were recorded using an EPC-7 or
AxoPatch 1D amplifier, filtered at 3 kHz, digitized at 44 kHz, stored
on VCR tape, and analyzed using custom software written for Igor Pro.
All voltages were corrected for a +10-mV liquid junction potential
between the bath and the gluconate-based patch pipette solution
(Neher 1992
).
The responses of perirhinal layer II/III pyramidal neurons to both
somatic current injection and trains of synaptic inputs were evaluated.
With current injection, neuronal firing characteristics and
subthreshold membrane responses were examined. Current-voltage (I-V) relationships were constructed by injecting small
hyperpolarizing and depolarizing current pulses (<25 pA) that resulted
in small voltage excursions (<10 mV) from the resting membrane
potential. Responses within this restricted range of linear and
symmetrical voltage excursions were used to obtain the neuronal input
resistance (RN) and the time constant
of the membrane voltage response (m), The
value of RN was calculated from the
slope of the best-fitting linear regression equation (least-squares
criterion). The time course of the membrane voltage response to small
current steps could always be well approximated by an exponential
function with a single time constant (least-squares criterion). Values
of
m were taken from either single pulses or
averages of 10-20 pulses.
Cells were studied only if they had a healthy visual appearance
(Moyer and Brown 1998), an uncorrected resting membrane
potential of
60 mV or more negative, an input resistance >120 M
,
and overshooting action potentials. Layer II/III pyramidal neurons were
first determined to be LS or RS based on their response to depolarizing
current steps (Faulkner and Brown 1999
). In response to
suprathreshold current steps, LS neurons in PR commonly delay the onset
of their spike trains for ~1 s or longer and continue firing for the
duration of the current step. In contrast, RS cells fire relatively
soon after onset of the current step and often exhibit strong
accommodation that terminates firing in spite of maintained
depolarization. LS neurons tend to exhibit less accommodation and can
even show "anti-accommodation" (Faulkner and Brown
1999
), a progressive acceleration in the firing rate, during
the early part of a current step.
Synaptic inputs were evoked using a concentric stimulating electrode whose 250-µm diameter tip was positioned into PR layer I, which contains afferents to layer II/III pyramids. Before studying the effects of synaptic trains, we first explored the responses to individual synaptic inputs (under current-clamp conditions) to get a baseline stimulation intensity (using monophasic current pulses of 0.2 ms duration). The stimulation intensity was gradually increased until an excitatory postsynaptic potential (EPSP) of ~2 mV amplitude was evoked.
The response of the postsynaptic neurons to long sequences (5-10 s) of EPSPs was examined by passing a train of 100-200 monophasic current pulses (0.2 ms duration/pulse) through the stimulating electrode at 20 or 25 Hz. These trains were repeated at 30-s intervals, each time increasing the stimulation from the baseline intensity until at least one action potential was produced in the postsynaptic neuron, thereby determining the threshold for synaptically produced orthodromic spiking in response to a long train. Once the threshold was found, the stimulation intensity was gradually increased to determine whether trains of EPSPs could produce sustained repetitive spiking in the postsynaptic neurons.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whole cell recordings were made under current-clamp conditions
from 61 layer II/III pyramidal neurons as previously described (Moyer and Brown 1998). Slightly more than half of the
neurons were LS cells (33 of 61; Table
1). Of these 61 neurons, a subset of 42 neurons was subjected to an extensive experimental protocol that
included stimulation of synaptic inputs to the neuron.
|
Figure 1 compares some general features
of LS and RS cells in response to depolarizing and hyperpolarizing
current steps. By definition, RS cells fired early in response to a
depolarizing current step, whereas LS cells fired late (Fig.
1A). On the other hand, most of the subthreshold
electrophysiological properties of the two cell types were
indistinguishable (summarized in Table 1). A notable exception is
m, which was slightly but significantly larger
in the LS than the RS neurons (t = 3.2, df = 53, P < 0.005; unpaired). Figure 1C shows
examples of the averaged responses to small (
10 pA) hyperpolarizing
current steps and semilogarithmic plots that illustrate
single-exponential fits to data from both a RS and a LS cell.
|
Figure 1D highlights two key aspects of a LS neuronal
response to a prolonged (60 s) depolarizing current step.
First, the beginning of spiking is clearly delayed from the
onset of the current step. In this case the delay is more than 2.3 s, and we have seen delays as long as 19 s. Second, once spiking
is initiated, it is sustained for the duration of the
current step, which lasted for 60 s in this example. Late spiking
cells often exhibit anti-accommodation early in the spike train (slight
tendency seen in Fig. 1A, bottom right trace)
(see also Faulkner and Brown 1999), but mild
accommodation is typically evident later in the spike train (Fig.
1D). By contrast, RS cells tend to show spike frequency
accommodation throughout the spike train (Fig. 1A,
bottom left trace).
When given a suprathreshold train of synaptic inputs, most LS cells (19 of 26) fired their first action potential more than 1 s after the
onset of the synaptic train; that is they exhibited delayed
spiking to synaptic stimulation. This delay is an order of
magnitude longer than m, the average value of
which was 76 ms in LS cells (Table 1; see also Figs. 1, A
and D, and 2A). An
example of delayed firing to a long (7.5 s) synaptic train (150 pulses
at 20 Hz) is illustrated in Fig. 2A. This set of traces shows the response of an LS cell to trains of progressively larger synaptic inputs. In the bottom two traces, the synaptic
stimulation was subthreshold for eliciting spiking in the pyramidal
neuron. In the top trace the synaptic stimulation elicited
repetitive spiking that began 4.2 s after the onset of the train
and continued for the duration of the train.
|
In contrast to the firing pattern observed in LS cells, RS neurons fired at a short latency from the onset of synaptic stimulation and then tended to show rapid accommodation (Fig. 2B). The top and bottom voltage traces in Fig. 2B show, respectively, the membrane response to supra- and sub-threshold trains of synaptic inputs. In both cases, the onset of the synaptic train caused an abrupt depolarization, but it was not sustained. The onset of synaptic stimulation also caused an abrupt depolarization in LS cells (Fig. 2A). A notable difference between cell types was that the depolarization in LS neurons was sustained for the duration of the synaptic train (Fig. 2A).
In contrast to the rapid depolarization observed at the onset of synaptic stimulation, the termination of stimulation was followed by a slow relaxation back to the resting potential (Fig. 2A). This did not occur when a depolarizing current step was used to fire the cell. In the latter case, termination of the current step was followed by a rapid relaxation of the membrane potential back to the resting level (Fig. 1, A and D). In the LS cell illustrated in Fig. 2A, the decay time constant following a suprathreshold current step was 92 ms, whereas the decay time constant following synaptic stimulation was 504 ms. These differences were reflected in the group data. In 12 LS cells that showed delayed and sustained spiking to a synaptic train, the mean decay time constants following a suprathreshold current step and a synaptic train were 76.4 ± 6.6 and 405 ± 74.1 (mean ± SE), respectively, a difference that was statistically significant (t = 4.43, df = 11, P < 0.005; paired comparisons).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This is the first demonstration of delayed and sustained spiking
in response to trains of synaptic inputs in vertebrate neurons. The
response of these cortical neurons to trains of synaptic inputs was
similar in certain respects to their response to a depolarizing current
step (compare Fig. 1, A and D, with Fig.
2A). The initial rapid depolarization was followed by a more
gradual depolarizing ramp until the spike threshold was reached. Once
above threshold, most LS neurons continued firing for the duration of
the synaptic train or current step. Computer simulations have shown
that this type of delayed and prolonged synaptic integration can
theoretically furnish a convenient and robust platform for interesting
forms of temporal encoding (McGann and Brown 2000;
Tieu et al. 1999
).
These interesting results naturally raise numerous questions regarding
the ionic mechanisms underlying the late spiking firing pattern we
observe. Recall that small current injections produced a rapid membrane
voltage response that could be well fit by a single exponential (Fig.
1; Table 1). However, larger current injections seemed to recruit
additional ionic conductances. The initial rapid membrane response in
LS cells was followed by a gradual depolarizing ramp (Figs.
1A and 2A). In hippocampal and striatal neurons,
a similar ramp has been suggested to result from a slowly inactivating
potassium conductance that is blocked by 4-aminopyridine (4-AP)
(Nisenbaum et al. 1994, 1996
;
Storm 1988
).
The conductance mechanism could be similar in perirhinal LS
cells, but there are two apparent differences. First, LS perirhinal cortical neurons do not require a strong hyperpolarizing prepulse to
exhibit delayed spiking, in contrast to findings in hippocampus (Storm 1988). Second, the delays we see in LS perirhinal
neurons are many times longer than those reported in striatal neurons (Nisenbaum et al. 1994
). Previous voltage-clamp
experiments performed on LS perirhinal neurons revealed a slowly
developing inward current when the membrane potential was stepped from
the resting potential to a just-subthreshold potential (Faulkner
and Brown 1999
). This inward relaxation, which has a time
course similar to the depolarizing ramp mentioned above, could reflect
a slowly inactivating potassium conductance. Although we have not yet
fully studied the pharmacology of this ionic current, preliminary
results indicate that bath application of 4-AP blocks both the
depolarizing ramp and the delayed spiking in perirhinal LS neurons
(Moyer et al. 2000
).
These preliminary findings encourage a full investigation into the
ionic mechanisms responsible for the firing properties of LS neurons in
perirhinal cortex. To date, we have observed at least three different
types of LS cells in rat perirhinal cortex. In addition to the LS layer
II/III pyramids discussed here, there are also small LS "cone
cells" in layer VI (Faulkner and Brown 1999) and large
LS pyramids in layer V (Moyer and Brown, unpublished observations).
These three cell types are morphologically quite distinct and are all
contained within our standard horizontal brain slice of perirhinal
cortex. It will be interesting and informative to compare
quantitatively their firing properties and to examine possible
similarities and differences in their pharmacology and ionic
conductance mechanisms.
The presence of LS neurons in PR layer II/III, combined with their
axonal projections (Faulkner and Brown 1999), have
suggested some interesting computational possibilities. If groups of
these cells were connected in series, they could form an array of delay lines capable of encoding and learning temporal relationships on the
order of seconds to tens of seconds (McGann and Brown
2000
; Tieu et al. 1999
). Because LS cells can
show sustained firing for tens of seconds, they might also play a role
in maintaining temporary stimulus representations when incorporated
into the appropriate recurrent circuit architecture (Tieu et al.
1999
). Consistent with this possibility, single-unit recordings
from rat PR have revealed neurons that are tonically active during the
delay period of an odor-guided, delayed
nonmatching-to-sample task (Young et al. 1997
).
![]() |
ACKNOWLEDGMENTS |
---|
This research is part of a dissertation submitted to the Psychology Department, Yale University, in partial fulfillment of the requirements for the PhD degree of J. M. Beggs.
This work was supported by National Institutes of Health Grants RO1 50948 to T. H. Brown and F32 NS-09992-01 (postdoctoral fellowship) to J. R. Moyer, as well as a National Science Foundation predoctoral fellowship to J. P. McGann.
Present address of J. M. Beggs: Laboratory of Systems Neuroscience, NIH, Unit of Neural Network Physiology, Bldg. 36, Rm. 2D-30, Bethesda, MD 20892-4075.
![]() |
FOOTNOTES |
---|
Address for reprint requests: T. H. Brown, Dept. of Psychology, Yale University, PO Box 208205, 2 Hillhouse Ave., New Haven, CT 06520.
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 5 October 1998; accepted in final form 23 February 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|