Download Horizontal Synaptic Connections in Monkey Prefrontal Cortex: An In

Horizontal Synaptic Connections in
Monkey Prefrontal Cortex: An In Vitro
Electrophysiological Study
Guillermo González-Burgos1, German Barrionuevo1,3 and
David A. Lewis1,2,3
In monkey dorsolateral prefrontal cortex (PFC), long-distance,
horizontally oriented intrinsic axon collaterals interconnect clusters
of pyramidal neurons in the supragranular layers. In order to study
the electrophysiological responses mediated by these long-distance
projections, an in vitro slice preparation of monkey PFC was used to
obtain whole-cell patch clamp recordings from layer 3 pyramidal
neurons. Using in vivo tracer injections, we found that long-distance
projections were well preserved in PFC slices cut in the coronal
plane. Postsynaptic currents were evoked by low-intensity electrical
extracellular stimulation applied successively to 20–30 discrete
sites located up to 2200 µm lateral to the recorded cell. Several
criteria were applied to discriminate between mono- and polysynaptic responses. Long-distance monosynaptic connections
were mediated by fibers with relatively slow conduction velocity
(0.14 m/s). Excitatory postsynaptic currents (EPSCs) evoked by
stimulation of short- or long-distance horizontal connections did not
differ in kinetic properties. The majority (77%) of the 35 layer 3 PFC
neurons studied were monosynaptic targets of long-distance
connections. EPSCs mediated by long-distance connections had
amplitudes that were similar or even larger than short-distance
EPSCs, suggesting that excitatory input provided by the former
was relatively robust. For most neurons (87.5%) in which a full
complement of monosynaptic EPSCs was evoked by multisite
stimulation, the EPSC amplitude as a function of stimulation distance
from the recorded cells exhibited statistically significant peaks.
The spacing between peaks was similar to the spacing between
interconnected clusters of neurons observed in previous anatomical
studies. The results show that long-distance excitatory connections
constitute a significant intrinsic pathway of synaptic communication
in layer 3 of monkey PFC.
As is the case for other regions of the macaque monkey neocortex, pyramidal neurons in the supragranular layers of the
dorsolateral prefrontal cortex (PFC) furnish intrinsic axon
collaterals that travel for substantial distances, up to several
millimeters, tangential to the pial surface (Levitt et al., 1993).
The transport of anterograde and retrograde tracers from focal
injections in monkey PFC (areas 9 and 46) has revealed that
clusters of pyramidal neurons in layers 2–3 give rise to intrinsic,
horizontal projections that terminate in discrete clusters of
labeled axon terminals in layers 1–3 (Levitt et al., 1993; Kritzer
and Goldman-Rakic, 1995; Pucak et al., 1996; Woo et al., 1997).
In primary sensory regions, the axons of these long-distance
horizontal connections terminate in patch-like clusters (Lund et
al., 1993; Yoshioka et al., 1996) and appear to preferentially link
columns of cells with similar functional properties (Gilbert
and Wiesel, 1989; Malach et al., 1993; Weliky and Katz, 1994;
Bosking et al., 1997; Yabuta and Callaway, 1998). In contrast, in
the monkey PFC, the clusters of labeled axon terminals and
neurons are elongated and form a series of regularly spaced
stripes (Levitt et al., 1993; Pucak et al., 1996). A lthough it
seems likely that the PFC neurons clustered in a given stripe
share certain functional characteristics (Goldman-Rakic, 1995),
this remains to be experimentally tested. Furthermore, in
monkey primary visual cortex, the axon terminals of horizontal
connections contact pyramidal and nonpyramidal neurons in
a proportion similar to the frequency of each cell type in the
cortex, suggesting that these connections do not have target
cell selectivity (McGuire et al., 1991). In contrast, although the
proportions of pyramidal to nonpyramidal neurons are similar in
the monkey visual and prefrontal cortices, >95% of the horizontal
connections in the PFC contact dendritic spines and thus appear
to preferentially target pyramidal neurons (Melchitzky et al.,
1998). Since these contacts form exclusively asymmetric synapses, these findings suggest that excitatory, long-distance,
horizontal connections in the PFC are biased to innervate other
pyramidal neurons.
Given that the stripes revealed by a given tracer injection in
the PFC appear to be reciprocally connected (Pucak et al., 1996),
it is reasonable to hypothesize that other pyramidal neurons in
the superficial layers are the principal synaptic targets of these
connections (Melchitzky et al., 1998). However, a proportion of
the dendritic spines found in the superficial layers are located on
the dendrites of neurons whose somata lie in deeper cortical
layers. Indeed, in rat somatosensory cortex, the axon collaterals
of layer 3 pyramidal neurons establish horizontal connections
with layer 3 and layer 5 pyramidal neurons with similar probabilities (Thomson and Deuchars, 1997; Markram, 1997; Thomson
and Bannister, 1998). In addition, the functional strength of the
long-distance horizontal connections presumed to exist between
supragranular pyramidal neurons in monkey PFC is not known.
Consequently, in the present study, we recorded visually
identified layer 3 pyramidal neurons using whole-cell patch
clamp methods applied in a living brain slice preparation from
monkey PFC. The improved signal-to-noise ratio of in vitro
tight seal whole-cell recordings allowed very sensitive detection
of small amplitude, fast synaptic events. Hence, it was possible to
employ low stimulation intensities, restricting the spread of the
stimulation current and thus improving the spatial resolution
of the stimulation procedure. Horizontal connections were activated by low-intensity stimulation applied at multiple sites in the
superficial layers via a multielectrode stimulation array, similar to
that employed previously for functional mapping of horizontal
connectivity in primary visual cortex (Weliky and Katz, 1994;
Weliky et al., 1995). This experimental approach was employed
to address the following questions: (i) Do pyramidal neurons in
layer 3 of monkey PFC receive monosynaptic, excitatory input
from horizontal, long-distance connections? (ii) If so, how robust
is this input as compared with input provided by short-distance
connections? (iii) What proportion of layer 3 cells receive this
type of synaptic input? (iv) Does the spatial pattern of excitatory
synaptic responses evoked by multisite stimulation ref lect the
© Oxford University Press 2000
Cerebral Cortex Jan 2000;10:82–92; 1047–3211/00/$4.00
Departments of 1Neuroscience and 2Psychiatry and 3Center for
the Neural Basis of Cognition, University of Pittsburgh,
Pittsburgh, PA 15260, USA
clustered organization of horizontal connectivity in the supragranular layers of monkey PFC?
Materials and Methods
PFC Slice Preparation
Tissue slices were obtained from 10 young adult male cynomolgus
monkeys (Macaca fascicularis). All animals were treated according to
the guidelines outlined in the NIH Guide to the Care and Use of Animals.
Following injections of ketamine hydrochloride (25 mg/kg), dexamethasone phosphate (0.5 mg/kg) and atropine sulfate (0.05 mg/kg),
an endotracheal tube was inserted and the animal was placed in a
stereotaxic frame. Anesthesia was maintained with 1% halothane in 28%
O2/air. A craniectomy was performed over the dorsal PFC and a small
block of tissue (∼4 × 6 × 4 mm) containing dorsal area 9 and, in some
cases, the medial bank of the principal sulcus (area 46) was removed. The
tissue block was immediately immersed in ice-cold modified artificial
cerebral spinal f luid (modified ACSF) (composition, in mM: sucrose 230,
KCl 1.9, Na2HPO4 1.2, NaHCO3 33, MgCl2 6, CaCl2 0.5, glucose 10,
kynurenic acid 2; pH 7.3–7.4 when bubbled with 95%/5% O2/CO2 gas
mixture). After hemostasis was achieved, the skin was closed and the
animal was treated postoperatively with analgesics and antibiotics as
previously described (Pucak et al., 1996). Animals recovered quickly and
no behavioral deficits were observed. Two to four weeks later, the
animals underwent the same procedure with the following modifications
to obtain tissue from the opposite hemisphere. After the craniectomy was
performed, the animal was given an overdose of pentobarbital (50 mg/kg)
and was perfused intracardially with ice-cold modified-ACSF. A tissue
block containing the portions of PFC areas 9 and 46 nonhomotopic to the
first biopsy was quickly excised and then the remainder of the brain was
removed for other studies.
The tissue blocks were glued to the stage of a vibratome and 400
or 500 µm thick slices were cut in the coronal plane while the block
was submerged in ice-cold, modified ACSF. After slicing, the tissue was
maintained at room temperature for at least 2 h in an incubation chamber
containing low Ca2+/high Mg2+ ACSF (low Ca2+ ACSF, composition, in mM:
NaCl 125, KCl 2, Na2HPO4 1.25, NaHCO3 26, CaCl2 1.0, MgCl2 6.0, glucose
10; pH 7.3–7.4 when bubbled with 95%/5% O2/CO2 gas mixture) before
being transferred to the recording chamber.
Slices transferred to a submersion recording chamber were superfused with ACSF (composition, in mM: NaCl 125, KCl 2, Na2HPO4 1.25,
NaHCO3 26, CaCl2 3.0, MgCl2 3.0, glucose 10; pH 7.3–7.4 when bubbled
with 95%/5% O2/CO2 gas mixture) at room temperature (20–24°C). The
relatively low K+ concentration in the external solution hyperpolarizes
the cells in the slice, and the high Ca2+ and Mg2+ concentrations produce
an increase in the spike threshold. Both factors decrease excitability and
reduce the probability of firing intercalated neurons, thus minimizing
polysynaptic responses (Berry and Pentreath, 1976).
Electrical Stimulation
Electrical stimulation was applied by means of a multiple electrode array
similar to that described by Weliky and Katz (Weliky and Katz, 1994).
The stimulation array was placed parallel to the pia (see Fig. 1) in
superficial–middle layer 3 (300–600 µm from the pial surface). The
electrode array consisted of 24–30 fine nichrome wires coated with
Formvar except at the tips (bare diameter 50 µm, coated diameter 65 µm;
A-M Systems Inc., Carlsberg, WA), which were glued together with
cyanoacrylate glue. The wire tips to be in contact with the tissue had
similar shapes and the mean (± SE) inter-tip distance for three different
arrays was 70 ± 5, 89 ± 10 and 60 ± 5 µm. Bipolar electrical stimulation was
applied between pairs of adjacent wires with the cathode always closer to
the recorded cell (Fig. 1). Bipolar square current pulses (20–100 µs,
20–200 µA) were applied using a constant current stimulus isolation unit
(Model SC-100, Winston Electronic Co., Millbrae, CA).
Electrical Characteristics of the Stimulation Array
For one stimulation array, the mean resistance between the input to
the stimulator and the tip of 20 of the wires was 36 Ω, and the mean
resistance between adjacent pairs of electrodes placed in the recording
solution or in contact with the tissue was 1.68 ± 0.17 kΩ. Since the
Figure 1. Procedures for stimulating and recording EPSCs in monkey PFC slices. (A)
Diagram illustrating the typical location of a multielectrode stimulation array (with
electrodes numbered 1–24) and whole-cell recording electrode in layer 3 of a coronal
slice. Bipolar stimulation was applied using adjacent wires, the cathode always being
closer to the cell. Recorded cells were located 400–600 µm in the horizontal direction
from the closest stimulation position. The stimulation array overlaps groups of pyramidal
cells with open (neurons connected with the recorded cell) or filled (neurons without
connections to the recorded cell) somata, representing the clustered organization of
horizontal connectivity in layer 3 of PFC. For simplicity, horizontal axon collaterals are not
shown. (B) A group of pyramidal cells identified visually in a monkey PFC slice using
infrared videomicroscopy and differential interference contrast optics. The large arrow
indicates the patch pipette used to obtain a whole-cell recording from one of these
cells; the small arrows indicate other pyramidal neurons, some of which located at
different depths in the slice with respect to the recorded neuron. Scale bar = 50 µm.
stimulus isolation unit delivers currents up to 10 mA with the output
connected to a load resistance of up to 10 kΩ, in our experiments the
current output was constant across the sections of tissue stimulated by
each bipolar position in the array. However, a factor that could introduce
variability in current output from the isolation unit is the electrode
capacitance. The total capacitance between adjacent pairs of electrodes
in one of our stimulation arrays was 1.4 ± 0.4 nF (range: 0.86–2.71 nF),
and time constants (R × C) for charging the electrodes were on the order
of microseconds (maximal value 2.8 µs). As a result, only a small fraction
of the total current delivered during the stimulation pulse was diverted
to charge the electrode capacitance. Thus, it is unlikely that there were
Cerebral Cortex Jan 2000, V 10 N 1 83
significant differences in electrical current stimulation f lowing between
the electrodes in the array due to differences in electrode capacitance.
Electrophysiological Recordings
Pyramidal neurons in layer 3 were identified visually using differential
interference contrast (DIC) and infrared video-microscopy with Leitz
Laborlux 12 or Zeiss Axioskop FS microscopes equipped with CCD
(CCD-72S, Dage-MTI) or New vicon image tube (NC-70, Dage-MTI)
cameras, respectively. Recorded cells were located 380–900 µm from the
pial surface (middle–deep layer 3) and were identified as pyramidal
neurons by the shape and size of the soma and the presence of an apical
dendrite (Fig. 1B). Pipettes were pulled from borosilicate glass and had
resistances between 3 and 7 MΩ when placed in the bath and filled with
the internal solution (composition, in mM: CsF, 120; CsCl, 10; HEPES,
10; EGTA, 5; DIDS, 1.0 mM, pH 7.2–7.4; osmolality: 270–290 mOsm).
Giga-Ohm seals (resistance >2 GΩ) were obtained using the ‘blow and
seal’ technique, as described elsewhere (Stuart et al., 1993). The access
resistance (Racc) was not compensated and had values between 8 and 25
MΩ. Racc was continuously monitored and recordings were rejected for
analysis when Racc changed by >20%. EPSCs were recorded at a nominal
holding potential of –80 to –70 mV (unless indicated) with an Axopatch1C amplifier (Axon Instruments Inc., Foster City, CA), filtered at 2–5 kHz,
digitized at 10 kHz and stored on disk for off-line analysis.
The parameters of the stimulation current delivered via the electrode
array (see above) were adjusted at the closest stimulation position
(position 1) to evoke large EPSCs without complex waveforms (Fig. 2A).
A fter stimulation from positions 1–2, the stimulus current was kept
constant and delivered successively to each of the other pairs in the
multielectrode array. At least 10 EPSCs were evoked from every electrode
pair in the array, intermittently returning to positions 1–2 to control for
stability of the recording conditions (see Fig. 2B). The stimulation
frequency was set at 0.1 Hz, because in preliminary experiments higher
frequencies produced variable degrees of depression of the response
amplitude.
Acquisition and analysis were performed using LabView (National
Instruments Corp., Austin, TX) running customized software. The
baseline noise was determined in each trace by measuring the peak
current in a time window with the same duration as that used to measure
peak EPSC amplitude, but positioned before the stimulation artifact.
In Vivo Extracellular Tracer Injection
Iontophoretic injections of 10% biotinylated dextran amine (BDA, mol.
wt 10 000) in 0.01 M phosphate buffer, pH 7.4, were made in layer 3 of
area 9 by passing positive current (5 µA, 7 s cycles) through glass pipettes
(tip diameter 20–50 µm) for 20 min (Pucak et al., 1996). These injections
were made in a subset of animals in order to determine the presence of
horizontal axons and clustered connections in thin (100–200 µm) coronal
slices that were adjacent to slices used for electrophysiological recording.
Statistical Analysis
To determine whether mean EPSC amplitude changed with stimulation
distance, one-way analysis of variance (ANOVA) was employed. When the
results of the ANOVA were significant (P < 0.05), peaks and troughs were
identified in the pattern of responses obtained for each cell following an
approach similar to that previously used by others (Weliky and Katz,
1994; Weliky et al., 1995). Peaks and troughs were defined as those
locations where EPSC amplitude was larger or smaller, respectively, than
those observed at the two adjacent locations. If the EPSC amplitude of a
peak was significantly different from both of the two closest f lanking
troughs using post-hoc HSD Neumann–Keuls comparisons between
means (P < 0.05), then it was considered a significant peak. Unless
otherwise stated, values are reported as mean ± SD.
Drugs
Bicuculline methiodide, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid
(DIDS), biocytin and kynurenic acid were purchased from Sigma (Saint
Louis, MO). BDA and 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) were
purchased from Molecular Probes (Eugene, OR).
84 Horizontal Synaptic Connections in Monkey PFC • González-Burgos et al.
Figure 2. Adjustment of stimulus current and control of response stability during
electrical stimulation. (A) The fixed stimulus current to be applied throughout the
stimulation array was adjusted while stimulating from the horizontal position closest to
the cell (i.e. electrode 1) to avoid evoking EPSCs with complex waveforms. In this
example a stimulus current of 150 µA was chosen, since a 170 µA stimulus evoked a
second component in the EPSCs (arrow). (B) To assess for the stability of EPSC
amplitude, stimulation was switched back to the first position every 10–15 min. The
upper graph shows superimposed traces recorded in response to stimulation at the
closest array position at five different time points during a representative experiment.
The mean (± SD) peak EPSC amplitude of the traces in the upper graph is plotted
against time in the lower graph. One-way ANOVA revealed no effect of time on EPSC
amplitude.
Results
Isolation of EPSCs Evoked by Stimulation of
Horizontally Oriented Connections
Preser vation of horizontally oriented axons in the PFC slice
preparation was demonstrated in animals in which extracellular
injections of anterograde tracers were placed in the superficial
cortical layers of area 9 in vivo. As shown in Figure 3, labeled
axons running parallel to the pia could be followed for up to
2000 µm, even in slices that were thinner (100 µm) than those
used for the electrophysiological recordings (400–500 µm). In
addition, clusters of labeled arborizing axons and terminals,
similar to those observed in previous anatomical studies (Levitt
et al., 1993; Pucak et al., 1996), were clearly evident in the slice
preparation.
To examine the electrophysiological properties of the
synaptic input furnished by horizontally oriented axons, wholecell recordings were obtained from visually identified layer 3
pyramidal neurons. Postsynaptic currents (PSCs) were evoked by
electrical stimulation applied in superficial layer 3, lateral to the
recording site (Fig. 1A). As seen in Figure 4A, initial experiments showed that the PSCs typically had mixed excitatory and
inhibitory components (EPSCs and IPSCs, respectively), with the
relative weights of the EPSCs and IPSCs varying significantly
between evoked responses. The IPSCs were more frequently
evoked by stimulation at the locations closest to the cell
Figure 3. Long-distance, horizontally oriented axons are preserved in coronal slices from monkey PFC. An extracellular injection of BDA was placed in vivo in PFC area 9 and, 10 days
later, coronal slices were cut from a tissue block including the injection site and surrounding region. The BDA label was visualized in a 100 µm thick slice located between those used
for the electrophysiological recordings. Labeled horizontal axons (arrows) were readily observed running parallel to the pial surface for >2000 µm. In addition, the BDA label was
organized into discrete clusters with high density of axon branches and synaptic varicosities. The size and laminar distribution of the labeled terminal clusters were similar to those
observed in previous anatomical studies (Pucak et al., 1996). Scale bar = 400 µm.
(300–500 µm away) and were probably di- or polysynaptic,
because they invariably had longer latencies than the EPSCs.
In addition, the IPSCs had fast time courses and reversed at
a membrane potential close to the equilibrium potential for
chloride ions (∼–65 mV in our experimental conditions, data
not shown) suggesting that they were mediated by activation of
GABAA receptor-gated chloride channels.
Because it is critical to study EPSCs in isolation, the presence
of the fast IPSC complicated the electrophysiological analysis of
the excitatory input. However, when the GABAA antagonist
bicuculline was bath-applied to the PFC slices, it induced epileptiform activity even at concentrations (0.5 µM) that are too
low to block the IPSCs (data not shown), as previously reported
(Gutnick et al., 1982; Chagnac-Amitai and Connors, 1989).
Recent studies have shown that, when applied intracellularly to
single cells in neocortical slices, cyanostilbene compounds like
DIDS block GABAA receptor channels without inducing such
hyperexcitability (Nelson et al., 1994; Dudek and Friedlander,
1996; Shao and Burkhalter, 1996). Indeed, after dialysis of PFC
pyramidal cells for at least 20 min with a CsF-based pipette solution containing 0.5–1.0 mM DIDS, no fast IPSCs were observed
(Fig. 4B). Slow IPSCs, such as those expected from the activation
of potassium channels by GABAB receptors (McCormick, 1989),
also were not observed when recording with the cesium-based
internal solution. Together, these data indicate that intracellular
blockade of fast and slow synaptic inhibition allowed us to study
isolated EPSCs in PFC pyramidal neurons.
Identification of Monosynaptic EPSCs Evoked by
Horizontally Oriented Connections
To determine if layer 3 pyramidal neurons were targets of
long-distance horizontal excitatory monosynaptic inputs, EPSCs
were evoked from different locations in the slice via the
multielectrode array. Whole-cell recordings were obtained from
a total of 47 layer 3 pyramidal neurons. In 12 of these 47 neurons,
Figure 4. Stimulation via the multielectrode array evoked both excitatory and inhibitory
postsynaptic currents in layer 3 PFC pyramidal neurons, and the latter were blocked by
dialysis with CsF-DIDS. (A) Postsynaptic currents (PSCs) were evoked by stimulation
with the electrode located closest to the cell. After 20 min of dialysis with CsF-based
internal solution, the holding potential was switched from –80 to –30 mV. Voltageclamping the soma above the equilibrium potential for chloride ions (close to –65 mV)
showed that the PSCs were usually composed of a short latency inward EPSC followed
by an outward IPSC with longer latency. The IPSC had a fast time course, suggesting
that it was mediated by activation of GABAA chloride channels. (B) A different cell was
dialyzed for 20 min with a CsF-based internal solution containing the GABAA chloride
channel blocker DIDS (1 mM). After dialysis, the holding potential was switched from
–80 to 0 mV and no outward current was observed, showing the absence of an IPSC.
Outward currents were observed only at positive holding potentials, above the reversal
potential for glutamate receptor-gated excitatory currents. At a holding potential of
+20 mV, the decay kinetics of the EPSCs were slower than that observed at –80 mV,
suggesting the presence of a small NMDA component. Calibration bars, vertical: 50 pA;
horizontal: 20 ms. Shown are the means of 10 consecutive current traces.
either the recordings were lost during the period of dialysis with
DIDS or a continuous and rapid rundown of the EPSCs precluded
an adequate interpretation of the results. In the remaining 35
Cerebral Cortex Jan 2000, V 10 N 1 85
Figure 5. A representative experiment showing EPSCs recorded from a layer 3 PFC
pyramidal cell in response to stimulation applied via the 24 electrodes of the stimulation
array. (A) Five consecutive EPSC traces were recorded in response to stimulation with
the array positions indicated by the numbers above each group of traces. Position 1 was
the closest to the recorded cell. The stimulation parameters (50 µs, 100 µA, 0.1 Hz)
were kept constant throughout the experiment. (B) Average of five traces recorded in
the experiment shown in panel A, showing that the mean EPSC amplitude was not
uniform, but varied with stimulation site. Overall, both the EPSC amplitude and the
probability to evoke a detectable response decayed with stimulation distance.
However, in most (30/35) neurons, long-distance stimulation (e.g. bipolar stimulation
applied at >600 µm from the cell) evoked detectable responses. In this example
neuron, all the multielectrode positions indicated by a number ≥5 were located at a
distance >600 µm. In addition, some stimulation positions (e.g. nos 7, 11 and 13)
evoked EPSCs with amplitudes greater than those observed at more proximal
stimulation sites. Calibration bars, vertical: 30 pA for A; 40 pA for B, horizontal: 20 ms.
neurons, stable EPSCs were evoked from 10 or more pairs of
stimulation positions in the slice (16.9 ± 5.1 positions per cell).
Figure 5 shows responses in one layer 3 pyramidal neuron
evoked from each of 24 electrode stimulation positions.
As stimulation was applied at positions increasingly distant
from the recorded neuron, EPSC amplitude diminished, and
fewer stimulations sites evoked detectable EPSCs. However, in
30 of the 35 neurons, EPSCs were detected when stimulation
was applied at long-distance (>600 µm from the recorded cell)
horizontal positions in the slice. As expected, low-intensity
stimulation did not evoke fast inward currents due to action
potentials in any of the 35 cells, indicating that EPSCs were
subthreshold, and that the probability of antidromic stimulation
of the axon collaterals was low.
86 Horizontal Synaptic Connections in Monkey PFC • González-Burgos et al.
Figure 6. Stimulation of the supragranular layers along the horizontal axes evoked
mono- and polysynaptic responses. On the left, five consecutive traces are shown
superimposed. On the right, schematic drawings show the combination of connections
presumably mediating the observed responses (R = recorded cell, S = stimulated
cells/elements). (A) Typical traces showing no EPSC detectable above the baseline
current. The drawing shows that stimulation may have activated some axons or cells,
but did not activate connections with the recorded cell. (B) Representative traces
showing EPSCs that exhibited a fairly constant latency and no failures. These EPSCs
likely resulted from activation of a purely monosynaptic long-distance pathway, as
suggested in the drawing. It is also possible that at least some of the responses could
derive from antidromic stimulation of the long-distance collaterals of cells that are
neighbors of the recorded neuron. However, this seems unlikely, because none of the
47 recorded neurons (and thus probably also none of their neighbors) was stimulated
antidromically. (C) Example of traces that showed EPSCs with complex waveforms,
formed by an early component with constant latency and no failures, and a late
component with variable latency and a significant failure rate. These EPSCs probably
resulted from activation of both mono- and polysynaptic pathways. (D) Example of an
EPSC waveform in which the single distinguishable component had both variable
latency and high failure rate. These EPSCs likely resulted from activation of a purely
polysynaptic pathway, as suggested in the drawing. Calibration bars: vertical, 50 pA;
horizontal, 20 ms.
As shown in Figure 6, four types of responses were evoked via
the stimulation array. One type of response (Fig. 6A) showed no
detectable EPSCs. Three other types of evoked responses
exhibited detectable EPSCs that were classified according to
waveform shape, incidence of observed failures, and variability
of latency between stimulus and EPSC onset. As shown in Figure
6B, one type of EPSC was characterized by monophasic waveforms which had no failures and a low trial-to-trial variability
in latency. Another type of EPSC had multiphasic waveforms
Figure 7. Analysis of the relation between latency to EPSC onset and stimulation
distance. (A) One example of the experiments in which, after eliminating the clearly
polysynaptic responses (see Fig. 6), there was a linear increase in EPSC latency with
stimulation distance, as expected if all the evoked EPSCs were monosynaptic. The
relation was well fit by a linear relation model, shown by the line. (B) An example of the
cells for which the EPSC latency did not clearly increase with stimulation distance and
the relation was poorly fit by a linear model, suggesting that at least some of the EPSCs
were polysynaptic.
(Fig. 6C) despite stimulation parameters that were adjusted to
evoke only monophasic EPSCs from array positions 1–2 (see
Fig. 2A). These complex EPSCs had an early component with
constant latency and no failures, and one or more late components with frequent failures and latency f luctuations of
several milliseconds. Finally, other EPSCs had monophasic
waveforms, but exhibited frequent failures and highly variable
latencies (Fig. 6D). In the 35 neurons examined in this study, the
mean (± SD) percentage of stimulation sites evoking each type
of recording trace was as follows: 56.5 ± 16.2% of the sites
evoked no detectable EPSCs; 27.7 ± 18.6% of the sites evoked
monophasic EPSCs; 13.4 ± 12.6% of the sites evoked complex
EPSCs; and 2.3 ± 5.0% of the sites evoked EPSCs with variable
latency and failures. We interpret the presence of failures and
variable latency as resulting from the involvement of intercalated
neurons firing intermittently and at variable latencies (Berry
and Pentreath, 1976). Therefore, the late components of the
complex EPSC waveforms (Fig. 6C) and the EPSCs with variable
latency and failures (Fig. 6D) were regarded as polysynaptic and
excluded from further data analysis, whereas the monophasic
EPSCs (Fig. 6B) and the early components of the complex EPSC
waveforms (Fig. 6C) were considered potentially monosynaptic
and examined further.
As a final test for monosynapticity, we plotted EPSC latency as
a function of horizontal distance between the stimulation and
recording sites for each neuron. Assuming that the synaptic
delay is nearly identical at all active synapses, then latency should
increase linearly with horizontal distance as a consequence of
the distance that the presynaptic action potential travels. For 18
of the 30 layer 3 pyramidal neurons in which long-distance
responses were observed, the plot of mean EPSC latency versus
horizontal distance was well fit by a linear regression line
(Fig. 7A), confirming that all the EPSCs in those neurons were
evoked monosynaptically. For the remaining 12 neurons, the
latency–distance plot was not well fit by a linear relation
(Fig. 7B), suggesting that, despite having low variability in
latency and lack of failures, the EPSCs evoked from some of the
stimulation sites probably were not monosynaptic.
Figure 8. Relations between the observed EPSC latencies and the latencies predicted
for monosynaptic EPSCs. The predicted latencies were calculated for each stimulation
distance using a model linear relation (conduction velocity = 0.139 m/s, synaptic delay
= 2.29 ms; see Results). (A) A scatter plot of observed versus predicted latency was
made for the 18 neurons in which all the observed EPSC were monosynaptic, according
to the latency–distance relation (see Fig. 7A). As expected, the points were close to the
theoretical line representing identity between observed and predicted latency. For
simplicity, plotted are only the latencies for EPSCs evoked by the farthest site that
evoked a detectable monosynaptic EPSC in each cell, all with the same symbol type.
These sites were always located at a distance >600 µm from the cell (range:
650–2035 µm) and their observed latencies had the largest deviations from the line of
unity. (B) A similar scatter plot to that of (A) was made for the 12 neurons in which the
EPSC latency–distance relation indicated that not all of the EPSCs were monosynaptic
(see Fig. 7B), but EPSCs were still evoked by long-distance stimulation (e.g. stimuli
applied at ≥600 µm from the cell). Plotted are the latencies of responses evoked by all
of the long-distance stimulation sites for each of the 12 cells. Each symbol type
represents a different neuron. The graph shows that some observed EPSC latencies
deviated largely from the identity line whereas for other latencies the deviation was
small. For nine of the 12 neurons, at least one of the long-distance EPSCs had a
deviation in latency that was less than or equal to the maximal deviation observed for
points in Figure 9A; therefore, it was concluded that those EPSCs were monosynaptic.
The Majority of Layer 3 Neurons Are Postsynaptic
Targets of Long-distance Horizontal Monosynaptic
Connections
A major goal of the present study was to establish the proportion
of PFC layer 3 pyramidal neurons that are targets of long-distance
horizontal monosynaptic connections. If monosynaptic EPSCs
were evoked in a neuron by at least one stimulation site located
>600 µm from the cell, then that neuron was assumed to be a
target of long-distance, horizontal connections. As mentioned
above, 18 cells exhibited EPSCs in response to long-distance
stimulation, with latency–distance relations indicating that the
evoked EPSCs were indeed monosynaptic. To determine if some
of the long-distance EPSCs evoked in the other neurons were also
monosynaptic, the experimentally observed EPSC latencies with
predicted latencies calculated for each stimulation distance were
correlated using a model linear relation. This linear model had
parameters equal to the average values obtained from the linear
latency–distance plots in the first group of 18 neurons. The
conduction velocity of the horizontal axon collaterals, estimated
from the slope of the linear regressions, was 0.14 ± 0.04 m/s and
the synaptic delay, estimated from the y-axis intercepts of the
linear regressions, was 2.29 ± 0.49 ms. As expected, when the
plot of observed versus predicted latency was calculated for
EPSCs in the first group of 18 cells, the points were always close
to the theoretical line of identity between observed and
predicted latency (Fig. 8A). For nine other cells (Fig. 8B), at least
Cerebral Cortex Jan 2000, V 10 N 1 87
one of the long-distance EPSCs had a deviation in latency that
was less than or equal to the maximal deviation from the identity
line observed for EPSCs in Figure 8A. Therefore, we concluded
that these nine pyramidal cells were also targets of long-distance
excitatory monosynaptic connections. Thus, 27 of the 35 (77%)
recorded layer 3 pyramidal neurons were targets of long-distance
monosynaptic inputs.
Although a complete characterization of the biophysical
properties of these monosynaptic EPSCs was beyond the scope
of this work, we compared some of these properties between
EPSCs evoked from short- vs. long-distance stimulation. The
mean (± SD) extrapolated reversal potential for monosynaptic
EPSCs evoked by long-distance stimulation was 9.9 ± 12.0 mV
(n = 8). In five of the 18 cells in which all evoked EPSCs were
monosynaptic we selected monophasic EPSC waveforms evoked
by stimulation at a short (510 ± 30 µm) or long (1340 ± 174 µm)
distance from the same cell. For these EPSCs, recorded at somatic
holding potentials of –80 to –70 mV, the observed kinetic
parameters were similar for responses evoked by long- and
short-distance stimulation. The EPSC 10–90% rise time was 4.89
± 1.97 ms for short-distance and 3.30 ± 1.74 ms for long-distance
stimulation, whereas the EPSC duration at half peak amplitude
was 17.7 ± 7.7 ms for short-distance and 11.4 ± 3.4 ms for
long-distance stimulation. In contrast to monosynaptic responses
evoked by long-distance stimulation (which must be mediated by
long-distance axonal projections), EPSCs evoked by short
-distance extracellular stimulation may not represent exclusively
short-distance inputs. Consequently, it is not clear to what extent
the kinetic properties described above represent two distinct
populations of synaptic contacts. The excitatory postsynaptic
potential (EPSP) obser ved between a pair of simultaneously
recorded layer 3 monkey PFC pyramidal neurons (with somata
separated by <100 µm) had a 10–90% rise time of 3.25 ms and
a width at half amplitude of 15.25 ms (unpublished results).
Therefore, these data suggest that the kinetics of monosynaptic
responses evoked by short-distance stimulation or by an actual
short-distance input are not significantly different. Moreover, no
significant differences in kinetics were observed between shortand long-distance responses.
The Strength of Evoked Monosynaptic EPSCs Varies as a
Function of Stimulation Distance
To activate horizontally oriented connections in the present
experiments, a constant low-intensity stimulation current was
applied throughout the multielectrode stimulation array. Therefore, differences between the strengths of responses evoked
by different stimulation sites are likely due to variations in the
number of horizontal connections available for activation at each
stimulation site. Indeed, a periodic variation in the evoked EPSC
amplitude as a function of stimulation distance is what one
would predict if the multisite electrical stimulation ref lects the
clustered organization of intrinsic, horizontal connections in the
PFC (Levitt et al., 1993; Pucak et al., 1996). Therefore, we
determined if the amplitude of the evoked EPSCs varied as a
function of distance from the recorded neuron for the 18
neurons in which all the observed EPSCs were monosynaptic. In
two of the neurons, the EPSC amplitude decayed with distance
in a monotonic fashion (Fig. 9A). In the other 16 cells (Fig.
9B–D), although the EPSC strength tended to decay with
distance (see also Fig. 5), the distribution of monosynaptic EPSC
amplitudes as a function of stimulation distance showed peaks
that were statistically significant. However, not all of the peaks
88 Horizontal Synaptic Connections in Monkey PFC • González-Burgos et al.
Figure 9. Spatial patterns of monosynaptic EPSC amplitude as a function of the
horizontal distance at which stimulation was applied. Stimulation parameters (current,
pulse duration and frequency) were constant throughout the different stimulation sites.
(A–D) Examples of the patterns of responses observed for the 18 PFC neurons in which
all the evoked EPSCs were monosynaptic, as defined by the fit of the latency–distance
plot by a linear relation (see Fig. 7 and Results). Filled circles show the mean (± SD;
n = 10) EPSC amplitudes and open circles the baseline noise. (A) A cell in which the
EPSC amplitude decreased monotonically with increasing horizontal distance. (B–D)
Three other examples showing that, although there was a tendency of the EPSC
amplitude to decay with distance, the EPSC strength showed peaks and troughs. For
some of the peaks (labeled by asterisks), the EPSC amplitude was significantly larger
(P < 0.05) than that of the adjacent troughs.
were experimentally equivalent, because some troughs represented baseline resting conductance (e.g. no detectable EPSC; see
Fig. 9B,D), whereas other troughs were actually small amplitude
EPSCs (see Fig. 9C). Interestingly, in two neurons (data not
shown) in which it was possible to test two different levels
of stimulation current throughout the array, the response size
evoked from each multielectrode position was greater with
the higher applied current (e.g. small EPSCs were observed for
positions that evoked no response at the lower current) without
altering the overall pattern of responses.
For 15 neurons, >1 peak was obser ved and the distances
separating consecutive peaks ranged from 135 to 810 µm
(n = 29). For most neurons, the amplitude of the peak EPSCs
tended to be smaller for more distant stimulation sites. However,
in several examples (e.g. Fig. 9C) EPSC strength was similar
for all spatial peaks, independent of the distance between the
cell and stimulation site, and in some cases EPSC strength
was even greater for stimulation sites that were more distant
(Fig. 9C).
Discussion
This study, in concert with an earlier report (Krimer and
Goldman-Rakic, 1997), demonstrates the feasibility and value
of a primate PFC slice preparation for electrophysiological,
anatomical and biochemical in vitro studies. Given the marked
structural and functional differences between the PFC of rodents
and primates (Preuss, 1995), this slice preparation makes it
possible to study cellular neurophysiological features that may
be unique to the primate PFC. In particular, this preparation
has enabled us, in the present report, to address the following
questions.
Do Pyramidal Neurons in Layer 3 of Monkey PFC
Receive Monosynaptic, Excitatory Input from
Horizontal, Long-distance Connections?
Our previous anatomical studies demonstrated that the horizontal, long-distance axon collaterals furnished by supragranular
pyramidal neurons in monkey PFC synapse, almost exclusively,
on the dendritic spines of other PFC pyramidal neurons
(Melchitzky et al., 1998). In the present study, we demonstrate
electrophysiologically that these long-distance axon collaterals
(originating up to ∼2.0 mm from the recorded layer 3 pyramidal
neuron) provide monosynaptic excitatory inputs to layer 3
pyramidal neurons. The monosynapticity of these connections
was verified using several criteria. First, the studied EPSCs had
to have a low failure rate, in contrast to the high failure rate
expected for polysynaptic inputs (Berry and Pentreath, 1976).
Second, the EPSCs had to have latencies that varied by <1 ms
between successive trials, because the latency of monosynaptic
connections between pairs of pyramidal neurons has been reported to f luctuate by ∼1 ms (Miles and Wong, 1986; Markram et
al., 1997; Hardingham and Larkman, 1998). Third, we required
that the increase in latency as a function of horizontal distance
be linear, and that the conduction velocity (calculated from the
slope of the linear regression) be compatible with that of thin
and unmyelinated axons (Andersen et al., 1978).
To improve the duration and stability of whole-cell
recordings, and the overall viability of the slices, the present
experiments were performed at room temperature, a condition
known to affect synaptic transmission and axonal conduction
velocity (Hardingham and Larkman, 1998). For example, in
neocortical slices maintained at 34–35°C (Thomson et al., 1988;
Lohmann and Rorig, 1994) conduction velocity was 0.1 and
0.3 m/s for pyramidal cell axon collaterals. In contrast, in the
present study we found that at room temperature (20–24°C)
conduction velocity was slower (mean: 0.14 m/s). This is
consistent with previous findings that low temperature markedly
slows action potential conduction velocity (Andersen et al.,
1978; Berg-Johnsen and Langmoen, 1992). Our estimations of
synaptic delay also are consistent with monosynaptic EPSCs
recorded at low temperatures. We estimated (from the intercepts
of the linear regressions) a synaptic delay of 2.29 ± 0.49 ms for
layer 3 horizontal connections at room temperature, a value
consistent with the report that the synaptic delay at mammalian
glutamate synapses is temperature-sensitive, increasing from
0.2 ms at 35°C to 3 ms at 20°C (Sabatini and Regehr, 1996). In the
present experiments, a long synaptic delay helped to discriminate between mono- and polysynaptic responses because the
latter should exhibit an additional latency of at least one synaptic
delay (see Fig. 6C,D).
In light of previously published results (Sabatini and Regehr,
1996; Hardingham and Larkman, 1998), two other effects of low
temperature to be considered are slow kinetics of the EPSC and
decreased probability of release. Although slow kinetics would
change the temporal window for synaptic integration, it should
not affect the present results because we did not study summation of synaptic events. A decrease in probability of release
would result in a high probability of observed failures, even if
the synaptic connections have multiple synaptic contacts (see
the following section).
In the present study, we did not examine the pharmacology of
the receptors mediating the EPSCs evoked by activation of
horizontal connections. Previous electron microscopy studies
showed that most, if not all, of the synapses established by
long-distance horizontal connections in PFC are asymmetric, and
thus very likely excitatory (Melchitzky et al., 1998). Consistent
with these results, in every cell tested, monosynaptic EPSCs
evoked by activation of long-distance horizontal connections
had an apparent reversal potential close to 0 mV, as expected if
synaptic excitatory glutamate receptor-gated channels mediated
the response (Hestrin et al., 1990).
We also measured the time course (rise time and duration) of
small, monosynaptic and monophasic EPSCs evoked by stimulation at different distances from a given neuron. We did not find
significant differences in the kinetics of EPSCs as a function of
this distance, suggesting that localization of synaptic contacts on
the dendritic arbor of layer 3 pyramidal neurons was similar for
short- and long-distance horizontal connections [however, see
the findings of Markram et al. (Markram et al., 1997)]. The time
course of the EPSCs also was similar to the time course of
short-distance EPSPs evoked by a single presynaptic pyramidal
neuron during simultaneous current-clamp recordings from a
pair of synaptically connected layer 3 pyramidal neurons in
monkey PFC. The similarity in waveform kinetics between
EPSCs and EPSPs also indicates that the synapses mediating the
EPSCs in the present study were located in a membrane region in
which voltage was poorly controlled by the somatic voltageclamp (Spruston et al., 1993). Finally, if the location of synaptic
contacts is in fact related to the effectiveness of a connection to
drive the postsynaptic cell, then the similarity in the time course
of EPSCs evoked from short- vs. long-distance stimulation sites
would indicate that differences in strength between short- and
long-distance connections are dependent only on the number of
synaptic contacts that each target cell receives from each source
(see below).
How Robust is the Input from the Long-distance as
Compared with the Short-distance Connections?
A large proportion of the excitatory input to cortical pyramidal
neurons is generally assumed to be provided by short-distance,
local axon collaterals of neighboring pyramidal cells (Douglas et
al., 1995; Markram, 1997). However, in many of our experiments, monosynaptic EPSCs elicited from distal stimulation sites
had a similar or larger amplitude than EPSCs elicited from more
proximal sites (Fig. 9). We assumed that the evoked EPSCs were
not unitary and that therefore the EPSC amplitude was directly
proportional to the number of stimulated synaptic connections.
If this assumption is correct (see below), then the presence
of large EPSCs evoked from distant sites is contrary to the
expectation that monosynaptic input would decline progressively with distance, due to the severing of horizontal axons
during the slicing procedure. Therefore, this finding suggests
that long-distance, horizontal, intrinsic projections are a
relatively strong source of excitatory input to layer 3 pyramidal
neurons.
The monosynaptic EPSCs recorded in the present study were
likely to be mediated by more than one synaptic connection
because (i) activation of unitary connections requires minimal
stimulation, which we did not use; and (ii) in neocortical slices,
unitary EPSCs evoked by minimal stimulation have been
reported to show high failure rates (Rumpel et al., 1998; Gil et
al., 1999), which we did not obser ve. In our experiments,
failures were detected only in EPSCs that, in concert, had a
highly variable latency and thus were polysynaptic. On the other
hand, the conclusion that unitary responses must exhibit
significant failure rates may not be correct. For example, in
neocortical slices at 32–34°C, unitary EPSPs had extremely
low failure rates (0–5%) during simultaneous recordings from
Cerebral Cortex Jan 2000, V 10 N 1 89
synaptically connected pyramidal cells (Markram et al., 1997;
Galarreta and Hestrin, 1998). This observation was explained
by the fact that pyramid-to-pyramid connections in neocortex
involve an average of six synaptic contacts (Markram et al.,
1997). Also, the sampling procedure in the present study was
biased to detect connections with a high probability of release
since only 10–20 traces were collected for each stimulation site.
Finally, the amplitudes of the EPSCs recorded in this study were
generally small and similar to that of unitary EPSCs observed in
paired recordings (Galarreta and Hestrin, 1998).
However, compared with paired recordings, a high failure
rate is to be expected with extracellular stimulation, due to
sources other than failures of release, e.g. stimulation failures
due to threshold stimulation intensity (Gil et al., 1999) and
conduction failures, which are more likely for cut axons than for
intact ones (Stratford et al., 1996). Moreover, unitary EPSPs in
paired recordings display almost no failures at 35°C but very
high failure rates when recorded at low temperatures, because
of a pronounced decrease in probability of release that seems to
overcome synapse multiplicity in pyramid-to-pyramid connections (Hardingham and Larkman, 1998). Therefore, it seems
reasonable to conclude that in our experimental conditions
(which include room temperature and non-minimal extracellular electrical stimulation) the absence of failures indicates a
multi-connection EPSC.
What Proportion of Layer 3 Pyramidal Cells Receive
Long-distance, Excitatory, Monosynaptic Inputs?
Our findings also suggest that most pyramidal neurons in layer 3
are targets of long-distance, horizontal projections. Specifically,
low-intensity stimulation at long distances from the recorded
layer 3 pyramidal cell evoked monosynaptic EPSCs in the
majority (77%) of these neurons. However, this proportion is
likely to be an underestimate since some long-distance axon
collaterals were probably severed by slicing of the tissue blocks.
Although the present study does not indicate whether horizontal
projections synapse selectively onto layer 3 pyramidal neurons,
our results show that these cells frequently receive this type of
synaptic input.
Alternatively, if our estimate is accurate, the fact that not all
the recorded layer 3 neurons appear to receive long-distance
input raises the possibility that a subpopulation (23%) of layer 3
pyramidal neurons does not participate in long-distance interactions. Interestingly, in monkey visual cortex, ∼25% of layer 2/3
pyramidal neurons give rise only to short-distance connections
(Yabuta and Callaway, 1998).
Does the Spatial Pattern of Excitatory Monosynaptic
Responses Evoked by Multisite Stimulation Ref lect the
Clustered Organization of Horizontal Connectivity in
Monkey PFC?
Previous anatomical studies showed that PFC supragranular
pyramidal neurons are organized into discrete clusters separated
by gaps, and that these clusters are reciprocally connected by
intrinsic, horizontally oriented axon collaterals (Levitt et al.,
1993; Kritzer and Goldman-Rakic, 1995; Pucak et al., 1996).
In addition, the labeled horizontal axons present in the gaps
between clusters are nonbranching and nonvaricose, suggesting
that these axons have a very low probability of connecting with
neurons located in the intervening gaps. Based on the spatial
arrangement of these horizontal connections, one would expect
90 Horizontal Synaptic Connections in Monkey PFC • González-Burgos et al.
a periodic variation in the strength of functional connectivity,
with peaks and troughs in connectivity strength occurring at the
locations of the clusters and gaps, respectively.
The functional significance of these findings depends, in part,
on the neural elements activated by the extracellular electrical
stimulation employed in this study. Experiments in rat visual
cortex slices suggest that axon branches, but not cell bodies,
dendrites or axon initial segments, are activated by electrical
stimulation (Nowak and Bullier, 1998a,b). In contrast, other
studies show that electrical or glutamate stimulation produce
similar spatial patterns of activation in slices from neocortex
(Dalva et al., 1997; Yuste et al., 1997), arguing against a
predominance of electrical stimulation of axons versus cell
somata. Since it is possible that the stimulation we used activated
both these types of presynaptic elements, it is important to consider how they would contribute to the observed profiles of
horizontal monosynaptic EPSCs. Specifically, if the stimulation
results solely in the activation of axons, one would expect a
simple decay of response strength with distance, because the
number of intact horizontal axons (and thus of inputs available
for stimulation) would progressively decrease as a function of
distance from the recorded cell, due to the slicing of the tissue
blocks. On the other hand, if the stimulation activates only cell
bodies (or axon initial segments), then stimulation at the
locations of the cell clusters would produce peaks in EPSC
amplitude, whereas stimulation at the gaps between clusters
would evoke no response.
The present study revealed two patterns in the distribution
of monosynaptic EPSC amplitudes evoked as a function of
horizontal distance in superficial layers of monkey PFC. In a
minority of cells (12.5%), EPSC amplitude decayed with distance
in a monotonic fashion (Fig. 9A). By contrast, in 87.5% of the
cells, the distribution of monosynaptic EPSC amplitudes (as a
function of horizontal distance from the recorded cell) showed
a series of amplitude peaks superimposed on an overall monotonic decay. As suggested elsewhere (Nowak and Bullier,
1998a,b), the decay in EPSC amplitude with stimulation distance
indicates that activation of axons is a major factor in determining
EPSC amplitude. However, the presence of peaks in the spatial
patterns of EPSC amplitude observed for the majority of PFC
layer 3 pyramidal cells would also be consistent with the
activation of discrete clusters of cell bodies whose intrinsic axon
collaterals innervate the recorded cell. Interestingly, the range of
horizontal distances (135–810 µm) between the stimulation sites
that evoked peaks in EPSC amplitude was very similar to the
range of distances (200–1200 µm) between centers of stripe-like
clusters in anatomical studies of monkey PFC (Levitt et al.,
1993). In fact, previous studies in other cortical regions suggest
that clustered connections are the basis of a peaked distribution
in EPSC amplitudes. For example, using whole-cell recordings
and multisite electrical stimulation in visual cortex slices, Weliky
and colleagues (Weliky and Katz, 1994; Weliky et al., 1995)
obser ved EPSC peaks when stimulation was applied at the
locations of clusters of neurons that shared orientation selectivity with the recorded cell. These iso-orientation clusters of visual
cortex neurons are known to be linked by intrinsic horizontal
axons (Gilbert and Wiesel, 1989). However, the present study
does not provide direct evidence for a correspondence between
location of clusters of connected neurons and stimulation sites
evoking peaks in EPSC amplitude. Therefore, whether the spatial
peaks in EPSC amplitude result from clustered connectivity in
the PFC slices remains to be demonstrated.
Functional Significance of Long-distance, Horizontal,
Excitatory inputs to Layer 3 Pyramidal Cells in Monkey
PFC
The present study demonstrates that long-distance horizontal
connections constitute a significant intrinsic pathway of synaptic communication in superficial layers of monkey PFC. Our
experimental approach does not, however, directly address the
functional role of these connections during dynamic operations
of the PFC in vivo. Our present in vitro results, in concert with
previous anatomical and in vivo electrophysiological studies,
may provide support for several possible functional roles for
these connections.
It is possible that, as in other regions of neocortex, PFC
neurons with similar functional properties are clustered and that
the horizontal connections link these clusters of cells. In spatial
delayed-response tasks, the delay period activity of PFC neurons
is selective for the location of the cue in the visual field,
suggesting that individual neurons have specific ‘memory fields’
(Funahashi et al., 1989; Rainer et al., 1998). By analogy to the
links between iso-orientation columns in the visual cortex, the
long-distance horizontal connections in the PFC have been
proposed to link clusters of cells sharing memory fields
(Goldman-Rakic, 1995). In addition, in visual cortex, horizontal
connections have been proposed to mediate the long-distance,
between-column synchronization of activity during visual
stimulation (Mountcastle, 1998). Because cells of different
columns usually have different receptive fields, horizontal
connections are thought to mediate the effects of stimulation
outside a given cell’s visual receptive field (Gilbert, 1998).
Therefore, by a strict analogy to the visual cortex, horizontal
connections in the PFC would mediate lateral interactions
between clusters of neurons with different memory fields but
which share some other functional property, such as object
selectivity (Rainer et al., 1998).
A lthough delay period activity of monkey PFC neurons is
thought to be the cellular basis of working memory (GoldmanRakic, 1995), the mechanisms generating the sustained firing of
PFC neurons during the delay period are still largely unknown.
It has been suggested that sustained activity may be subserved,
at least in part, by reverberating excitatory circuits (Funahashi
and Kubota, 1994), but the organization of this circuitry is not
known. The presence of reciprocal, long-distance, horizontal
connections between clusters of supragranular pyramidal
neurons (Pucak et al., 1996), the selectivity of these axon
collaterals for pyramidal cell targets (Melchitzky et al., 1998) and
the observation that the vast majority of layer 3 pyramidal
neurons receive robust, long-distance, monosynaptic inputs
(present study) suggest that, in the monkey PFC, these horizontal
connections could ser ve an important role in promoting
recurrent activation of supragranular pyramidal cells. That is,
the mutual excitation of these cell clusters via reciprocal,
horizontally oriented inputs could contribute to the sustained
activity of PFC cells during the delay period of delayed-response
tasks (Lewis and Anderson, 1995). However, sustained neuronal
activity is also present in parietal and temporal cortices during
the delay period of working memory tasks (Fuster and Jervey,
1981; Miller and Desimone, 1994; Zhou and Fuster, 1996).
Interestingly, compared with the smaller patch-like arrangement
of horizontally connected cell groups in posterior cortical
regions (Lund et al., 1993), the stripe-like organization of cell
clusters in the PFC suggests that a larger number of pyramidal
cells per cluster participate in horizontal interconnections. If
these reciprocal, horizontal connections subserve reverberating
activity, then the larger the number of pyramidal cells recruited,
the more robust their sustained firing is likely to be. Therefore,
the stripe-like architecture of the PFC could explain why the
delay-related activity of PFC cells is less likely to be interrupted
by intervening stimuli than the delay-related firing of neurons in
temporal or parietal cortices (Miller et al., 1996).
Notes
We thank Darrell Henze and Nathan Urban for providing the acquisition
and analysis software and for the helpful discussions. We also thank Brad
Booth, Mar y Brady and Darlene Melchitzky for their excellent technical
assistance. This work was supported by NIMH grants MH51234 and
MH45156 and Independent Scientist Award MH00519.
Address correspondence to David A. Lewis, MD, University of
Pittsburgh, 3811 O’Hara Street, W1650 BST, Pittsburgh, PA 15213, USA.
Email: [email protected].
References
Andersen P, Silfvenius H, Sundberg SH, Sveen O, Wigstrom, H (1978)
Functional characteristics of unmyelinated fibres in the hippocampal
cortex. Brain Res 144:11–18.
Berg-Johnsen J, Langmoen IA (1992) Temperature sensitivity of thin
unmyelinated fibers in rat hippocampal cortex. Brain Res 576:
319–321.
Berry MS, Pentreath V W (1976) Criteria for distinguishing between
monosynaptic and polysynaptic transmission. Brain Res 105:1–20.
Bosking WH, Zhang Y, Schofield B, Fitzpatrick D (1997) Orientation
selectivity and the arrangement of horizontal connections in tree
shrew striate cortex. J Neurosci 17:2112–2127.
Chagnac-Amitai Y, Connors BW (1989) Horizontal spread of synchronized activity in neocortex and its control by GABA-mediated
inhibition. J Neurophysiol 61:747–758.
Dalva MB, Weliky M, Katz LC (1997) Relationships between local synaptic
connections and orientation domains in primary visual cortex.
Neuron 19:871–880.
Douglas RJ, Koch C, Mahowald M, Martin K A, Suarez HH (1995)
Recurrent excitation in neocortical circuits. Science 269:981–985.
Dudek SM, Friedlander MJ (1996) Intracellular blockade of inhibitory
synaptic responses in visual cortical layer IV neurons. J Neurophysiol
75:2167–2173.
Funahashi S, Kubota K (1994) Working memory and prefrontal cortex.
Neurosci Res 21:1–11.
Funahashi S, Bruce CJ, Goldman-Rakic PS (1989) Mnemonic coding
of visual space in the monkey’s dorsolateral prefrontal cortex. J
Neurophysiol 61:331–349.
Fuster JM, Jervey JP (1981) Inferotemporal neurons distinguish and retain
behaviorally relevant features of visual stimuli. Science 212:952–955.
Galarreta M, Hestrin S (1998) Frequency-dependent synaptic depression
and the balance of excitation and inhibition in the neocortex. Nature
Neurosci 1:587–594.
Gil Z, Connors BW, Amitai Y (1999) Efficacy of thalamocortical and
intracortical synaptic connections: quanta, inner vation, and reliability. Neuron 23:385–397.
Gilbert CD (1998) Adult cortical dynamics. Physiol Rev 78:467–485.
Gilbert CD, Wiesel TN (1989) Columnar specificity of intrinsic horizontal
and corticocortical connections in cat visual cortex. J Neurosci
9:2432–2442.
Goldman-Rakic PS (1995) Cellular basis of working memor y. Neuron
14:477–485.
Gutnick MJ, Connors BW, Prince DA (1982) Mechanisms of neocortical
epileptogenesis in vitro. J Neurophysiol 48:1321–1335.
Hardingham NR, Larkman AU (1998) The reliability of excitatory synaptic
transmission in slices of rat visual cortex in vitro is temperature
dependent. J Physiol 507:249–256.
Hestrin S, Perkel DJ, Sah P, Manabe T, Renner P, Nicoll R A (1990)
Physiological properties of excitatory synaptic transmission in the
central nervous system. Cold Spring Harbor Symp Quant Biol 55:
87–93.
Krimer LS, Goldman-Rakic PS (1997) An interface holding chamber for
anatomical and physiological studies of living brain slices. J Neurosci
Methods 75:55–58.
Kritzer MF, Goldman-Rakic PS (1995) Intrinsic circuit organization of the
Cerebral Cortex Jan 2000, V 10 N 1 91
major layers and sublayers of the dorsolateral prefrontal cortex in the
rhesus monkey. J Comp Neurol 359:131–143.
Levitt JB, Lewis DA, Yoshioka T, Lund JS (1993) Topography of pyramidal
neuron intrinsic connections in macaque monkey prefrontal cortex
(areas 9 and 46). J Comp Neurol 338:360–376.
Lewis DA, Anderson SA (1995) The functional architecture of the
prefrontal cortex and schizophrenia. Psychol Med 25:887–894.
Lohmann H, Rorig B (1994) Long-range horizontal connections between
supragranular pyramidal cells in the extrastriate visual cortex of the
rat. J Comp Neurol 344:543–558.
Lund JS, Yoshioka T, Levitt JB (1993) Comparison of intrinsic connectivity
in different areas of macaque monkey cerebral cortex. Cereb Cortex
3:148–162.
Malach R, Amir Y, Harel M, Grinvald A (1993) Relationship between
intrinsic connections and functional architecture revealed by optical
imaging and in vivo targeted biocytin injections in primate striate
cortex. Proc Natl Acad Sci USA 90:10469–10473.
Markram H (1997) A network of tufted layer 5 pyramidal neurons. Cereb
Cortex 7:523–533.
Markram H, Lubke J, Frotscher M, Roth A, Sakmann B (1997) Physiology
and anatomy of synaptic connections between thick tufted pyramidal
neurones in the developing rat neocortex. J Physiol 500:409–440.
McCormick DA (1989) GABA as an inhibitory neurotransmitter in human
cerebral cortex. J Neurophysiol 62:1018–1027.
McGuire BA, Gilbert CD, R ivlin PK, Wiesel TN (1991) Targets of
horizontal connections in macaque primary visual cortex. J Comp
Neurol 305:370–392.
Melchitzky DS, Sesack SR, Pucak ML, Lewis DA (1998) Synaptic targets of
pyramidal neurons providing intrinsic horizontal connections in
monkey prefrontal cortex. J Comp Neurol 390:221–224.
Miles R, Wong RK (1986) Excitatory synaptic interactions between CA3
neurones in the guinea-pig hippocampus. J Physiol 373:397–418.
Miller EK, Desimone R (1994) Parallel neuronal mechanisms for shortterm memory. Science 263:520–522.
Miller EK, Erickson CA, Desimone R (1996) Neural mechanisms of visual
working memory in prefrontal cortex of the macaque. J Neurosci
16:5154–5167.
Mountcastle VB (1998) Perceptual neuroscience. Cambridge, MA:
Har vard University Press.
Nelson S, Toth L, Sheth B, Sur M (1994) Orientation selectivity of cortical
neurons during intracellular blockade of inhibition. Science 265:
774–777.
Nowak LG, Bullier J (1998a) Axons, but not cell bodies, are activated by
electrical stimulation in cortical gray matter. I. Evidence from chronaxie measurements. Exp Brain Res 118:477–488.
Nowak LG, Bullier J (1998b) Axons, but not cell bodies, are activated
by electrical stimulation in cortical gray matter. II. Evidence from
selective inactivation of cell bodies and axon initial segments. Exp
Brain Res 118:489–500.
Preuss TM (1995) Do rats have prefrontal cortex? The Rose–Woolsey–
Akert program reconsidered. J Cogn Neurosci 7:1–24.
Pucak ML, Levitt JB, Lund JS, Lewis DA (1996) Patterns of intrinsic and
92 Horizontal Synaptic Connections in Monkey PFC • González-Burgos et al.
associational circuitry in monkey prefrontal cortex. J Comp Neurol
376:614–630.
Rainer G, Asaad WF, Miller EK (1998) Memory fields of neurons in the
primate prefrontal cortex. Proc Natl Acad Sci USA 95:15008–15013.
Rumpel S, Hatt H, Gottmann K (1998) Silent synapses in the developing
rat visual cortex — evidence for postsynaptic expression of synaptic
plasticity. J Neurosci 18:8863–8874.
Sabatini BL, Regehr WG (1996) Timing of neurotransmission at fast
synapses in the mammalian brain. Nature 384:170–172.
Shao Z, Burkhalter A (1996) Different balance of excitation and inhibition
in forward and feedback circuits of rat visual cortex. J Neurosci
16:7353–7365.
Spruston N, Jaffe DB, Williams SH, Johnston D (1993) Voltage- and spaceclamp errors associated with the measurement of electrotonically
remote synaptic events. J Neurophysiol 70:781–802.
Stratford KJ, Tarczy Hornoch K, Martin K AC, Bannister NJ, Jack JJB
(1996) Excitatory synaptic inputs to spiny stellate cells in cat visual
cortex. Nature 382:258–261.
Stuart GJ, Dodt HU, Sakmann B (1993) Patch-clamp recordings from the
soma and dendrites of neurons in brain slices using infrared video
microscopy. Pf luger’s Arch Eur J Physiol 423:511–518.
Thomson A M, Bannister AP (1998) Postsynaptic pyramidal target
selection by descending layer III pyramidal axons: dual intracellular
recordings and biocytin filling in slices of rat neocortex. Neuroscience 84:669–683.
Thomson A M, Deuchars J (1997) Synaptic interactions in neocortical
local circuits: dual intracellular recordings in vitro. Cereb Cortex
7:510–522.
Thomson A M, Girdlestone D, West DC (1988) Voltage-dependent
currents prolong single-axon postsynaptic potentials in layer III
pyramidal neurons in rat neocortical slices. J Neurophysiol 60:
1896–1907.
Weliky M, Katz LC (1994) Functional mapping of horizontal connections
in developing ferret visual cortex: experiments and modeling. J
Neurosci 14:7291–7305.
Weliky M, Kandler K, Fitzpatrick D, Katz LC (1995) Patterns of excitation
and inhibition evoked by horizontal connections in visual cortex share
a common relationship to orientation columns. Neuron 15:541–552.
Woo TU, Pucak ML, Kye CH, Matus CV, Lewis DA (1997) Peripubertal
refinement of the intrinsic and associational circuitry in monkey
prefrontal cortex. Neuroscience 80:1149–1158.
Yabuta NH, Callaway EM (1998) Cytochrome-oxidase blobs and intrinsic
horizontal connections of layer 2/3 pyramidal neurons in primate V1.
Vis Neurosci 15:1007–1027.
Yoshioka T, Blasdel GG, Levitt JB, Lund JS (1996) Relation between
patterns of intrinsic lateral connectivity, ocular dominance, and
cytochrome oxidase-reactive regions in macaque monkey striate
cortex. Cereb Cortex 6:297–310.
Yuste R, Tank DW, Kleinfeld D (1997) Functional study of the rat cortical
microcircuitry with voltage-sensitive dye imaging of neocortical
slices. Cereb Cortex7:546–558.
Zhou YD, Fuster JM (1996) Mnemonic neuronal activity in somatosensory
cortex. Proc Natl Acad Sci USA 93:10533–10537.