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The Journal of Neuroscience, May 1, 2003, 23(9):3930
Gating of Hippocampal-Evoked Activity in Prefrontal Cortical
Neurons by Inputs from the Mediodorsal Thalamus and Ventral
Tegmental Area
Stan B.
Floresco and
Anthony A.
Grace
Department of Neuroscience, University of Pittsburgh, Pittsburgh,
Pennsylvania 15206
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ABSTRACT |
Projections from the hippocampus, the mediodorsal thalamus (MD),
and the ventral tegmental area (VTA) form interconnected neural
circuits that converge in the prefrontal cortex (PFC) to participate in
the regulation of executive functions. The present study assessed the
roles that the MD and VTA play in regulating the hippocampal-PFC
pathway using extracellular single-unit recordings in
urethane-anesthetized rats. MD stimulation inhibited PFC neuron firing
(~100 msec duration) evoked by fimbria/fornix (FF) stimulation in a
majority of neurons tested. However, this effect was reduced if
activation of thalamocortical inputs occurred almost simultaneously (10 msec) with stimulation of the FF. In a separate population of neurons,
burst stimulation of the MD produced a short-term (~100 msec)
inhibition or facilitation of FF-evoked firing in 66 and 33% of PFC
neurons, respectively. Moreover, tetanic stimulation of the MD caused a
longer-lasting (~5 min) potentiation of FF-evoked firing. Burst
stimulation of the VTA inhibited FF-evoked firing in a
frequency-dependent manner: firing evoked by higher-frequency trains of
pulses to the FF was less inhibited than firing evoked by single-pulse
stimulation. The inhibitory actions of VTA stimulation were augmented
by D1 receptor antagonism and attenuated by D2 and D4 antagonists. Moreover, stimulation of the MD 10 msec
before stimulation of the FF attenuated the VTA-mediated inhibition of evoked firing. Thus, both the MD and VTA exert a complex gating action
over PFC neural activity, either facilitating or inhibiting firing in
the hippocampal-PFC pathway depending on the frequency and relative
timing of the arrival of afferent input.
Key words:
prefrontal cortex; hippocampus; ventral
tegmental area; mediodorsal thalamus; dopamine; in vivo
electrophysiology; gating; rat
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Introduction |
Studies in both animals and humans
have implicated the prefrontal cortex (PFC) and related subcortical
afferent connections in mediating executive functions such as
prospective coding, set-shifting, and working memory (Kolb, 1984 ;
Hauser, 1999; Robbins, 1996; Goldman-Rakic, 1998; Miller, 2000). A
contemporary theory of PFC function posits that the frontal lobes play
an essential role in integrating different types of information,
subserved by distinct cortical and subcortical networks, to organize
behavior for the attainment of future goals (Miller, 2000; Fuster,
2001). Hippocampal projections to the PFC form one transcortical
network that mediates cognitive processes such as working memory. In
the rat, the ventral CA1/subicular region of the hippocampus sends
glutamatergic projections to both pyramidal neurons and GABAergic
interneurons of the prelimbic region of the PFC (Conde et al., 1995;
Carr and Sesack, 1996; Gabbott et al., 2002). Stimulation of
hippocampal afferents evokes excitatory and inhibitory responses in PFC
neurons (Gigg et al., 1994; Jay et al., 1995, Mulder et al., 1997;
Lewis and O'Donnell, 2000). The importance of hippocampal-PFC
circuits in working memory is underscored by the finding that
disconnection between these regions selectively disrupts retrieval of
information during delayed responding (Floresco et al., 1997; Aujla and
Beninger, 2001).
Two other subcortical nuclei that play a role in cognitive processes
mediated by the hippocampal-PFC circuits include the ventral tegmental
area (VTA) and the mediodorsal nucleus of the thalamus (MD). The VTA
sends a dopaminergic projection to the PFC (Van Eden et al., 1987), and
dopamine (DA) terminals are found often in close proximity to
hippocampal terminals on PFC neurons (Carr and Sesack, 1996).
Intracellular in vivo recordings have shown that stimulation
of the VTA can produce a prolonged depolarization in PFC neurons
accompanied by a reduction in spike firing (Lewis and O'Donnell,
2000). Moreover, VTA activation can exert two differential effects on
hippocampal-evoked activity of PFC neurons: (1) an inhibition of firing
evoked by low-frequency stimulation of the ventral subiculum and (2) an
enhancement of long-term potentiation of the hippocampal-PFC pathway
induced by high-frequency stimulation (Jay et al., 1995; Gurdren et
al., 1999). These data suggest that DA input plays an important
neuromodulatory role over activity in the hippocampal-PFC pathway, a
contention supported by the findings that selective disruption of DA
D1 receptor modulation of hippocampal inputs to
the PFC impairs working memory mediated by hippocampal-PFC circuits
(Seamans et al., 1998; Aujla and Beninger, 2001). The functional role
of D2-like (D2,
D4) receptors in the PFC remains to be
established; however, activation of these receptors tends to inhibit
PFC neural activity, either by direct actions on PFC pyramidal neurons
or by facilitating GABAergic transmission (Sesack and Bunney, 1989;
Rubinstein et al., 2001; Wedzony et al., 2001).
The MD shares a reciprocal glutamatergic projection with the PFC with
thalamocortical inputs synapsing on layer III and V neurons, and
possibly GABAergic interneurons as well (Krettek and Price, 1977;
Groenewegen, 1988; Ray and Price, 1992; Pirot et al., 1994; Kuroda et
al., 1998). Behavioral studies have implicated the MD in a broad range
of PFC-related processes, including prospective coding (Joyce and
Robbins, 1991; Daum and Ackermann, 1994) and strategy selection (Hunt
and Aggleton, 1998). Of particular relevance is the fact that
disconnection between the MD and PFC disrupts working memory processes
dependent on both hippocampal-PFC circuits and mesocortical DA
(Floresco et al., 1999).
The above-mentioned findings suggest that converging corticopetal
inputs originating from the hippocampus, the MD, and the VTA may
interact in a cooperative manner to regulate executive functions
governed by the PFC. Despite this, it is surprising that there is a
paucity of research investigating the mechanisms by which these inputs
interact to influence PFC neural activity. As such, the present study
was undertaken to assess the modulatory actions that inputs from the MD
and the VTA exert over hippocampal-evoked firing of PFC neurons, using
in vivo extracellular single-unit recordings.
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Materials and Methods |
Subjects and surgery
Male Sprague Dawley rats (300-400 gm; Hilltop,
Scottsdale, PA) were anesthetized with urethane (1.5 gm/kg, i.p.) and
mounted in a stereotaxic frame, with the incisor bar set at  3.3 mm.
Body temperature was maintained at 37°C with a temperature-controlled heating pad. In all surgical preparations, the scalp was incised and
holes were drilled in the skull overlying the prelimbic region of the
medial PFC, the fornix/fimbria (FF), the MD, and the VTA. Concentric
bipolar electrical stimulating electrodes (SNE-100; Kopf, Tujunga,
CA) were implanted into the three afferent regions of the PFC.
The stereotaxic coordinates were as follows (flat skull): FF
electrode = anteroposterior (AP) 1.3 mm (bregma), mediolateral
(ML) +1.6 mm, dorsoventral (DV) 4.0 mm (cortex); MD electrode = AP 2.9 mm, ML +0.7 mm, DV 5.3 mm; VTA electrode = AP 5.4 mm,
ML +0.7 mm, DV 7.8 mm. Animal care and surgical procedures were
performed in accordance with the guidelines outlined in the NIH
Guide for the Care and Use of Laboratory Animals and were approved
by the Institutional Animal Care and Use Committee of the University of Pittsburgh.
Extracellular recordings and cell-searching procedures
Extracellular recording microelectrodes were constructed from
2.0 mm outer diameter borosilicate glass capillary tubing
(WPI) using a vertical micropipette puller
(Narishige, Tokyo, Japan). The tips of the electrodes were
broken back against a glass rod to ~1 µm tip diameter and filled
with 2 M NaCl containing 2% Pontamine sky blue dye. The
in vitro impedance of the microelectrodes ranged from 5 to
10 M as measured at 135 Hz using a Winston Electronics BL-1000 impedance meter. After a burr hole was drilled overlying the
PFC, the dura was resected, and the electrode was lowered into the PFC
(coordinates: +3.5-2.7 mm anterior from bregma, 0.6-0.8 mm lateral
from the midline, 2.2-5.0 mm ventral from brain surface) with a
hydraulic microdrive (Kopf Model 640). The electrode signal was
amplified, filtered (400-4000 Hz), and discriminated from noise using
a combination amplification and window discrimination unit for
extracellular recording (Fintronics, Orange, CT) and displayed on an
oscilloscope (Tektronics, Wilsonville, OR). The data were
acquired, stored, and analyzed using custom-designed computer software
(Neuroscope) running on an Intel-based personal computer with a data
acquisition board interface (Microstar Laboratories, Bellevue, WA).
After the glass microelectrodes had been lowered to the dorsal border
of the PFC, a cell-searching procedure began. In this procedure, the
microelectrode was lowered incrementally through the PFC while
alternating stimuli were delivered to the FF and the MD (1500 µA) at
1 sec intervals (i.e., each afferent was stimulated at 0.5 Hz).
Although previous in vitro studies in hippocampus neurons
have shown that prolonged low-frequency stimulation can induce
long-term depression (Mulkey and Malenka, 1992), other studies in
vivo have shown that extended bouts of low-frequency stimulation
(1 Hz, 15 min) of the hippocampus does not produce any reliable change
in synaptic efficacy in the PFC (Burette et al., 1997). Cathodal
constant current pulses (0.2 msec duration) were delivered to the FF
and MD through an Iso-Flex optical isolator (A.M.P.I.,
Jerusalem, Israel) via a Master-8 programmable pulse generator
(A.M.P.I.) using the parameters noted below. Once a cell
was detected, the position of the microelectrode was adjusted to
maximize the spike amplitude relative to background noise. Neurons that
responded only to FF stimulation or MD stimulation or received
converging input from both regions were identified by their robust
excitatory response after stimulation of the respective afferent
region. Only neurons that responded with an orthodromic, monosynaptic
response and displayed a signal-to-noise ratio of at least 3:1 were
used in the data analysis. Evoked firing was characterized as
monosynaptic/orthodromic if the response displayed spike jitter of at
least 2 msec and a shift in spike latency with increasing current
amplitude, followed by paired-pulse stimulation at 50 Hz (otherwise
characterized as polysynaptic), but failed to follow 400 Hz
paired-pulse stimulation (otherwise characterized at antidromic) (Pirot
et al., 1994; Mulder et al., 1997).
With respect to MD-evoked responses, previous studies have demonstrated
that electrical stimulation of the MD can evoke two types of
monosynaptic responses in PFC neurons. Single-pulse stimulation can
evoke a short latency (<10 msec) action potential that likely represents orthodromic activation of thalamocortical axons. However, higher-frequency (10 Hz) stimulation yields longer latency (>13 msec)
spikes that are thought to be caused by antidromic activation of axons
of PFC projection neurons that terminate in the MD but also have
collaterals that synapse onto other PFC neurons (Pirot et al., 1994).
Experiments assessing the conduction velocities of MD neurons
projecting to the PFC have revealed that antidromically evoked firing
of MD neurons occurs 2-11 msec after PFC stimulation (Pirot et al.,
1994). In the present study, we observed that orthodromic monosynaptic
action potentials evoked by single-pulse stimulation of the MD
displayed a latency range of 6-22 msec (mean = 13.4 msec).
However, to maximize the possibility that these responses were driven
by activation of thalamocortical projections and not axon collaterals
of PFC projection neurons, we included only PFC neurons that displayed
MD-evoked orthodromic firing with latencies of  12 msec. Whenever a
PFC neuron was encountered that displayed an antidromic spike after MD
stimulation (but no response to FF stimulation), the location of that
neuron was noted, but no other data were taken.
Stimulation protocols
After establishing that firing evoked by stimulation of the FF
or MD was monosyaptic and orthodromic, stimulation currents were
adjusted to submaximal stimulation intensity (range 100-1800 µA) so
that stimulation of the FF or MD would evoke an action potential
~60% of the time (range 40-75%, depending on the experiment) in
response to single-pulse stimulation delivered at 0.2 Hz. In each of
these experiments, data were compiled using a minimum of 25 sweeps. We
used various stimulation protocols designed to investigate different
types of interactions among hippocampal, MD, and VTA inputs to the PFC.
Interactions between converging inputs from the hippocampal and
MD in the PFC: sequential-pulse protocols. In PFC neurons that
displayed a monosynaptic orthodromic spike after stimulation of both
the FF and MD, we conducted a series of sequential single-pulse stimulation experiments to assess how stimulation of one input could
influence firing evoked by stimulation of the second input. In these
experiments, each neuron received a conditioning pulse of one input
followed by a test pulse of the other input, at intervals ranging
between 10 and 500 msec, in a counterbalanced order, using the same
stimulation intensity for each interstimulus interval (ISI).
Paired-pulse facilitation/depression was also examined for each
individual input, using 25-100 msec intervals between the conditioning
pulse and the test pulse.
Modulation of hippocampal-evoked firing by burst stimulation of
the MD. For some PFC neurons that only fired in response to FF and
not MD stimulation, a series of experiments assessed the effect of
burst stimulation of the MD on firing evoked by FF stimulation. In
these studies, the stimulation intensity of the FF was adjusted so that
the firing probability was ~50% and kept constant throughout the
course of the experiment. The firing probability was maintained at
~50% so that we would be able to observe either inhibition or
facilitation of FF-evoked firing by MD burst stimulation. After establishing baseline firing probabilities, we applied a four-pulse, 20 Hz train to the MD (800-1000 µA) 10, 25, or 50 msec before the FF
pulse, with 25 sweeps collected for each interstimulus interval. This
firing pattern mimics that displayed by MD neurons during learning
(Oyoshi et al., 1996). Any neuron that displayed reliable firing in
response to MD burst stimulation was not included in the data analysis.
After each series of 25 sweeps, FF-evoked firing probability was
monitored at the same stimulation intensity for 3-5 min using single
pulses to the FF delivered at 0.2 Hz. This continued until the
FF-evoked firing probability returned to ~50%.
We also assessed effects of tetanic stimulation of the MD on longer
lasting (i.e., minutes) changes of FF-evoked firing in PFC neurons. In
this experiment, baseline probability of evoked firing in response to
single pulses delivered to the FF were recorded over ~10 min, using
repeated sweeps administered every 2.5 min. Once stable levels of
evoked-firing activity were observed (15% variation in spike
probability over 5-10 min, three to four sweeps), 25 trains (four
pulses, 20 Hz ISI) were delivered to the MD at a frequency of 0.2 Hz.
Thirty seconds after this tetanus (assigned as time 0), the FF was
again stimulated with single pulses at 0.2 Hz using the same
stimulation current as before tetanus, and changes in FF-evoked firing
probability were assessed for another 10 min with repeated sweeps
recorded every 2.5 min. Data were normalized to the average firing
probability observed over 5 min before tetanus and analyzed in terms of
percentage change in firing probability. For both of the
above-mentioned protocols, any neuron displaying reliable firing in
response to MD burst stimulation was not included in the data analysis.
VTA modulation of FF-evoked firing of PFC neurons. For these
experiments, FF stimulation intensities were adjusted to evoke an
action potential ~60-75% of the time after single-pulse stimulation of the FF delivered at 0.2 Hz. Once baseline firing probabilities were
established, the VTA was stimulated in a burst pattern (two four-pulse
20 Hz trains of pulses; interburst interval = 200 msec) 10 or 50 msec before stimulation of the FF. Usually we stimulated the VTA with
an intensity of 600 µA, but in some instances, the intensity was
increased to up to 800 µA to induce a noticeable inhibition of
FF-evoked firing. To assess whether activation of the VTA exerted a
frequency-dependent modulation of evoked firing, we stimulated the FF
first with single pulses and then with five-pulse, 20 Hz trains, each
delivered at 0.2 Hz. The 20 Hz stimulation frequency mimics the way
hippocampal projection neurons fire during working memory tasks
(Hampson et al., 2000). We used a minimum of 25 sweeps per interburst
interval to compile the data for all experiments assessing the effects
of VTA stimulation.
Pharmacological manipulations
For some experiments involving burst stimulation of the VTA,
separate groups of rats were implanted with intravenous jugular catheters, consisting of PE 10 tubing attached to a 30 ga needle and a
1 ml syringe. These experiments used a between-subjects design, and
every animal received only one drug injection. After isolation of each
PFC neuron, we confirmed that burst stimulation of the VTA inhibited
hippocampal-evoked firing induced by single-pulse stimulation of the FF
before drug administration. Baseline levels of FF-evoked firing were
established, after which drugs were administered. We would then wait
10-20 min after drug injection before stimulating the VTA again. All
drugs were purchased from Sigma (St. Louis, MO), with the
exception of CP 293,019, which was donated by Pfizer (Groton, CT). The selective D1 receptor
antagonist SCH23390 (0.2 mg/kg), the selective D2
antagonist eticlopride (0.25 mg/kg), and the selective NMDA receptor
antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid
(CPP) (2.0 mg/kg) were dissolved in physiological saline. The
D2/D4 antagonist
haloperidol (0.5 mg/kg) was dissolved in dilute lactic acid. The
selective D4 antagonist CP 293,019 (10 mg/kg) was
immersed in a drop of NaOH and 40% solution of cyclodextrin and
sonicated until dissolved. The concentrations of these solutions were
set so that injection volumes would range between 0.15 and 0.30 ml, and
no more than one drug injection was given per animal. The doses of
these drugs were chosen from previous studies (Gioanni et al., 1998;
Mansbach et al., 1998; Floresco et al., 2001).
Histology
At the end of each experiment, the recording site in the PFC was
marked via iontophoretic ejection of Pontamine sky blue dye from the
tip of the recording electrode (30 µA constant current for 20-30
min). Iron deposits were made in the fimbria, the MD, and the VTA
stimulation sites by passing DC current (100 µA for 10 sec) through
the stimulating electrode. After dye ejection, brains were
removed and fixed in formalin containing 0.1% potassium ferricyanide
for at least 24 hr. The brains were then immersed in phosphate-buffered
sucrose solution (25%) until saturated. The tissue was sectioned into
40 µM coronal slices, mounted, and stained with cresyl
violet to enable histological determination of recording and
stimulating electrode sites. Representative locations of different
classes of PFC neurons and stimulating electrodes are presented in
Figure 1.
Data analysis
The data were analyzed in terms of evoked firing probability in
response to stimulation of the FF or MD, or percentage change of
baseline-evoked firing probability. Evoked firing probabilities were
calculated by dividing the number of action potentials observed by the
number of stimuli administered (typically 25-50 pulses) × 100. For the sequential-pulse experiments that measured the interactions
between hippocampal and MD inputs, the baseline firing probabilities
evoked by each input were calculated by taking the mean firing
probability evoked when that input was stimulated as the conditioning
pulse. Changes in these probabilities were used as an index of the
influence that MD or VTA inputs exerted over hippocampal-evoked firing
of PFC neurons. The type of statistical analysis depended on the
particular experiment, but typically entailed two- or three-way
repeated measures ANOVA, with the exception of the pharmacological
studies, which also included drug as a between-subjects factor.
Multiple comparisons were made using two-tailed Dunnett's test for
repeated measures.
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Results |
General properties of PFC Hipp neurons: convergence of inputs and
projections to the MD and VTA
The data from a total of 114 PFC neurons that displayed a
monosynaptic orthodromic spike after stimulation of the FF are
presented here (hereafter referred to as PFCHipp neurons). These
cells displayed a mean basal spontaneous firing rate of 3.1 ± 0.5 Hz (range 0.0-17.2 Hz; median firing rate 2.9 Hz) and had a mean evoked firing latency of 11 ± 0.8 msec (range 7-17 msec),
consistent with previous findings (Mulder et al., 1997). Most of these
114 neurons that fired in response to FF stimulation were also tested for an antidromic response to MD or VTA stimulation. Ten of these cells
(10 of 109; 9.2%) were confirmed PFC MD projection neurons (mean
antidromic spike latency = 11.2 ± 0.9 msec; range 7-18
msec), whereas 24.6% of these neurons (17 of 69) responded with an
antidromic spike after VTA stimulation (mean antidromic spike
latency = 9.6 ± 1.0 msec; range 4-19 msec). In addition, 4 of 60 neurons (6.7%) responded with an antidromic spike after
stimulation of both the VTA and MD, suggesting that a
subpopulation of PFC neurons that receive hippocampal input send axon
collaterals to both regions (Fig. 1E). It
is notable that many of the PFCHipp neurons were located in the same
vertical electrode track where other PFC neurons that were
antidromically activated by MD stimulation were observed. This suggests
that most of the PFCHipp neurons that were recorded in the present
study were located in the projection layers of the PFC. Last, and most
pertinent to the present study, 13 of 105 PFCHipp neurons (12.4%)
also displayed converging orthodromic input from the MD, exhibiting a
monosynaptic action potential after MD stimulation that was within the
latency range of our inclusion criterion (mean spike latency = 7.6 ± 0.6 msec). Representative locations of these neurons
are shown in Figure 1. PFCHipp neurons that projected to the MD were
clustered in the more rostral regions of the PFC and found in both
prelimbic and infralimbic areas, whereas PFCHipp neurons that
projected to the VTA clustered more rostrally and were located in the
dorsal prelimbic region.
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Figure 1.
Histology. A, Schematic of
coronal sections of the rat brain showing representative placements of
recording electrodes where different PFC neurons were observed.
PFCHipp neurons, black square; neurons that responded with an
orthodromic action potential after MD stimulation, gray circles;
PFCHipp cells that were confirmed to project to the MD, open
circles; PFCHipp cells that were confirmed to project to the VTA;
PFCHipp cells that were antidromically activated by both the VTA and
the MD, stars; PFCMD projection neurons that did not respond to FF
stimulation, triangles. Numbers correspond to millimeters from bregma.
B-D, Photographs of a representative
placement of a stimulating electrode in the FF
(B), the MD (C),
and the VTA (D). Arrows highlight the
location of stimulating electrode placements. cc, Corpus callosum;
hipp, hippocampus. E, Individual data. Ten overlaid
traces recorded from a PFC neuron that displayed an antidromic (AD)
spike after MD stimulation and an orthodromic (ortho) spike after FF
stimulation (E1). This same neuron could also be
activated antidromically by stimulation of the VTA
(E2) (5 traces).
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Interactions between converging hippocampal and MD inputs to
the PFC
In six PFC neurons that displayed a monosynaptic spike in response
to stimulation of both the FF and the MD (six rats), we administered a
sequential-pulse stimulation protocol to assess how stimulation of one
input could influence firing driven by the other input. In these cells,
the basal firing probabilities evoked by either FF or MD single-pulse
stimulation did not differ (FF = 55.7 ± 4/7%; MD = 53.6 ± 7.3%; F(1,5) = 0.04;
NS). Application of a conditioning pulse to the FF 10-500 msec before
a test pulse to the MD resulted in a profound and significant
(p < 0.05) depression of MD evoked firing
probability (Fig. 2A,
black squares) compared with single-pulse stimulation of the MD alone
(Fig. 2A, gray square) (F(6,30) = 3.5, p < 0.01; and Dunnett's, p < 0.05).
The suppression of MD-evoked firing probability was maximal when the
conditioning pulse to the FF was administered 10 msec before MD
stimulation (94 ± 3%), whereas longer intervals (25-500 msec)
produced suppression of firing to between 45 and 75% of the baseline
MD-evoked firing probability. In these same neurons, a similar profile
was observed when a conditioning pulse was applied to the MD before a
test pulse to the FF. In this instance, FF-evoked firing probability was significantly reduced by ~60% when a conditioning pulse was administered 25-100 msec before FF stimulation (Fig.
2B, black circles) when compared with single pulse of
the FF alone (Fig. 2B, gray circle). However,
application of a conditioning pulse to the MD either 10 or 250-500
msec before FF stimulation did not significantly effect the FF evoked
firing probability (Fig. 2B, white circles)
(F(6,30) = 5.6; p < 0.01). The duration of the inhibition of MD-evoked spiking by the FF
was substantially longer (~500 msec) than the reduction in FF-evoked
firing mediated by the MD (~100 msec). This difference may have been
caused by differences in the duration of feed-forward inhibition evoked by each afferent. To investigate this difference further, we analyzed the duration of inhibition of spontaneous activity evoked by
single-pulse stimulation of each afferent. In accordance with our
findings using sequential-pulse protocols, single-pulse stimulation of the MD evoked an initial monosynaptic spike, which was followed by a
period of inhibition of spontaneous firing that lasted 160 ± 21 msec. In contrast, inhibition of spontaneous firing evoked by FF
stimulation had a substantially longer duration (403 ± 27 msec).
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Figure 2.
Examples of sequential-pulse interactions of the
PFC and MD inputs to the PFC. A, In neurons that fired
in response to stimulation of both the FF and MD, a conditioning pulse
applied to the FF inhibited firing evoked by a test pulse to the MD
(black squares; mean + SEM) compared with a single MD pulse alone with
the same intensity (gray square, hatched line). B, In
these same neurons, a conditioning pulse applied to the MD
significantly inhibited firing evoked by an FF test pulse compared with
firing evoked by single pulses to the FF (gray circles). Here, the
inhibition was significant (p < 0.05) only
at intervals of 25-100 msec (black circles) but not at intervals of 10 or 250-500 msec (white circles) when compared with the firing
probability observed using single-pulse stimulation (gray circle).
Bottom panels diagram the stimulation protocols used for
A and B, respectively.
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Subsequent analyses of these data were conducted to assess whether the
neuron fired an action potential to the conditioning pulse to either MD
or FF stimulation had any effect on the firing probability observed in
response to the test pulse to the FF or the MD. Analysis of
these data revealed that FF stimulation caused the same level of
depression of MD-evoked spike firing independent of whether the FF
conditioning pulse evoked a spike
(F(1,5) = 0.2; NS). The reverse was
also true: MD stimulation caused a similar depression in the response
to FF stimulation independent of whether the MD condition pulse evoked
an action potential (F(1,5) = 2.7; NS). Thus, activity in the hippocampal-PFC pathway 10-500 msec before
impulse activity in the MD-PFC pathway can profoundly reduce the
probability of firing induced by thalamocortical inputs, an effect that
is not related to FF-evoked firing. The MD-PFC pathway exerts a
similar gating action over hippocampal-evoked firing, although this
inhibitory action occurs at a more temporally discrete range (25-100
msec). Importantly, the inhibitory influence that this
thalamocortical pathway exerts over hippocampal-evoked firing of PFC
neurons can be offset if impulse activity from the hippocampus arrives
almost simultaneously with inputs from the MD (i.e., within ~10 msec).
We also assessed paired-pulse facilitation/depression in the
hippocampal-PFC or MD-PFC pathway individually, using 25, 50, and 100 msec interstimulus intervals between conditioning and test pulses
(Table 1). For PFCHipp neurons
(n = 10; six rats), delivering paired pulses to the FF
at intervals of 50 or 100 msec, but not at 25 msec, resulted in
significant paired-pulse facilitation (p < 0.01). In contrast, for neurons PFCMD (n = 9; five
rats), there was a significant (p < 0.05)
paired-pulse depression of the test pulse at a 100 msec interval, but
not at the 25 or 50 msec intervals (Fig. 2B)
(F(2,34) = 6.9; p < 0.01).
Burst activation of the MD produces differential short-term gating
of hippocampal-evoked firing in subpopulations of PFC neurons that are
not activated by MD stimulation
In light of the dense thalamocortical projection to the PFC, we
were surprised to observe that a relatively small proportion (<15%)
of PFCHipp neurons fired a monosynaptic action potential after
stimulation of the MD. However, this may be an underestimation of the
total number of PFCHipp neurons that may be modulated by
activity in the MD-PFC pathway. For example, it is possible that a
greater proportion of PFCHipp neurons also received direct or
indirect excitatory input from the MD, but the strength of these inputs
was only sufficient to evoke a subthreshold EPSP unobservable using
extracellular recordings. Alternatively, a proportion of MD axons may
make connections with GABAergic interneurons in the PFC, which in turn
synapse on PFCHipp neurons. With this in mind, we assessed how
activation of the MD would affect hippocampal-evoked firing in PFC
neurons that only displayed an extracellular spike after FF stimulation
but not after MD stimulation.
Eighteen PFCHipp neurons that showed no reliable excitatory or
inhibitory response to single-pulse MD stimulation were tested in this
manner. In these cells, single-pulse stimulation of the MD 10-50 msec
before a single pulse to the FF had no discernable effect over
FF-evoked firing (Fig. 3C1).
We then applied repeated four-pulse, 20 Hz train to the MD 10-50 msec
before single-pulse stimulation of the FF. In all PFC neurons that were
tested in this manner (n = 18; eight rats), burst
stimulation of the MD had some effect over FF-evoked firing, revealing
two distinct populations of PFC neurons. In 12 of these cells (67%),
burst stimulation of the MD caused a pronounced reduction in the firing probability evoked by FF stimulation at all intervals tested, when
compared with the firing probability observed after single-pulse stimulation of the FF alone (F(2,22) = 3.7, p < 0.05; and Dunnett's, p < 0.01) (Fig. 3A,C2). Four of these
cells were tested further using intervals of 100 and 250 msec between
the MD train and the single pulse to the FF. This protocol revealed
that the inhibitory effects of MD stimulation were still apparent at
intervals of 250 msec after an MD burst (data not shown). In contrast
to the above-mention effects, in another six PFCHipp neurons (33%), burst stimulation of the MD had the opposite effect: an increased FF-evoked firing probability (F(1,5) = 6.92; p < 0.05) (Fig. 3B). The location of
these two types of PFC neurons is shown in Figure 3D. We
observed no consistent pattern of localization of these two types of
neurons within the PFC; both types were found in the dorsal and ventral
regions of the deep layers of the prelimbic and infralimbic cortex.
Thus, these data imply that natural-type bursting activity in the
thalamocortical pathway can exert differential gating actions on PFC
neural activity driven by hippocampal inputs. Most PFC neurons are
inhibited by bursting activity in the thalamocortical pathway, whereas
another group of neurons displays a facilitation of hippocampal-evoked
firing after MD burst stimulation.
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Figure 3.
Burst stimulation of the MD modulates
hippocampal-evoked firing in PFC neurons. A, In 12 of 18 PFCHipp neurons that did not fire in response to MD stimulation,
burst stimulation of the MD inhibited FF-evoked firing (black bars;
mean + SEM), relative to the firing probability observed after
single-pulse stimulation of the FF alone at the same stimulation
intensity (gray bars; mean + SEM). B, In another 6 of 18 PFCHipp neurons, burst stimulation of the MD facilitated FF-evoked
firing. C, Representative data from individual neurons.
C1, Ten overlaid traces showing the effect of
single-pulse stimulation of the MD (100 µA) 25 msec before
stimulation of the FF (680 µA). C2, In the same
neuron, 10 traces showing the effect of burst stimulation of the MD
(last 2 pulses shown) before FF stimulation. Over 10 sweeps,
single-pulse stimulation of the MD had no effect over FF-evoked firing,
but burst stimulation substantially reduced the probability of firing
over the same number of trails. D, Location of
PFCHipp neurons the evoked firing of which was inhibited (open
squares and minus sign) and facilitated (black squares) by burst
stimulation of the MD. E, Diagrams of the stimulation
protocol used in this experiment. Here, the MD was stimulated with a
four-pulse, 20 Hz train (1) 10-50 msec before single-pulse stimulation
of the FF (2). MD stimulation did not evoke firing in any of these
neurons. Significant difference versus single-pulse stimulation of the
FF: **p < 0.01.
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Repeated burst stimulation of the MD produces a longer
lasting potentiation of hippocampal-evoked firing in PFC neurons
Over the course of the above-mentioned series of experiments, we
noticed an increase in the FF-evoked firing probability after repetitive burst stimulation of the MD (i.e., 25 bursts delivered at
0.2 Hz frequency), regardless of whether MD activation inhibited or
facilitated FF-evoked firing, an effect that lasted a number of
minutes. Therefore, we conducted another experiment to formally assess
this heterosynaptic potentiation of FF-evoked firing by burst
stimulation of the MD. Repeated burst stimulation of the MD resulted in
a robust potentiation of FF-evoked firing probability in all neurons
tested (n = 8; four rats) (Fig.
4A,B1,B2).
This potentiation of hippocampal evoked firing probability reached a
peak at 1 min after tetanus (+46.7 ± 14%) (Fig.
3B1,B2) and remained significantly elevated for
another 6-8 min before returning to baseline levels of evoked firing
probability (F(7,91) = 4.7, p < 0.01; and Dunnett's, p < 0.05, 0.01). To confirm that the potentiation of hippocampal-evoked firing
was not merely caused by repetitive single-pulse stimulation of the FF,
a separate group of neurons (n = 6; three rats) were
tested under conditions in which the MD was not stimulated. These
neurons displayed no significant change in the probability of firing
evoked by repetitive single-pulse FF stimulations over a 15 min period
(F(7,91) = 0.32, NS) (Fig. 4A, gray circles). Thus, repetitive bursting activity
in this thalamocortical pathway can produce a robust, short-term
potentiation of hippocampal-evoked firing of PFC neurons, thereby
priming PFC neurons to be more responsive to information conveyed by
the hippocampus.
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Figure 4.
Repeated burst stimulation of the MD potentiates
FF-evoked firing in PFC neurons. A, In neurons that did
not fire in response to MD stimulation, tetanic burst stimulation of
the MD (25 4-pulse 20 Hz trains; interburst interval of 5 sec; arrow)
potentiated FF-evoked firing for ~10 min (black squares). In
contrast, repeated single-pulse stimulation of the FF alone had no
effect (gray circles). Symbols represent mean percentage change (+SEM)
in baseline FF-evoked firing. B, Peristimulus time
histograms showing a typical response from a single PFC neuron at
baseline (B1) 2 min before and 1 min after MD tetanus
(B2) (25 sweeps each). In this cell, MD tetanus
substantially potentiated FF-evoked firing probability. Arrows
represent time points at which single pulses were administered to the
FF (450 µA stimulation intensity for both). * p < 0.05 and ** p < 0.01, respectively, versus
baseline.
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Interactions between hippocampal and VTA inputs to
the PFC
Single-pulse stimulation of the FF
In a separate group of PFCHipp neurons, we assessed the effect
of burst stimulation of the VTA on FF-evoked firing of PFC neurons. All
of the neurons tested (n = 54; 51 rats) fired in response to FF stimulation but showed no monosynaptic response after MD
stimulation. These data are presented in Figure
5. In accordance with previous findings
(Jay et al., 1995; Gurden et al., 1999), burst stimulation of the VTA
produced a drastic inhibition of FF-evoked firing
(F(1,22) = 80.0, p < 0.001; and Dunnett's, p < 0.01) (Fig. 5). This
inhibitory effect of VTA stimulation was apparent when the interval
between the last pulse in the VTA burst and the single stimulation of
the FF was either 10 or 50 msec
(F(1,22) = 8.7; p < 0.01). Others have shown that single-pulse stimulation of the VTA
inhibits hippocampal-evoked activity only at intervals of <25 msec
(Jay et al., 1995; Gurden et al., 1999). Thus, burst stimulation of the
VTA, which is known to be more effective at releasing mesolimbic DA
than single pulses (Garris and Wightman, 1994), inhibits firing of PFC
neurons evoked by single-pulse stimulation of the hippocampus, and this
effect lasts at least 50 msec after the end of the VTA burst.
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Figure 5.
Burst stimulation of the VTA inhibits FF-evoked
firing in PFC neurons. Mean firing probability (+SEM) evoked by
single-pulse stimulation of the FF alone (gray bars) or after burst
stimulation of the VTA (black bars) either 10 or 50 msec before FF
stimulation. Bottom panel diagrams stimulation protocol used in this
experiment. Here, the VTA was stimulated in a burst pattern (1) (2 4-pulse 20 Hz trains; interburst interval of 200 msec) 10 or 50 msec
before single-pulse stimulation of the FF (2).
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Train stimulation (20 Hz) of the FF
A number of recent studies have shown that the actions of DA on
evoked activity in PFC neurons are dependent on the frequency at which
excitatory inputs are stimulated (Jay et al., 1995; Otani et al., 1998;
Gurden et al., 1999; Seamans et al., 2001). In light of these data, we
assessed the effect of burst stimulation of the VTA on
hippocampal-evoked firing of PFC neurons when the FF was stimulated
with a higher-frequency train of pulses. FF stimulation consisted of a
five-pulse, 20 Hz train. Stimulation of the FF in this manner resulted
in an equivalent probability of firing (75-90%) in PFC neurons
throughout each pulse in the train (Figs. 6A, gray circles, 7 A1). However, activation of the VTA before 20 Hz train
stimulation of the FF altered this firing
profile in an interesting manner. Burst stimulation of the VTA produced a profound reduction in the firing probability observed after the first
pulse in the five-pulse train delivered to the FF, when compared with
the firing probability observed at the same time point when no VTA
stimulation was given (75.8 ± 4 vs 7.4 ± 3%) (Fig.
6A, gray square). However, the firing probability
observed in the latter parts of the train was significantly less
attenuated compared with the firing probability evoked by the first
pulse (Figs. 6A, black squares, 7A2)
(mean = 52.3 ± 7%; F(4,36) = 7.1; p < 0.01). This effect emerged by the second
pulse in the train, and the firing probability evoked by pulses 2-5
did not differ throughout the course of the train. It is important to
note that burst stimulation of the VTA still caused a significant
reduction (p < 0.01) in the firing probability
evoked by later pulses in the train compared with the firing
probability at the same time points of the train when no VTA
stimulation was administered, but the magnitude of this inhibition was
substantially reduced compared with that observed during the first
pulse (first pulse firing probability = 7.4 ± 3%; pulses
2-5 = 52.3 ± 7%; p < 0.01). From these data it is apparent that the inhibitory actions that the VTA
exerts over hippocampal-evoked firing in PFC neurons are frequency
dependent. Burst stimulation of the VTA can drastically inhibit firing
evoked by single-pulse stimulation of the hippocampus. However, if
impulse traffic in the hippocampal-PFC pathway occurs in the form of a
higher-frequency train of action potentials, the VTA-mediated
inhibition is reduced substantially in the latter portions of
the train.
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Figure 6.
The effects of D1,
D2, D4, and NMDA receptor
blockaded on VTA-induced inhibition of FF-evoked firing in PFC neurons.
For all panels, symbols represent mean (+SEM) firing probability evoked
by each pulse in a five-pulse train delivered to the FF. Gray circles
represent FF-evoked firing probability for each pulse in the five-pulse
train, when no VTA stimulation was given. Squares represent FF-evoked
firing probability after a VTA burst. Black squares denote significant
within-group difference (p < 0.05), and
white squares denote no significant within-group difference in firing
probability observed for latter pulses in the train compared with the
firing probability observed during the first pulse (gray square). For
comparative purposes, the gray line in
B-F represents firing probability of the
control condition (VTA + FF). *p < 0.05, **p < 0.01, significant difference versus
probabilities within groups, observed at the same time point in the
train with and without VTA stimulation;
 p < 0.05, significant difference in
firing probabilities between groups versus those observed at the same
time point in the control condition (gray line). A,
Burst stimulation of the VTA dramatically reduced firing evoked by the
first pulse in the train but was less effective for pulses 2-5.
B, Administration of the D1 antagonist
SCH23390 augmented the VTA-induced inhibition. C, The
D2 antagonist eticlopride abolished the inhibition of
firing observed in the latter parts of the train, whereas haloperidol
(D) almost completely attenuated the inhibition.
The D4 antagonist CP 293,019 (E) also
attenuated the inhibitory effect of VTA stimulation. The NMDA receptor
antagonist CPP (F) was without effect.
G, Diagrams of the stimulation protocol used in this
experiment. Here, the VTA was stimulated in a burst pattern (1) before
five-pulse, 20 Hz train stimulation of the FF (2).
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Figure 7.
Representative data from individual neurons
recorded from experiments using 20 Hz train stimulation of the FF. Each
panel represents 10 overlaid traces, and gray arrows denote time points
where the FF was stimulated. A, Example of a neuron in
the control condition (stimulation intensities: FF = 1000 µA,
VTA = 750 µA). Train stimulation of the FF by itself reliably
evoked firing over all five pulses of the train (A1).
Burst stimulation of the VTA 10 msec before train stimulation of the FF
drastically reduced firing evoked by the first pulse in the train,
whereas firing evoked by the latter pulses was relatively unaffected
(A2). For clarity, only the last pulse of the VTA burst
(VTA stim) is shown. B, Example of a neuron pretreated
with the D1 receptor antagonist SCH23390 (stimulation
intensities: FF = 680 µA, VTA = 600 µA). As was observed
in control neurons, train stimulation of the FF by itself reliably
evoked firing over all five pulses of the train (B1).
However, in the presence of SCH23390, burst stimulation of the VTA
completely abolished firing evoked by pulses 1-2 and substantially
reduced firing evoked by latter pulses of the train
(B2).
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Selective DA receptor antagonists alter the effects of VTA
stimulation on FF-evoked firing
To ascertain the role that DA receptors play in the effects of VTA
stimulation, we administered selective DA receptor antagonists before
burst stimulation of the VTA. Analysis of these data revealed significant drug × pulse number × VTA stimulation
interaction (F(20,192) = 1.9;
p < 0.05). Simple main effects analysis revealed that
when no VTA stimulation was administered, there were no significant differences between drug treatment groups with respect to the firing
probabilities evoked by train stimulation of the FF
(F(5,48) = 0.1; NS). This finding
indicates that any differences between the control condition and the
drug treatment group could not be attributed to group differences in
the firing probabilities evoked by train stimulation alone.
Furthermore, the fact that blockade of D1,
D2, or D4 receptors did not
alter firing probabilities evoked by 20 Hz train stimulation implies
that any increase in mesocortical DA release that may be caused by
stimulation of hippocampal afferents (Gurden et al., 2000) does not
influence spike firing evoked during train stimulation.
D1 receptor blockade
Administration of the D1 antagonist SCH
23390 (0.2 mg/kg, i.v.; n = 9) markedly enhanced the
inhibitory actions of VTA stimulation on firing evoked by train
stimulation of the FF (Figs. 6B, 7B). Burst stimulation of the VTA caused a pronounced inhibition of firing
evoked by the second and third pulse of the five-pulse FF train. In
fact, the firing probabilities at these time points did not differ from
the firing probability evoked by first pulse (Fig.
6B, white squares). Moreover, the firing probability
at the middle portion of the train was significantly inhibited compared with the probability observed at the same time points for control neurons. The firing probability evoked by the last two pulses in the
train was significantly reduced (p < 0.01) when
compared with the firing probability at the same time points of the
train when no VTA stimulation was administered but was significantly higher (p < 0.01) than the firing probability
evoked by the first pulse of the train. Thus, D1
receptor blockade revealed an underlying potent VTA-mediated inhibition
during the latter parts of the train.
D2 receptor blockade
In contrast to the effects of D1 receptor
blockade, intravenous administration of the D2
receptor antagonist eticlopride (0.25 mg/kg; n = 10)
abolished the inhibitory actions of VTA stimulation on firing evoked by
the latter parts (pulses 3-5) of train stimulation of the FF but had
no significant effect on the inhibition observed during the early parts
of the train (Fig. 6C). This attenuation of the VTA-induced
inhibition was even more pronounced after pretreatment with the
D2/D4 antagonist
haloperidol (0.5 mg/kg; n = 8). In these cases, VTA
stimulation had no effect over the firing probabilities evoked by
pulses 2-5, when compared with the probabilities observed when no VTA
stimulation was given (Fig. 6D). Moreover, in the presence of haloperidol, VTA stimulation was less effective at inhibiting firing induced by the first pulse in the train, when compared with the control condition (p < 0.05).
D4 receptor blockade
Haloperidol has a higher affinity for DA D4
receptors than eticlopride (Durcan et al., 1995; Seeman and Van Tol,
1995; Seeman et al., 1997) and was more effective than eticlopride in
attenuating the inhibitory actions of VTA stimulation on FF-evoked
activity. In addition, application of D4
antagonists can increase the excitability of PFC neurons (Rubinstein et
al., 2001). In keeping with these observations, administration of the
selective DA D4 receptor antagonist CP 293,019 (10 mg/kg; n = 9) caused an effect like that of
haloperidol but unlike that of eticlopride. D4
receptor blockade attenuated significantly (p < 0.05) the VTA-induced inhibition of the firing probability evoked by
the first pulse of the five-pulse FF train, when compared with neurons
in the control condition (Fig. 6E). The effect of CP
293,019 on the firing probability evoked in the latter parts of the
train were mixed; D4 receptor blockade appeared to reduce the VTA-induced inhibition of firing evoked by pulses 2-4,
but the firing probability at these time points was still significantly
lower when compared with the probabilities observed when no VTA
stimulation was given. VTA stimulation did not affect the firing
probability evoked by the fifth pulse in the train when compared with
the same time point when no VTA stimulation was administered.
NMDA receptor blockade
Previous studies in vitro have shown that the
facilitatory effects of D1 receptor activity on
synaptic transmission in the PFC may be co-mediated by the NMDA
receptor (Seamans et al., 2001). Therefore, we conducted an experiment
to assess whether the frequency-dependent effects observed in the
present study were mediated by NMDA receptors. Surprisingly, in eight
cells tested, administration of the NMDA receptor antagonist CPP (2 mg/kg) caused no change in the firing probability evoked by train
stimulation of the hippocampus after VTA stimulation (Fig.
6F). As observed in the control condition, burst
stimulation of the VTA drastically reduced the firing probability evoked by the first pulse in the train, whereas firing probability evoked by latter pulses in the train was significantly higher when
compared with the first pulse. The discrepancy between the present
in vivo study and the effects reported by Seamans et al. (2001) in vitro may be attributed to a number of procedural
differences, such as blockade of Na+
channels in the study by Seamans and colleagues, the duration of the
train used (5 vs 15 pulses), the dependent variable (spike firing vs
subthreshold EPSPs), and method of DA receptor activation (VTA
stimulation vs bath application of a selective D1
receptor agonist).
The protocol used in the pharmacological experiments used
between-subjects comparisons between animals that received injections of DA antagonists and those that received no drug treatment. However, as noted above, the effects of VTA stimulation on firing evoked by
single-pulse stimulation of the FF was assessed for each individual cell, before any drug administration. Thus, we were able to conduct a
within-subjects analysis of the effects of DA receptor blockade on the
VTA-induced inhibition of firing evoked by single-pulse stimulation of
the FF. Analysis of these data revealed that before drug
administration, burst stimulation of the VTA drastically inhibited
FF-evoked firing of all neurons in all groups [>80% reduction in
firing probability (Fig. 8, gray bars) when compared with single-pulse
stimulation alone (Fig. 8, white
bar)]. Similar to the effects
observed with train stimulation of the FF, the VTA-induced inhibition
of firing evoked by single-pulse stimulation of the FF was not affected
by SCH 23390 and was significantly (but not completely) attenuated by
eticlopride, haloperidol, and CP 293,019 (F(4,32) = 3.0, p < 0.05; and Dunnett's, p < 0.05, p < 0.01) (Fig. 8, black bars). Thus, the finding that blockers for
D2 and D4 receptors reduced
the effects of burst stimulation of the VTA suggests that the
inhibitory actions of VTA stimulation on evoked firing of PFCHipp
neurons is mediated at least in part by the mesocortical DA
projection.
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Figure 8.
D2 and D4 but not
D1 receptor blockade attenuates VTA-mediated inhibition of
firing evoked by single-pulse stimulation of the FF. Bars represent the
percentage change in firing probability in response to single pulses to
the FF after burst stimulation of the VTA before drug administration
(gray bar) and 10-20 min after drug administration (black bars). These
values are expressed as a percentage change relative to the
evoked-firing probability observed when the FF was stimulated alone
(open bar; for comparative purposes). Thus, a score of 0% indicates
that no spike firing is elicited by FF stimulation after VTA
stimulation, whereas FF stimulation alone is always 100%. Only the
D2 antagonist eticlopride, the
D2/D4 antagonist haloperidol, and the
D4 antagonist CP 293,019 were effective in attenuating the
VTA-induced inhibition, whereas the D1 antagonist SCH23390
and the NMDA antagonist CPP were without effect. Significance at
*p < 0.05, **p < 0.01 versus pre-drug
condition.
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To summarize, activation of the VTA exerts a frequency-dependent
inhibition over firing driven by hippocampal inputs to the PFC. VTA
stimulation potently inhibits hippocampal-evoked firing evoked by
single-pulse stimulation. However, when this pathway is activated at a
higher frequency, the inhibition of hippocampal-evoked firing is
attenuated, and inputs from the temporal lobe are much more likely to
evoke spike firing. The inhibitory actions of the VTA were reduced
after blockade of DA D2 and
D4 receptors. Moreover, the DA-mediated
inhibition induced by stimulation of the VTA appears to have both an
early and a late component. Inhibition that occurs <100 msec after the
end of a VTA burst is reduced by D4 receptor blockade (CP 293,019) (Fig. 6E), whereas a later
onset inhibition (100-250 msec after a VTA burst) appears to be
mediated more prominently by D2 receptors
(eticlopride) (Fig. 6C). Accordingly, treatment with an
antagonist that has affinity for both D2 and
D4 receptors (haloperidol) (Fig.
6D) almost completely abolished the inhibitory actions of VTA stimulation. The finding that antagonists for both D2 and D4 receptors did
alleviate much of the inhibitory actions of VTA stimulation implies
that these effects are mediated primarily by DA and not the
mesocortical GABA projection (Carr and Sesack, 2000). Last, blockade of
D1 receptors disrupted the frequency-dependent modulation of impulse activity in this pathway, augmenting the VTA-mediated inhibition of FF-evoked firing.
Interactions among hippocampal, MD, and VTA inputs to the PFC
The above-mentioned findings indicate that bursting activity of
the VTA can exert a frequency-dependent gating action over PFC neuron
firing driven by inputs from the hippocampus. In light of these data,
we were interested in assessing what role the VTA may play in
modulating the interactions between MD and hippocampal inputs to the
PFC. In nine PFCHipp neurons (nine rats) that also displayed a
monosynaptic action potential in response to MD stimulation, we
assessed how burst stimulation of the VTA modulated MD gating exerted
over hippocampal-evoked firing, using our sequential-pulse stimulation
protocol. For these experiments, we only used interpulse intervals of
10-50 msec, because we did not observe any significant effect of MD
conditioning pulses applied >100 msec before FF test pulses, and the
magnitude of inhibition of firing by MD conditioning pulses was
equivalent at intervals of 25-100 msec.
Analysis of these data revealed a significant MD pulse × VTA
stimulation interaction (F(2,16) = 5.8; p < 0.05) (Fig.
9A). In these cells, the
baseline firing probability evoked by MD stimulation and FF stimulation
did not differ (FF mean = 59.5 ± 4%; MD mean = 63.9 ± 6%; F(1,8) = 0.7; NS).
As observed previously (Fig. 2), delivery of a conditioning pulse to
the MD 10 or 50 msec before stimulation of the FF significantly
inhibited FF-evoked firing, when compared with firing evoked by
single-pulse stimulation of the FF alone. The inhibition of FF-evoked
firing by a conditioning pulse to the MD was significantly greater
(p < 0.05) when the interval between pulses was
50 versus 10 msec (Fig. 9A). Burst stimulation of the VTA 10 msec before single-pulse stimulation of the FF also inhibited FF-evoked
spike firing (p < 0.05). However, application
of a conditioning pulse to the MD 10 msec after VTA stimulation, and 10 msec before FF stimulation (i.e., a VTA burst-MD pulse-FF pulse
sequence), attenuated the VTA-mediated inhibition of FF-evoked firing.
The hippocampal-evoked firing probability was significantly higher
(p < 0.05) when compared with firing probability observed after VTA burst-FF stimulation alone. Moreover, at the 10 msec interval between MD and FF stimulation, there was no
difference in the firing probabilities observed after a VTA-MD-FF sequence when compared with an MD-FF paired-pulse sequence. This effect was not observed when a longer, 50 msec interval between the MD
conditioning pulse and FF test pulse was used. In this instance, a
conditioning pulse to the MD 10 msec after VTA stimulation and 50 msec
before FF stimulation caused no significant change in the FF-evoked
firing probability, when compared with the reduced firing probability
displayed after VTA stimulation alone. Thus, as observed previously,
activation of either VTA or MD inputs to the PFC can inhibit evoked
firing in PFCHipp neurons. However, during periods of VTA bursting
activity, activation of PFC neurons by the MD that occurs nearly
simultaneously with activation by hippocampal inputs converging on the
same neuron can reduce the inhibitory actions that the VTA exerts over
spike firing driven by the hippocampus.
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Figure 9.
Interactions among hippocampal, MD, and VTA inputs
converging on the same PFC neurons. A, FF-evoked firing
probabilities in PFCHipp neurons that also responded with an
orthodromic monosynaptic action potential after stimulation of the MD.
Application of a conditioning pulse to the MD 10 or 50 msec before a
test pulse to the MD (gray bars) significantly reduced the evoked
firing probability relative to that observed when the FF was stimulated
alone (gray hatched bar). Burst stimulation of the VTA also attenuated
FF-evoked firing (black bars). However, application of a conditioning
pulse to the MD 10 msec before an FF test pulse significantly
(*p < 0.05) attenuated the VTA-mediated
inhibition. Stars denote significant difference versus firing
probability evoked by FF single-pulse stimulation.
B, Location of all neurons observed in this study (open
stars) that responded with an orthodromic, monosynaptic action
potential after stimulation of the FF and the MD. Numbers beside each
plate correspond to millimeters from bregma. C, Diagram
of the stimulation protocol used in this experiment. Here the VTA was
stimulated in a burst pattern (1) 10 msec before administering MD-FF
sequential-pulse stimulation (2, 3). Both the MD and FF were stimulated
with single pulses that were separated by 10 or 50 msec.
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Discussion |
Using different stimulation protocols, we report four primary
observations: (1) in the majority of PFCHipp neurons tested, inputs
from the MD exert a powerful inhibitory gating action over hippocampal-evoked firing; (2) repetitive burst stimulation of the MD
potentiates hippocampal-evoked activity that lasts several minutes; (3)
stimulation of the VTA inhibits hippocampal-evoked firing with a
preferential attenuation of low-frequency inputs, an effect that was
altered after selective blockade of DA receptors. D1 receptors facilitate higher-frequency
transmission in the hippocampal-PFC pathway, whereas
D2 and D4 receptors inhibit
firing, and (4) the relative timing of inputs from the MD or VTA
determines their impact on PFC neuron responses to hippocampal inputs.
Activation of the MD ~10 msec before activation of hippocampal inputs
was less likely to inhibit hippocampal-evoked firing than at longer intervals. This last finding suggests that inputs from the MD may play
a role in heterosynaptic coincidence detection (Usrey, 2002),
inhibiting inputs from the temporal lobe unless they arrive synchronously with diencephalic inputs.
Modulation of the hippocampal-PFC pathway by the MD
Thalamocortical inputs from the MD typically exerted a pronounced
inhibition of hippocampal-evoked firing in PFC neurons. Although there
is only preliminary ultrastructural evidence that inputs from the MD
synapse on PFC GABAergic interneurons (Kuroda et al., 1998), such an
arrangement is commonly reported in other thalamocortical projection
systems (Freund et al., 1985; Staiger et al., 1996), and activation of
thalamocortical axons yields a threefold larger amplitude EPSP on
fast-spiking interneurons as compared with pyramidal neurons (Beierlein
and Connors, 2002). Furthermore, stimulation of the MD produces an
EPSP-IPSP sequence in PFC neurons (Gigg et al., 1994; Lewis and
O'Donnell, 2000), and chemical stimulation of the MD activates
c-fos immunoreactivity in PFC GABA-containing neurons
(Bubser et al., 1998). In the present study, the inhibition of
hippocampal-evoked firing after activation of glutamatergic MD
afferents is likely mediated by feedforward inhibitory circuits. The
fact that this effect was observed in the majority of PFCHipp
neurons suggests that a large proportion of information that arrives
from the hippocampus is under the modulatory control of the MD.
For PFCHipp neurons that did not fire in response to MD stimulation,
activation of thalamocortical afferents with a train of stimuli (but
not single pulses) attenuated hippocampal-evoked firing in a majority
of neurons. This finding suggests that although relatively few
PFCHipp neurons appeared to receive direct monosynaptic input from
the MD, a larger proportion of these cells can still be modulated by MD
inputs in a frequency-dependent manner. Reyes et al. (1998) reported
that activity of neocortical GABAergic interneurons was facilitated
when excitatory inputs were stimulated with a 10 Hz train. Thus, an
increase in activity of MD inputs to the PFC in the 10-40 Hz range can
filter a large ensemble of PFCHipp neurons, attenuating firing
driven by temporal lobe inputs. This finding parallels phenomena
observed in other thalamocortical systems, whereby thalamic nuclei can
gate the flow of sensory inputs to the neocortex, producing widespread
suppression of activity, thereby focusing cortical sensory
representations (Castro-Alamancos, 2002; Castro-Alamancos and Oldford,
2002).
In contrast to the above-mentioned effects, 33% of the PFCHipp
neurons displayed a facilitation of hippocampal-evoked firing after
train stimulation of the MD, although MD stimulation alone did not
evoke firing. Moreover, repeated trains of thalamic stimulation facilitated hippocampal-evoked firing for minutes after tetanus. These
data, combined with those described above, suggests the presence of two
distinct populations of PFCHipp neurons: one receives a
predominantly inhibitory (presumably polysynaptic) input from the MD,
and a second receives a weak excitatory input that by itself is
insufficient to evoke spike firing. With respect to this latter
population, intracellular recordings have shown that train stimulation
of thalamic nuclei can produce an "augmenting response" in
sensorimotor cortex, causing a sustained depolarization lasting
hundreds of milliseconds (Castro-Alamancos and Connors, 1996; Steriade
et al., 1998). This phenomenon is thought to originate in deep cortical
layers and activates reverberatory circuits via intralaminar and
horizontal collaterals. A similar mechanism may explain the effects
observed here: high-frequency activation of subthreshold inputs to the
MD would have depolarized a subpopulation proportion of PFC neurons
(distinct from those that are inhibited by MD activation) and made them
more responsive to hippocampal input.
VTA modulation of hippocampal-evoked firing of PFC neurons
Burst stimulation of the VTA induced a frequency-dependent
inhibition over evoked firing in PFCHipp neurons; these effects were
altered by systemic administration of DA antagonists. This observation
complements previous findings in which activation of DA receptors
promotes a frequency-dependent inhibition over synaptically evoked
activity of PFC neurons (Jay et al., 1995; Gurden et al., 1999; Seamans
et al., 2001). The faster-onset (>100 msec)
D4-mediated inhibition and a longer-lasting
D2-mediated attenuation of spike firing (~300
msec) after a VTA burst may be related to the location of these
receptors in the PFC. In addition to being localized on some pyramidal
neurons, D2-like receptors in the PFC also reside
on GABAergic interneurons (Mrzijak et al., 1996; Wedzony et al., 2001)
and on excitatory presynaptic terminals (Sesack et al., 1995). Thus,
multiple cellular mechanisms exist by which D2
and D4 receptors could decrease the responsivity
of PFC neurons to hippocampal inputs, including facilitation of GABA release (Retaux et al., 1991; Grobin and Deutch, 1998; Zhou and Hablitz, 1999), modulation of the resting membrane potential (Gulledge and Jaffe, 1998, Yang et al., 1999), and presynaptic inhibition of
hippocampal inputs. Indeed, in the nucleus accumbens, application of
D2 agonists altered hippocampal terminal
excitability, implying that DA inhibits hippocampal inputs
presynaptically (Yang and Mogenson, 1986). We observed a dramatic
alteration in the paired-pulse ratio of FF-evoked firing after VTA
stimulation (Fig. 6A), suggesting that mesocortical
DA can also inhibit hippocampal inputs presynaptically.
Blockade of D1 receptors augmented the
VTA-mediated inhibition of hippocampal-evoked firing, abolishing the
frequency-dependent inhibition that was observed under control
conditions. This finding implies that D1 receptor
activity can offset t |
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ABSTRACT
Introduction
