Department of Experimental Neurophysiology, Istituto Nazionale
Neurologico, 20133 Milan, Italy
 |
INTRODUCTION |
Clinical reports and animal
studies demonstrated that the parahippocampal region supports complex
cognitive functions, in particular related to memory formation,
consolidation, and retrieval (Alvarez and Squire 1994
;
Amaral 1999
; Braak and Braak 1993
;
Brown and Aggleton 2001
; Brown and Xiang
1998
; Gaffan and Parker 1996
; Suzuki
1996
; Young et al. 1997
; Zola-Morgan et
al. 1989
). The two major sub-regions of the parahippocampus,
the entorhinal cortex (ERC) and the perirhinal cortex (PRC), do not
serve as mere cortical transfer areas of incoming inputs to the
hippocampus, but retain integrative properties that are essential for
memory processing. It has been demonstrated that selective ablations of
the PRC in rats and monkeys affect visual discrimination
(Buckley and Gaffan 1997
, 1998
;
Meunier et al. 1993
; Myhrer and Wangen
1996
; Riches et al. 1991
; Suzuki et al.
1993
; Wan et al. 1999
), associative memory
(Murray and Bussey 1999
), spatial memory (Liu and
Bilkey 1998a
,b
; Wiig and Bilkey 1994
), and odor
recognition (Otto and Eichenbaum 1992a
; Young et
al. 1997
) and exacerbated memory impairment induced by
hippocampal lesions (Ennaceur and Aggleton 1997
;
Wiig and Bilkey 1995
; Zola-Morgan et al.
1993
). In addition, the demonstration that responses of PRC
neurons can be enhanced or depressed during repetitive presentation of
complex sensory stimuli (Otto and Eichenbaum 1992b
;
Xiang and Brown 1999
; Young et al. 1997
;
Zhu and Brown 1995
) suggested that the PRC is involved
in memory representation and in some form of recognition memory
(Brown and Xiang 1998
; Bussey et al.
1999
). The integrative role of the PRC in memory function is
presumably sustained by the associative interactions among neurons
within the parahippocampal network. The study of the connections within
the PRC has been object of extensive anatomical investigations in
different mammalian species (Burwell and Amaral 1998a
,b
;
Burwell et al. 1995
; Lavenex and Amaral
2000
; Lopes da Silva et al. 1990
; Van
Hoesen and Pandya 1975
). According to these works, neocortical
inputs from the somatosensory, auditory, and visual uni-polimodal
cortical areas project to the PRC (Burwell et al. 1995
;
Suzuki 1996
; Suzuki and Amaral 1994
),
from where they are transmitted to the hippocampus proper either
directly (Naber et al. 1997
, 1999
;
Witter et al. 2000
) or via a pathway mediated by the ERC
(Burwell and Amaral 1998a
; Lopes da Silva et al.
1990
; Witter 1993
). In spite of the extensive
anatomical studies, very little is known about the physiological
features of the responses generated within the PRC by either
neocortical or intrinsic PRC stimulation (Bilkey 1996
;
Cho et al. 2000
; Ziakopoulos et al.
1999
). In the present study we performed a detailed
electrophysiological study of the associative cortico-cortical
interactions between neocortex and area 36 in the isolated guinea pig
brain maintained in vitro, a preparation that allows a facilitated
approach to the intact rhinal area through a direct visual control of
the placement of the recording and stimulating electrodes. The present results have been communicated in abstract form (Biella et al. 2000
).
 |
METHODS |
After barbiturate anesthesia (sodium pentothal, 20 mg/kg ip) the
brain of young adult guinea pigs (150-200 g, 4-6 wk old) was
dissected out according to the standard procedure previously described
in details (de Curtis et al. 1991
, 1994a
,
1998
). The isolated brain was arterially perfused at 5.5 ml/min with a solution (composition, in mM: 126 NaCl, 3 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, 15 glucose, and 2.1 HEPES, with 3%
dextran M.W. 70.000) oxygenated with a 95%
O2-5% CO2 gas mixture (pH
7.3). Experiments were performed at 32°C. Stimuli were delivered via tungsten electrodes arrays formed by two or three electrode pairs vertically separated by 500-1,000 µm (FHC, Bowdoinham, ME). The tungsten electrode pairs were positioned at different depths within the
cortical structures. Extracellular recordings were performed with
either glass micropipettes (filled with 1 M NaCl) or stainless steel
electrodes. Electrodes arrays formed by four to six stainless steel
rods separated by 410 µm (provided by FHC) were also utilized for
extracellular recordings. Laminar profiles of evoked activity were
performed by averaging four to seven responses recorded with 16-lead
silicon probes (single recording sites separated by 50 µm on a single
vertical shaft; kindly provided by Jamille Hetke of the Center of
Neural Communication and Technology of the University of Michigan, Ann
Arbor, MI). The position of the electrodes could be easily and rapidly
modified during the experiment under direct visual control with a
stereoscopic microscope. The field potentials were amplified
(extracellular amplifier by Biomedical Engineering, Thornwood, NY),
digitized via an ATMIO-64E3 National Instruments board, and stored on
tape (Biologic, Claix, France) for off-line analysis.
Intracellular recordings were performed with micropipettes filled with
2 M potassium acetate and 2% biocytine. The intracellular signals were
recorded with a Neurodata amplifier (New York, NY). Data acquisition
and analysis was performed by using CLAMPVIEW, a software developed in
our Department by Gerardo Biella in collaboration with the Italian
branch of National Instruments. Current source density analysis (CSD)
was performed with a 200-µm differentiation grid on laminar profiles
recorded with the 16-channel silicon probes (50-µm inter-electrode
spacing) according to the standard procedure previously described
(Biella and de Curtis 1995
, 2000
; Ketchum and Haberly 1993
).
Electrolytic lesions performed at the end of the experiments were
utilized to mark the position of both the stimulating electrodes and
the silicon probes (see METHODS in Biella and de
Curtis 2000
). When intracellular biocytine injections were
performed, brains were processed for biocytine-horseradish peroxidase
visualization. After fixation in 4% paraformaldehyde, 75-µm sections
were cut by vibratome, and intracellular biocytine was revealed by
processing the sections with ABC kit (Vector Laboratories). Sections
were counterstained with thionin to identify cortical layers.
 |
RESULTS |
Experiments were carried out on 38 isolated guinea pig brains. The
borders between the neocortex (NC) and the PRC were identified on
coronal sections of the guinea pig brain stained with thionin on the
basis of cytoarchitectonic criteria previously described in the rat
(Burwell and Amaral 1998b
; Insausti et al.
1997
). In comparison to NC, area 36 in the PRC shows a broader
layer II and a less distinct subdivision in six layers. No recordings
were performed in the most caudal part of the rhinal region, named postrhinal cortex.
The general pattern of propagation of associative excitation from the
NC to area 36 was investigated in 16 experiments by positioning 2 array
of 3 electrodes, each separated by 410 µm, along the rhinal sulcus in
area 36 (see schematic drawings in Fig.
1). Stimuli were applied to the temporal
NC region (2 mm dorsal to area 36) with pairs of stainless steel
electrodes inserted at 100- to 300-µm depth. Area 36 recordings close
to the NC stimulation site (electrodes 6 and 5 in
Fig. 1A) showed an early negative potential followed by a
positive potential (recordings performed at 600-µm depth). At
recording sites >800 µm caudal to the stimulus location, an isolated
late depth-positive response, not preceded by the negative component,
was observed (electrodes 1-4 in Fig. 1A). A
similar pattern, with opposite rostro-to-caudal distribution, was
observed when the NC stimulating electrode was positioned at the caudal
end of the recording electrode array (close to electrode 1;
not shown). Stimulation of superficial layers in area 36 itself induced
biphasic negative-positive responses just caudal and rostral to the
stimulation site (electrodes 3 and 4 in Fig.
1B), whereas late depth-positive potentials were recorded at
more remote locations (electrodes 1, 2, 5, and 6 in Fig. 1B). Stimulation of deep NC layers induced either no
response or small amplitude potentials in area 36 (n = 8). No consistent local response was induced within area 36 by local
stimulation of deep layers (not shown; n = 3).

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Fig. 1.
Field potentials recorded at different sites in area 36 in response to
superficial neocortex (NC) stimulation (A) and area 36 stimulation (B). Schematic drawings of the electrode
placement are illustrated. The recording sites were separated by 410 µm. All recordings were performed at 500- to 600-µm depth. An early
depth-negative response was recorded at sites 5 and
6 after NC stimulation (A) and at sites
3 and 4 after area 36 stimulation (B).
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|
To evaluate the pattern of local network activation in area 36, CSD
analysis of field potential laminar profiles was performed (n = 7) (Biella and de Curtis 1995
;
de Curtis et al. 1994b
). Figure 2A illustrates a typical field
potential profile recorded with the 16-channel silicon probe after
superficial NC stimulation (FP profile; left column) and the
relative extracellular current profile (CSD profile; right
column). The distribution in time and space of the extracellular
currents induced by NC stimulation are shown in the contour plot in
Fig. 2B, where the field potential traces at different
depths are superimposed on top of the contour plot. The results
demonstrated that at recording sites close to NC stimulation an early
current sink located at 250- to 400-µm depth with a 10- to 12-ms
delay from the stimulus artifact correlated to the early
surface-positive/depth-negative potential (Fig. 2B; compare
the delays of the potentials to the current sinks in the contour plot).
Such a sink was coupled to a superficial source and was followed by a
large current dipole that peaked at 15-20 ms, in coincidence with the
surface-negative/depth-positive component of the field response. The
largest sink at this delay was located in the surface; a sink in deep
layers (>500-µm depth) was observed in 3 of 10 CSD profiles.
NC-induced active current events were restricted to the superficial 700 µm in area 36. The absence of local sinks or sources below 700-µm
depth was verified in four experiments, in which the laminar profile
was extended to 1,300 µm either by lowering the 50-µm inter-lead
silicon probe or by using 16-channel probes with inter-lead separation
of 100 µm. CSD analysis of laminar profiles performed in area 36 at
the recording site rostral to the coronal level in which the NC
stimulus was delivered (see schematic drawing in Fig. 2A)
revealed a superficial sink with a delay >20 ms that correlated with a
late field response (Fig. 2C). At recording sites both near
and far from the stimulation, field potential components and associated
sinks with a long delay from the stimulus artifact were seldom observed
(see sinks with a >40-ms delays in the contour plots in Figs. 2,
B and C, and 3A). Such responses were
not analyzed in detail in the present study and were restricted to the
network mechanisms that generate activity in the early 40-50 ms after
the stimulus.

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Fig. 2.
Perirhinal cortex potentials and currents evoked by NC stimulation.
Current source density (CSD) analysis of NC-evoked field potential
laminar profiles performed with a 16-channel silicon probe (50-µm
inter-lead spacing) positioned in area 36, close (36c) and far (36f)
from the stimulating electrode (st. NC in the drawing in
A). In the left column in
A, the laminar field potential profile evoked in area
36c is shown. The relative CSD profile is illustrated in the
right panel. In B, the contour plot
obtained from the CSD traces shows the distribution of current sinks
and sources in the cortical layers in the position 36c (see
Biella and de Curtis 2000 for details on contour plots).
The correlation between the sinks/sources dipoles and the superimposed
field responses recorded with the multi-channel silicon probe are
shown. The dotted line marks the stimulus artifact. In
C, recordings performed in the same experiment and
relative CSD contour plot obtained in position 36f are shown.
Separation between isocurrent lines 10 mV/mm2.
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Figure 3 illustrates typical profiles
evoked in area 36 by stimulation of area 36 itself (n = 12). As for the response to NC stimulation, at cortical sites close to
the stimulation electrode (see scheme), a sink at 200- to 300-µm
depth followed by a larger and longer-lasting sink-source dipole
between 100 and 200 µm was observed (Fig. 3A). The late
potential at 15-20 ms observed in area 36 at a distance higher than
800 µm from the coronal level of the stimulating electrode correlated
to a superficial current sink that had the same depth location of the
late sink in the close recording site (Fig. 3B; probe
positioned at 1 mm from the stimulating electrode). No differences in
current distribution pattern was observed if the stimulating electrode
was positioned either caudal or rostral to the recording electrodes.
The results obtained with CSD analysis demonstrated that quite
stereotyped responses were generated in area 36 following both NC and
local cortical stimulation.

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Fig. 3.
PRC potentials and currents evoked by area 36 stimulation. In the
drawing the positions of the stimulating electrode and recording
electrodes are schematically represented. In A, the
superimposed field traces recorded with the 16-channel silicon probe
and the CSD contour plot obtained from area 36c (close to the
stimulation site) are illustrated. In B, the same is
shown for a recording site in area 36f, 1 mm rostral to the stimulation
site. Recordings were performed in the same experiment. Separation
between isocurrent lines 10 mV/mm2.
|
|
CSD analysis allows to detect laminar-segregated excitatory synaptic
potentials and population events but is not ideal to detect inhibitory
synaptic potentials and spatially distributed events. Therefore, on the
basis of the CSD analysis, only a partial picture of the events induced
by NC and local area 36 stimulation can be extrapolated (Ketchum
and Haberly 1993
; Mitzdorf 1985
). To further
characterize the network activation pattern in area 36, extracellular
laminar profile recordings with the 16-channel probe were coupled to
intracellular recordings from principal neurons in layer II and III
neurons. Of 18 neurons recorded, 6 stained neurons showed either
multipolar or pyramidal morphology. Similar intracellular/extracellular
correlation patterns were observed following both superficial NC
(n = 4) and local area 36 stimulation
(n = 18). As illustrated in Fig.
4, stimulation induced two
small-amplitude depolarizing potentials separated by approximately 7-8
ms that increased in amplitude on membrane hyperpolarization (Fig.
4B). On the basis of their membrane potential dependence,
both potentials were interpreted as excitatory postsynaptic potentials
(EPSPs). The first EPSP (e-EPSP) preceded the onset of a biphasic
inhibitory postsynaptic potential (IPSP), whereas the second EPSP
(l-EPSP, marked by the asterisk) occurred during the onset phase of the
IPSP. In the large majority of recorded neurons, the early IPSP was
depolarizing at resting membrane potential [average resting membrane
potential (RMP) =
69 ± 5.9 mV, mean ± SE;
dotted line in the neuron illustrated in Fig. 4]. The time course and
the membrane potential reversal of the early and late IPSPs (e-IPSP:
64.6 ± 4.3 mV; l-IPSP:
89 ± 2.1 mV; n = 7) were compatible with the activation of GABAa and GABAb
receptor-mediated potentials, respectively.

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Fig. 4.
Correlation between intracellular potentials and field responses
simultaneously recorded in area 36 after superficial NC stimulation.
A: the early component of the field potential (recorded
at 500-µm depth) correlates with an excitatory postsynaptic potential
(EPSP) in a layer II principal cell in area 36; the late depth-positive
potential is associated with the intracellularly recorded fast
inhibitory postsynaptic potential (e-IPSP) and a late EPSP marked by
the asterisk. The membrane potential reversal of the fast and slow
components of the IPSP is also illustrated. The EPSP-IPSP and rebound
spike are illustrated with a slower time scale in the inset.
B: the amplitudes of the early (e-EPSP) and late EPSPs (l-EPSP)
increased with membrane hyperpolarization. Note that the IPSP is
depolarizing at resting membrane potential (RMP; horizontal dotted
line = 73 mV).
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The e-EPSP correlated with both the early depth-negative extracellular
potential and the early sink located in layers II-III (Fig.
5). The late depth-positive wave and the
sink-source dipole that peaked at 15-20 ms correlated to both the
l-EPSP (marked by the asterisk) and the onset of the e-IPSP. The
disynaptic nature of the e-IPSP and the l-EPSP was demonstrated by
applying a pairing stimulation test with 30- to 50-ms stimulus
intervals (Fig. 6; n = 4). The subtraction of the paired response at 40 ms (b) from the response evoked by a single shock (a) demonstrated the
abolition of the e-IPSP and the l-EPSP in the conditioned response
(trace b-a in Fig. 6). Not a single neuron recorded in
layers II and III discharged an action potential before the IPSP on
either NC or area 36 stimulation at resting conditions, suggesting that the IPSPs are generated by a feed-forward circuit (see
DISCUSSION). As illustrated in Fig.
7, action potential could be generated exclusively by stimuli of very low intensity (30-40 µA).
Interestingly, such a spike was followed by a very small amplitude
IPSP. When the biphasic IPSP appeared by further increasing the
stimulus intensity (>50 µA), the spike was abolished and the
duration of the e-EPSP was shortened (n = 3).

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Fig. 5.
Correlation between intracellular synaptic responses recorded at 2 different membrane potentials in a layer III neuron, field potentials,
and the CSD current dipoles following closeby NC stimulation. The
superimposed extracellular potentials recorded simultaneously with the
16-channel silicon probe are shown in the middle part of
the figure. The early field component and the intracellular e-EPSP
correlate to the early CSD sink. The l-EPSP and the e-IPSP correlate
with the superficial sink at 50- to 200-µm depth. Isocurrent
lines = 10 mV/mm2.
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Fig. 6.
Mono- and polysynaptic responses in the intracellular responses evoked
by area 36 stimulation in a principal cell of layer II. The recording
was performed at resting membrane potential ( 75 mV); the e-IPSP is
depolarizing. Single stimuli to the NC (a) and paired
stimuli at 40-ms (b) and 600-ms (c)
intervals. The digital subtraction of the single from the paired
responses (b-a and c-a) shows that the
l-EPSP and the e-IPSP in the conditioned response are abolished,
whereas the monosynaptic e-EPSP is preserved.
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Fig. 7.
Action potential firing in layer II-III cells is observed exclusively
at low stimulation intensity. Intracellular recording from a multipolar
neuron in layer II following NC stimulation at different intensities
(values on the left). All recordings were performed at
60 mV (RMP = 75 mV). Weak stimuli induced an EPSP and a spike.
Stronger stimuli determined the appearance of the IPSP, associated with
the abolition of the action potential discharge.
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In five experiments, local stimulation in area 36 at different
rostral-caudal levels was delivered to mimic two separate inputs far
and close to the recording site (see schematic drawing of the electrode
placement in the inset in Fig.
8A). Stimulation of the far
site evoked a late EPSP that increased in amplitude and duration by
injecting into the cell a depolarizing current (Fig. 8A),
whereas stimulation at the electrode positioned close to the
intracellular recording site induced a typical EPSP-IPSP sequence
(dotted line) that showed a shorter delay than the EPSP induced by far
site stimulation. In all the cells recorded far from the stimulation
site, no IPSPs were observed (n = 5), even when the
membrane reached action potential threshold, as illustrated in Fig.
8A. Paired-pulse test using the stimulation site far from the recording electrode in area 36 neurons induced a complete abolition
of the EPSP in the conditioned response (trace b-a in Fig.
8B).

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Fig. 8.
Simultaneous intracellular and extracellular recording in area 36 in
response to area 36 stimulation at a sites close to (st. 36c) and far
(st. 36f) from the recording spot (see inset).
A: responses to st. 36f induced a late EPSP (and a late
potential in the extracellular recording showed in the bottom
trace); by modifying membrane potential an increase in both
amplitude and duration of the EPSP was observed. An action potential
could be generated on the EPSP for membrane depolarization above
threshold. The dotted line illustrates the response obtained by st. 36c
in the same cell; a typical EPSP/IPSP sequence is observed. RMP = 70 mV. B: in a different neuron, intrinsic (area 36)
stimulation at the far site (st. 36f) induced a late EPSP
(a). In the other traces the responses to paired stimuli
at 30 ms (b), 70 ms (c) and 600 ms
(d) are illustrated. In the bottom panel
the subtracted traces show that the l-EPSP is abolished at brief
inter-stimulus intervals (trace b-a) and recovers in
response to conditioned stimuli at longer delays.
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 |
DISCUSSION |
In the present study, simultaneous, combined evaluation of
intracellular potentials and extracellular laminar profiles was utilized to characterize the propagation pattern of synaptic activity in area 36 of the PRC, in response to both NC stimulation and intrinsic
stimulation within area 36 itself. The electrophysiological study of
associative interactions within the rhinal cortex has been largely
facilitated by the ability to perform simultaneous extra/intracellular
recordings and CSD analysis of field potential laminar profiles in the
in vitro isolated guinea pig brain, a unique preparation to study
limbic system physiology (Biella and de Curtis 2000
;
de Curtis et al. 1994b
; Dickson et al.
2001
; Paré et al. 1992
). We demonstrate
that synaptic excitation in area 36 1) is curtailed by a
prominent inhibitory disynaptic activity close to the stimulation site
and 2) is effectively propagated at distance within area 36. These results confirm the observation recently reported in guinea pig
horizontal PRC slices, which demonstrated the presence of EPSP-IPSP
sequences and pure late EPSPs in PRC neurons located, respectively,
close to and remote from the NC stimulating electrode (Martina
et al. 2001
).
In our experiments, similar responses were observed following
superficial layer stimulation in the temporal NC and in area 36, which
most probably activated the output fibers of layer II-III neurons that
terminate in area 36 (Burwell and Amaral 1998a
;
Burwell et al. 1995
; Deacon et al. 1983
).
NC/intrinsic stimulation of deep layers, which contain cells that
participate to a lesser extent to the PRC projection, did not evoke a
consistent activation pattern. CSD analysis of extracellular field
profiles demonstrated that the associative inputs (both neocortical and
intrinsic) induce a monosynaptic sink centered at 250- to 400-µm
depth, presumably on the basal and apical dendrites of layer II-III
neurons (Faulkner and Brown 1999
). The experiments
performed with multi-electrode arrays demonstrated that the propagation
of the early excitatory component (i.e., the depth-negative component
of the field response) is spatially restricted in the rostrocaudal
dimension of the PRC. As expected, intracellular recordings performed
in layer II-III principal neurons in the region in which the early
depth-negative potential was observed demonstrated that the
monosynaptic CSD sink was associated to a small-amplitude monosynaptic
EPSP, likely mediated by glutamatergic synapses (Cho et al.
2000
; Ziakopoulos et al. 1999
). The duration of
the intracellularly recorded e-EPSP was consistently shorter then the
extracellular monosynaptic current sink. This finding is not
surprising, since it is well known that the extracellular sink directly
reflects the time course of synaptic activation at its site of
generation, presumably in the dendrites, whereas the intracellular
e-EPSP is influenced by simultaneously active membrane conductances,
such as the one coupled with the e-IPSP possibly generated close to the
soma, that accelerate the EPSP decay.
The monosynaptic EPSP was followed by a biphasic IPSP. The early IPSP
was depolarizing at resting membrane potential and showed a potential
reversal compatible with the activation of GABAa postsynaptic receptors
in a cortical structure (Connors et al. 1988
;
Scharfman and Sarvey 1987
). The time course and the
reversal potential of the late IPSP was consistent with a GABAb
receptor-mediated response (Connors et al. 1988
;
Scharfman and Sarvey 1987
). The IPSPs were presumably
generated by the stimulus-evoked discharge of inhibitory interneurons
with a superficial axonal arborization, similar to the neurons
described in the layers I-II of the rat PRC (Faulkner and Brown
1999
) or in the neighboring entorhinal cortex (Funahashi and Stewart 1998
; Jones and Buhl 1993
). The
pairing test demonstrated that the IPSPs observed in our experiments
are polysynaptic and excluded the possibility that they could be due to
a monosynaptic activation mediated by the direct stimulation of
inhibitory interneurons via extracellular diffusion of the current
coupled to the electrical stimulus. Several indications suggest that a
feed-forward circuit, and not a feed-back inhibition, mediates the
IPSPs recorded in our experiments. First, the IPSP was evoked in the
absence of action potential firing by principal layer II-III neurons.
Indeed, we showed that these cells generate a spike on synaptic
activation at low stimulus intensity exclusively; when the NC/intrinsic
stimulus intensity was increased, the neurons cease to fire in
coincidence with the appearance of the IPSP. Second, when a single
action potential was activated at low stimulus intensity (see Figs. 7 and 8), no clear IPSP was observed, suggesting that recurrent inhibition generated by neuronal firing in this region is possibly weak. Accordingly, the spikes evoked in an area 36 neuron by remote NC
stimulation was followed by a small-amplitude, if any, hyperpolarizing potential, whereas stimulation of the same cell with an electrode located close by induced a typical large-amplitude and biphasic IPSP
(see Fig. 8). Interestingly, spikes were often generated with a long
delay after the onset of the depolarizing EPSP. This observation may be
related to the demonstration that PRC layer II-III neurons with
variable morphology show typical delayed firing in response to a just
supra-threshold intracellular current injection (Faulkner and
Brown 1999
). Finally, the time-to-onset of the IPSP is faster
than the onset of the late EPSP, which is supposedly due to the
activation of a recurrent excitatory circuit. These data suggest that
feed-forward inhibition mediated through the activation of inhibitory
cells by afferent stimulation controls the excitability of area 36 neurons. Moreover, the results of the experiments with remote and close
by stimulations suggest that, as for the e-EPSP, the feed-forward IPSPs
do not propagate at distance in the rostrocaudal dimension. Therefore
we infer that the axonal arborization of area 36 interneurons that
generate the feed-forward IPSP is spatially restricted in the
rostrocaudal dimension. The presence of a prominent feed-forward
activation turned on by strong incoming inputs could account for the
recent demonstration of a marked paired-pulse depression of the
NC-induced response reported in PRC rat slices in vitro
(Ziakopoulos et al. 1999
), a mechanism that has been
associated with the decrease of PRC neuronal responses to familiar
visual stimuli in a recognition memory task (Cho et al.
2000
).
The superficial current sink demonstrated by CSD analysis in area 36 at
both sites close to and distant from the stimulating electrode
correlated to the e-IPSP and the late EPSP. It is likely that, at least
at sites close to the stimulation electrode, the inhibitory synaptic
events might contribute to the generation of the disynaptic superficial
sink, since in our experiments the e-IPSPs were depolarizing at resting
membrane potential; an outward current, i.e., an extracellular sink, is
expected during the e-IPSP in these conditions. In addition, the
presence of a sizable late superficial sink at sites remote from the
influence of the feed-forward e-IPSP demonstrated that the late
excitatory potential also contributes substantially to the
extracellular current. Indeed, a late EPSP was identified in area 36 neurons following both NC and local stimulation in spatial vicinity and
at a distance from the stimulation site. The disynaptic nature of both
the e-IPSP and the l-EPSP was assessed by performing a pairing test;
indeed, the reactivation of such potentials in the conditioned response
was prevented when a shunting e-IPSP was induced by the first
conditioning stimulus delivered 20-30 ms earlier (Biella et al.
1996
). Since the large majority of the neurons in layer II-III
did not generate an action potential at stimulus intensities that
evoked the e-IPSP, what is the substrate for the generation of the
disynaptic l-EPSP? Following our previous assumption that IPSPs do not
propagate at distance, the demonstration that layer II-III principal
cells discharge an action potential only at low stimulus intensity
whereas disynaptic EPSPs can be activated also at high stimulus
intensity (in the presence of a pronounced IPSP) lead us to speculate
that the disynaptic EPSPs could be due to recurrent excitation mediated by the activation of neurons located at the periphery of the region of
influence of the feed-forward inhibitory circuit, that fire an action potential.
This study was sponsored by Human Frontier Science Program
Organization Grant RG 109/96 and by European Community Grant VSAMUEL (IST 99-1-1-A). The multi-channel silicon probes were kindly provided by the University of Michigan Center for Neural Communication Technology, sponsored by National Center for Research Resources Grant
P41-RR-09754.
Address for reprint requests: M. de Curtis, Dept. of Experimental
Neurophysiology, Istituto Nazionale Neurologico, via Celoria 11, 20133 Milan, Italy (E-mail: decurtis{at}istituto-besta.it).