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The Journal of Neuroscience, September 15, 2002, 22(18):8238-8250
Serotonergic Modulation of Supragranular Neurons in Rat
Sensorimotor Cortex
R. C.
Foehring3,
J. F. M.
van Brederode1, 2,
G. A.
Kinney1, 2, and
W. J.
Spain1, 2
1 Department of Neurology, University of Washington,
Seattle, Washington 98195, 2 Department of
Neurology, Veterans Affairs Medical Center, Seattle, Washington
98108, and 3 Department of Anatomy and Neurobiology,
University of Tennessee, Memphis, Memphis, Tennessee 38163
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ABSTRACT |
Numerous observations suggest diverse and modulatory roles for
serotonin (5-HT) in cortex. Because of the diversity of cell types and
multiple receptor subtypes and actions of 5-HT, it has proven difficult
to determine the overall role of 5-HT in cortical function. To provide
a broader perspective of cellular actions, we studied the effects of
5-HT on morphologically and physiologically identified pyramidal and
nonpyramidal neurons from layers I-III of primary somatosensory
and motor cortex. We found cell type-specific differences in response
to 5-HT. Four cell types were observed in layer I: Cajal Retzius, pia
surface, vertical axon, and horizontal axon cells. The physiology of
these cells ranged from fast spiking (FS) to regular spiking (RS). In
layers II-III, we observed interneurons with FS, RS, and late spiking
physiology. Morphologically, these cells varied from bipolar to
multipolar and included basket-like and chandelier cells. 5-HT
depolarized or hyperpolarized pyramidal neurons and reduced the slow
afterhyperpolarization and spike frequency. Consistent with a role in
facilitating tonic inhibition, 5-HT2 receptor activation
increased the frequency of spontaneous IPSCs in pyramidal neurons. In
layers II-III, 70% of interneurons were depolarized by 5-HT. In layer
I, 57% of cells with axonal projections to layers II-III (vertical
axon) were depolarized by 5-HT, whereas 63% of cells whose axons
remain in layer I (horizontal axon) were hyperpolarized by 5-HT. We
propose a functional segregation of 5-HT effects on cortical
information processing, based on the pattern of axonal arborization.
Key words:
5-HT; interneuron; potassium; cortex; pyramidal cell; biocytin
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INTRODUCTION |
Normal fluctuations in cortical
states, mood, attention, and information processing rely on activation
of neocortical serotonin (5-HT) receptors (Waterhouse et al. 1986b ;
Jacobs et al., 1990; Reuter and Jacobs, 1996; Marek and Aghajanian,
1998). Altered serotonergic transmission is implicated in anxiety and
depression, seizures, action of psychotropic hallucinogens, and
schizophrenia (Statnick et al., 1996; Marek and Aghajanian, 1998).
Frontoparietal cortex receives serotonergic projections from the dorsal
and median raphe nuclei (Waterhouse et al. 1986a; Tork, 1990). More
than one dozen 5-HT receptor types have been described (Hoyer et al., 1994), several of which are expressed in frontoparietal cortex (Bruinvels et al., 1994; Pompeiano et al., 1994; Burnet et al., 1995;
Wright et al., 1995; Morales and Bloom, 1997; Vysokanov et al., 1998).
With the exception of the 5-HT3 receptor
(ligand-gated ion channel), 5-HT receptors couple to G-proteins to
exert their effects (Hoyer et al., 1994). These observations suggest
diverse modulatory roles for 5-HT in cortex.
In vivo, firing rates of pyramidal neurons are generally
decreased by iontophoresis of 5-HT (Krnjevic and Phillis, 1963; Roberts and Straughan, 1967; Jordan et al., 1972; Reader et al., 1979; Lakoski
and Aghajanian, 1985). 5-HT also reduced the responses of somatosensory
cortical neurons to afferent input (Waterhouse et al. 1986b) and
altered memory fields in prefrontal cortex (Williams et al., 2002).
Previous in vitro studies of the actions of 5-HT in cortex
concentrated on layer V pyramidal neurons. In rat cortex, these cells
were hyperpolarized or depolarized by 5-HT, with most cells exhibiting
both responses (Davies et al., 1987; Araneda and Andrade, 1991; Tanaka
and North, 1993; Spain, 1994; Marek and Aghajanian, 1998). The
hyperpolarizations involved activation of 5-HT1A
receptors and increased potassium conductance
(GK). Depolarizations were reported to
involve 5-HT2A receptors and a decrease in
GK. 5-HT also reduced the slow
afterhyperpolarization (sAHP) (Araneda and Andrade, 1991; Tanaka and
North, 1993) and induced an afterdepolarization (ADP) (Araneda and
Andrade, 1991). In cat sensorimotor cortex, the 5-HT response
correlated with pyramidal cell firing type (Spain, 1994).
Understanding serotonergic effects on cortical function requires
knowledge of the actions of 5-HT on other cell types in cortical local
circuits. GABAergic interneurons in the neocortex are morphologically and physiologically diverse (White, 1989; Kawaguchi, 1993, 1995; Cauli
et al., 1997; Kawaguchi and Kubota, 1997, 1998), and GABAergic inhibition has been proposed to be a target of serotonergic modulation (DeFelipe et al., 1991; Smiley and Goldman-Rakic, 1996; Abi-Saab et al., 1999). Little is known, however, about the responses of specific interneuron types to 5-HT. A few layer V nonpyramidal cells
were included in the Tanaka and North (1993) study, and a preliminary
report suggests 5-HT3 responses in neocortical
layer V interneurons (Xiang et al., 1999). Altered tuning of memory fields was reported for thin spiking neurons (putative interneurons) in
primate prefrontal cortex (Williams et al., 2002).
5-HT2A receptor activation increased the
frequency of spontaneous EPSCs (sEPSCs) and sIPSCs on both pyramidal
cells and layer I interneurons (Zhou and Hablitz, 1999b). Effects of
other transmitters on cortical interneurons are cell type specific
(Kawaguchi and Shindou, 1998; Para et al., 1998; Xiang et al.,
1999; Zhou and Hablitz, 1999a).
We examined serotonergic effects on identified interneurons and
pyramidal cells from rat sensorimotor cortex (layers I-III). Our major
finding was that 5-HT had different effects on interneurons, and these
differences corresponded to laminar differences in axonal projections.
Some of these data have been presented previously in abstract form
(Foehring et al., 1996).
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MATERIALS AND METHODS |
Sprague Dawley rats [postnatal day (P) 6-24; most were
P18-23] were deeply anesthetized with an intraperitoneal injection (1.8 ml/kg) of a mixture of 1:3 xylazine/ketamine dissolved in an equal
volume of saline. These ages correspond to the peak expression of
5-HT2A receptors in cortex (approximately P13)
(Roth et al., 1991). The density of serotonergic fibers (Foote and
Morrison, 1984; Nakazawa et al., 1992),
5-HT1A receptors (Zifa et al., 1988), and
5-HT levels (Edagawa et al., 2001) continue to increase with age, at least through 5 weeks postnatally. 5-HT7
receptors are expressed in low abundance in this cortical region
(Gustafson et al., 1996), with developmental regulation of receptor
abundance (Vizuete et al., 1997). It is possible that
changes in relative abundance of receptor types could alter the effects
of 5-HT at other ages. We therefore restrict our conclusions to ~2-3
weeks postnatally, an especially important time for generation of new synapses in neocortex (Armstrong-James and Johnson, 1970; Wise and Jones, 1976).
Once the animals were areflexic, a shallow ventral incision severed the
carotid arteries. The skull was opened dorsally, and the brain was
exposed. The dura was reflected, and cold artificial CSF (ACSF) (see
below) was applied to the brain surface while four scalpel cuts
isolated the left somatosensory and motor cortex (sensorimotor cortex)
and underlying forebrain. The isolated block of tissue was then lifted
clear of the brain with cottonoid and glued to the stage of a modified
Vibratome (Pelco 101 series 1000; Ted Pella, Inc., Redding, CA). The
tissue was immersed in cutting solution (see below) at 4°C, and
coronal sections were cut at 300 µM. The slices were
maintained in ACSF at 35°C for 1 hr and thereafter stored at room temperature.
Solutions. All chemicals were obtained from Sigma (St.
Louis, MO), unless noted otherwise. The cutting solution contained (in
mM): KCl 5, NaH2PO4 1.25, NaHCO3 26, MgCl2 5, TEA-Cl
20, Choline-Cl 105, sucrose 20, and dextrose 10 [320 mOsm/l, pH
maintained at 7.4 by bubbling with carbogen (95%
O2/5% CO2)]. ACSF
contained (in mM): NaCl 125, KCl, 3, NaH2PO4 1.25, NaHCO3 26, dextrose 20, MgCl2 2, and CaCl2 2 (310 mOsm/l). The pH was maintained at 7.4 by bubbling with carbogen. For
whole-cell recordings, the internal recording solution contained (in
mM):
KCH3SO4 135, MgCl2 2, KCl 5, HEPES 10, ATP 2, Na3GTP 0.5, biocytin (0.5%), and EGTA 0.1, pH
7.2 (270 mOsm/l). For recording spontaneous IPSCs,
CsCH3SO4 replaced
KMeSO4 in the internal solution 5-HT and
serotonergic agonists and antagonists were obtained from RBI. They were
prepared in d,d H2O and frozen in aliquots until
use. Agonists were only tested in cells with stable membrane potential
for >2 min in control solution. Na-metabisulfite was included (final
concentration 50-100 µM) with 5-HT and
agonists to prevent oxidation (Sutor and ten Bruggencate, 1990).
Na-metabisulfite caused no effects on its own (n = 4).
Solutions were held in separate bottles at 35°C. A manifold and
valves were used to control which solution flowed into the common line
to the recording chamber.
Recordings. A single slice was transferred to a recording
chamber on the fixed stage of a Zeiss Axioscope. Slices were submerged and perfused continuously in ACSF (~2 ml/min). Cortical layers were
located under low power, and then a 40× water immersion lens with
differential interference contrast (DIC) optics, and near infrared
(bandpass 750-800 nm) illumination was used to identify individual
neurons. The image was projected onto a video camera sensitive to
infrared light and displayed on a video monitor (Dodt and
Zieglgansberger, 1994). Solutions were bubbled with carbogen and
preheated to 37°C in a water bath. The solutions were fed by gravity
to the recording chamber. An in-line heater and temperature probe were
used to regulate bath temperature at 32 ± 2°C. In most cases,
5-HT and agonists were applied in the bath solution (10-60 µM). In a few cases, drugs were applied
directly to visualized neurons using pressure (Picospritzer;
General Valve, Fairfield, NJ). Antagonists were added in the bath for
5-10 min before application of agonist plus antagonist.
Whole-cell electrodes were fabricated from VWR (VWR Scientific
Products, Westchester, PA) hematocrit glass (4-8 M ) with a two-stage pull on a List vertical puller. Electrodes were not polished
or coated for current-clamp recordings. We coated the electrodes with
Sylgard (Dow Corning Corporation, Midland, MI) for recording of IPSCs.
Recordings were made in current clamp (Axoclamp 2A, bridge mode; Axon
Instruments, Union City, CA) or voltage clamp (Axopatch 1C or 200B).
The electrodes were visually guided onto the cell bodies while positive
pressure was being applied. On cell contact, negative pressure was
applied until a tight seal (>1 G) was formed. Further negative
pressure was used to attain the whole-cell configuration. Whole-cell
current and membrane potential were filtered (<3 kHz), amplified, and recorded on a video recorder with pulse code modulation (Neurocorder, 44 kHz sampling rate; Neurodata Instrument Corp., New York, NY) for
later analysis or digitized on-line with pClamp 6 (Axon Instruments). A
chart recorder was used to monitor slow changes in membrane currents
and potentials. Data were analyzed off-line using Axograph (Axon
Instruments). Voltage records for current clamp were corrected for the
liquid junction potential [determined using the method of Neher
(1992)], which was 10 mV. Series resistance was typically 10-20 M.
Cells were eliminated if series resistance increased greatly during the recording.
Spontaneous synaptic currents were detected and analyzed as described
previously (van Brederode et al., 2001). Current segments (1-2 min)
were scanned for synaptic events using a threshold detection method
(Mini Analysis, Synaptosoft, Decatur, GA).
Morphology. Neurons were filled with Biocytin during the
recording and identified as interneurons or pyramidal cells on the basis of morphology. After recording, the electrode was slowly retracted from the cell. The slice was then placed in 4%
paraformaldehyde for 24 hr. We typically recorded one cell per slice,
or if a second cell was recorded from, it was chosen to be in a
different layer and location in the slice. The fixed slices were then
washed in PBS and then processed according to the avidin-biotin method
(Vectastain) of Horikawa and Armstrong (1988). Biocytin was visualized
using DAB/peroxidase. Filled cells were drawn under high power (40 or 100× oil immersion lens) with the aid of a drawing tube on a
microscope. These drawings were later traced in ink, transferred to
acetate, and photographed. Selected cells were drawn in detail using
the Eutectics system (Eutectics Corp.). The cell dimensions were not corrected for tissue shrinkage or two-dimensional projection errors.
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RESULTS |
We recorded from 106 cells (52 pyramidal and 54 nonpyramidal) that
were tested with bath-applied 5-HT and that met our criteria of
overshooting action potentials (APs) and stable resting potential (negative to  50 mV) and input resistance (current-clamp recordings). Additional cells were examined with direct pressure application of 5-HT
or 5-HT3 agonist (see below). All of the
recordings were obtained with whole-cell patch clamp of cells
visualized with infrared illumination and DIC optics (Stuart et al.,
1993; Dodt and Zieglgansberger, 1994) in the supragranular
layers of sensorimotor cortex. Data are presented as mean ± SEM,
unless noted otherwise. Table 1 lists the
nonpyramidal neurons included in the analyses, including cortical
layer, age of animal, and morphological and physiological
classification (see below). Table 2
contains representative data from 42 interneurons (21 from layer I, 21 from layer II) for which complete physiological data were obtained.
Our goal was to determine the overall, dominant effect of 5-HT on
different cell types. Unless stated otherwise, all data presented below
for pyramidal and nonpyramidal neurons reflect bath
application of transmitter or agonists. Both hyperpolarizations and
depolarizations were fully reversible if the agonists were applied for
more than ~1 min (see Fig. 2C2,E) and recovery
often required long (>10 min) washes in control solutions
(n = 25 cells).
Our bath application data are biased against rapidly desensitizing
responses (cf. 5-HT3 receptor mediated). We also
used direct pressure application (Picospritzer) of 30 µM
5-HT to 13 cells (7 pyramidal, 6 interneurons). Of these cells, three
were depolarized, four hyperpolarized, and six showed no response to
5-HT. Multiple 5-HT receptor types are likely colocalized on individual
cells (Martin-Ruiz et al., 2001), and dominance of a particular
response does not rule out expression of other receptor subtypes
(Araneda and Andrade, 1991).
Pyramidal neurons
To determine whether responses of supragranular pyramidal neurons
to 5-HT were similar to those reported for layer V pyramidal neurons
(Davies et al., 1987; Araneda and Andrade, 1991; Tanaka and North,
1993), we examined the effects of 5-HT on layers II-III pyramidal
neurons (n = 38 cells) (Fig.
1). In superficial layers II-III (near
I-II border; layer II), 14 of 28 (50%) cells were depolarized by 5-HT
(Fig. 1A). The other 14 cells (50%) were
hyperpolarized (Fig. 1B). In deep layers II-III
(layer III), 9 of 10 cells (90%) were depolarized by 5-HT, and 1 cell
was hyperpolarized (Table 3). Many (18 of
28) of the layer II pyramidal cells had atypical orientation of the
apical dendrite: the dendrite was horizontal, oblique, or even inverted
(van Brederode et al., 2000). Of these 18 cells, 9 were depolarized and
9 were hyperpolarized by 5-HT.
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Figure 1.
Response of layers II-III pyramidal neurons to
5-HT. Time of 5-HT application is indicated by solid bar
above traces. A, Twenty-three of 38 pyramidal cells were depolarized by bath application of 50 µM 5-HT. Downward deflections represent response to
current ramps repeated at 5 sec intervals (see inset,
below). In this cell, input conductance decreased in 5-HT. This cell
was from layer III and was depolarized to firing. B,
Other pyramidal cells were hyperpolarized by 5-HT (15 of 38). This cell
was from layer II. Downward deflections are response to 500 msec, 200 pA current injections. In this cell, the peak hyperpolarization was 2 mV. C, Firing of layer II inverted pyramidal neuron (van
Brederode et al., 2000) in response to 1 sec, 1.3 nA current injection.
Note prominent spike frequency adaptation. D, Firing of
the same cell as in B, except in the presence of 60 µM 5-HT (bath application). The membrane potential was
manually returned to the original resting potential by DC injection. In
5-HT, the cell fired more action potentials, although the current
stimulus was reduced (1.2 nA). In addition, 5-HT reduced the sAHP.
E, The top trace is the response of a
horizontally oriented pyramidal neuron from layer II (van Brederode et
al., 2000) to a 500 msec, 145 pA current injection. The bottom
trace shows the response of the same cell in the presence of
bath-applied 30 µM 5-HT to a 500 msec, 92 pA current
injection. The membrane potential was manually returned to the original
resting potential by DC injection. The current stimuli were chosen to
elicit the same numbers of spikes (10) in both control and 5-HT
solutions. Note the different patterns of firing, with marked spike
frequency adaptation in control solution and reduced adaptation in
5-HT. F, Same cell and traces as in
E, with expanded time base and superimposition of
traces to show the reduction in the sAHP by 5-HT.
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Although not studied in detail, the sAHP was significantly reduced
(from 4.5 ± 2 to 1.6 ± 2 mV; 10-100% reduction) by 5-HT in 12 of 12 layers II-III cells tested (Figs.
1E,F), with associated reduction in spike frequency adaptation (Fig. 1, compare C,
D) (n = 12) (Araneda and Andrade, 1991;
Spain, 1994). The sAHPs were elicited by repetitive firing in response
to 500 msec constant current injections. Currents were adjusted to
match numbers of spikes (3-21) and DC current injection was used to
match resting potential, between 5-HT and control solutions. The sAHPs
were measured at 500 msec after firing (after the mAHP has fully
decayed) (Lorenzon and Foehring, 1993). In two cells, an ADP was
induced by bath-applied 30 µM 5-HT (data not
shown) (Araneda and Andrade, 1991; Spain, 1994).
Nonpyramidal neurons
Stable recordings and 5-HT application were obtained for 54 cells
from layers I-III that were later identified by morphology as
nonpyramidal. Overall, 28 of 54 nonpyramidal cells (52%) were depolarized by 5-HT (Fig. 2, Table 3).
Eight cells (15%) showed no response to 5-HT, and 18 of 54 cells
(33%) were hyperpolarized (Fig. 2, Table 3).
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Figure 2.
Membrane potential responses of
interneurons to 5-HT. Time of agonist application is indicated by
solid bar above traces. A,
Bath application of 5-HT hyperpolarized a subpopulation of
supragranular interneurons. The dotted line indicates
the original membrane potential (66 mV). This cell (layer I
horizontal axon cell) was hyperpolarized 2.7 mV by 5-HT. The negative
deflections are responses to a 500 msec input conductance stimulus
(200 pA; inset). The box plots (Tukey, 1977) indicate
the median values (vertical line within
box) and inner quartile (box margin) and
outer quartile (horizontal lines extending from
box) of the responses for layers II
(n = 14 cells) and II-III (n = 3). B, Other interneurons were depolarized by 5-HT. This
cell (layer I, vertical axon) was depolarized to spiking. More
typically, depolarizations did not reach spike threshold. The box plots
summarize the data for response amplitude by layer (layer I:
n = 12 cells; layers II-III: n = 16 cells). C, The membrane potential responses to 5-HT
were retained in the presence of 1 µM TTX.
C1, 5-HT was added to the bath. This layer II
nonpyramidal cell was depolarized by 15 mV. Downward deflections
represent response to current ramps (350 pA, 500 msec).
C2, This basket-like cell (layer II) was hyperpolarized
~3 mV by 5-HT in the presence of TTX. Downward deflections represent
response to current ramps (400 pA, 500 msec) repeated at 5 sec
intervals. Note reversal of effect in control wash. D,
Receptor pharmacology. D1, The hyperpolarization was
mimicked by bath application of the 5-HT1A receptor agonist
10 µM 8-OHDPAT in this layer II FS cell. Dotted
line indicates original resting potential. Downward deflections
represent response to current steps (300 pA; see
inset). D2, The application of the
5-HT2A/2C receptor agonist 30 µM DOI
led to a 15 mV depolarization in this layer I FS cell (pia surface
cell; 1 µM TTX present). Downward deflections represent
response to current ramps (350 pA, 500 msec). E,
Depolarizing effects of 5-HT were fully reversible. In this layers
II-III interneuron, 5-HT was applied in the bath for 30 sec. The cell
depolarized by 7 mV, and this depolarization was reversed after wash in
control solution.
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Neocortical nonpyramidal cells are highly diverse with respect to
morphology, firing behavior, and expression of peptides and calcium
binding proteins (White, 1989; Kawaguchi and Kubota 1996, 1997, 1998;
Cauli et al., 1997). We therefore separated the data by layer and cell
type. For each layer, we classified cells by axonal and dendritic
morphology. Secondary classification was on the basis of the
combination of firing pattern (amount of spike frequency adaptation,
regularity of firing to near rheobasic currents), AP width, and input resistance.
Layer I receives a dense serotonergic projection (Mulligan and Tork,
1988; DeFelipe et al., 1991; Hornung and Celio, 1992; Smiley and
Goldman-Rakic, 1996). We recorded from 31 morphologically identified
interneurons in layer I (see Fig. 4, Table 1). Twelve of these cells
(39%) were depolarized by 5-HT. Five cells (16%) showed no response
to 5-HT, and 14 cells (45%) were hyperpolarized (Table 2). We recorded
from 23 cells in layers II-III (Table 3) that were tested with 5-HT
and (1) could either be classified on the basis of morphology into a
particular cell type or (2) were clearly not pyramidal in morphology
and showed fast spiking (FS) or late spiking (LS) physiology (McCormick
et al., 1985; Cauli et al., 1997; Kawaguchi and Kubota 1997, 1998) (see
Figs. 4, 5). Of these 23 cells, 16 (70%) were depolarized by 5-HT.
Three cells did not respond to 5-HT (13%), and four were
hyperpolarized (17%) (Fig. 2, Table 3).
Pharmacology
Both hyperpolarizations (n = 6; 4 pyramidal, 2 nonpyramidal) and depolarizations (n = 8 pyramidal, 4 nonpyramidal) were observed in the presence of TTX, suggesting that the
effects were not dependent on presynaptic or postsynaptic action
potentials (Fig. 2C). We also tested the response to 30 µM 5-HT in several cells in the presence of 100 µM picrotoxin, 50 µM
AP5, and 10 µM CNQX to block transmission via
GABAA, NMDA, and AMPA receptors, respectively (data not shown). We observed depolarizations (two of four pyramidal and four of six nonpyramidal cells) and hyperpolarizations (two of four
pyramidal and two of six nonpyramidal cells) in the presence of these
blockers, suggesting that the membrane potential effects were direct on
the cells recorded from rather than caused by altered synaptic input.
Although we did not quantify dose-response relationships for the
responses to 5-HT, we tested two or more doses in seven cells. For
depolarizations (n = 3) and hyperpolarizations
(n = 4), the higher dose always caused a greater
response (data not shown). Repeated application of the same dose
of agonist resulted in similar response to 5-HT (five depolarization,
three hyperpolarization), 2,5-dimethoxy-4-iodoamphetamine
hydrochloride (DOI) (see below; four cells), and
8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) (two cells).
Receptors
Hyperpolarizations
To gain insight into which receptor types were responsible for the
membrane potential changes in response to 5-HT, we tested responses to
specific 5-HT receptor agonists and antagonists. On the basis of
previous work in neocortex (Davies et al., 1987; Araneda and Andrade,
1991; Tanaka and North 1993; Spain, 1994) and other cell types (Andrade
and Nicoll, 1987; Collino and Halliwell, 1987), we first examined
responses to the bath application of 10 µM of the
5-HT1A/5-HT7 agonist
8-OHDPAT (Fig. 2D1). In layer I cells, 8-OHDPAT (10 µM) hyperpolarized six of eight cells tested (3 ± 2 mV; n = 6). Two horizontal axon cells
showed no response. In layer II, five interneurons were tested with
8-OHDPAT; two cells were hyperpolarized, and three cells did not
respond. 8-OHDPAT also hyperpolarized four of six layers II-III
pyramidal cells (4 ± 1 mV; data not shown; two cells did not respond).
5-HT elicited hyperpolarizations in three of three interneurons and
three of three pyramidal neurons tested in the presence of the
5-HT2/5-HT7 antagonists
ritanserin or ketanserin (2 µM; data not shown). Combined
with the 8-OHDPAT data, these data suggest that the hyperpolarizations
are mediated by 5-HT1A receptors, although
further study with more specific agonists/antagonists will be required.
Depolarizations
On the basis of receptor localization studies (Pompeiano et al.,
1994; Wright et al., 1995; Morales et al., 1996; Morales and
Bloom, 1997; Jakab and Goldman-Rakic, 1998) and physiology in various
cell types (Lakoski and Aghajanian, 1985; Davies et al., 1987; Jackson
and White, 1990; Araneda and Andrade, 1991; Bobker, 1994; Gellman and
Aghajanian, 1994; Kawa, 1994; Spain, 1994; McMahon and Kauer, 1997;
Roerig et al., 1997; Marek and Aghajanian, 1998), we expected the
depolarizations to be mediated by 5-HT2 or
5-HT3 receptors [5-HT7
receptors are another possibility (Gustafson et al., 1996)]. Because
5-HT3 receptor-mediated events are reported to
rapidly desensitize (Jackson and Yakel, 1995), we thought it unlikely
that the depolarizations that we observed after bath application of
5-HT would be 5-HT3 mediated. Consistent with
this hypothesis, bath application of the 5-HT3
agonist 1-(m-chlorophenyl) methyl biguanide (biguanide; 10-30
µM) caused no response in both interneurons tested (data
not shown). Pressure application of 30 µM biguanide to 3 interneurons and 10 pyramidal cells elicited no membrane potential
response in 12 cells; 1 interneuron was depolarized (data not shown).
Zhou and Hablitz (1999b) reported inward currents in response to local
application of 5-HT3 agonists in just 4 of 43 layer I cells. In contrast, bath application of the specific
5-HT2A/5-HT2C agonist DOI
(10-30 µM) (Hoyer et al., 1994) caused a depolarization
in four of four layer I interneurons tested (5 ± 2 mV) and two of
four layer II cells (two cells showed no response) (Fig.
2D2). The specific
5-HT2A/2C/7
antagonist ketanserin prevented the depolarization by 5-HT in three of
four interneurons tested (data not shown). Ketanserin alone had no
effect on membrane potential (n = 4). Zhou and Hablitz
(1999b) did not observe inward currents in layer I (n = 20) or layer II (n = 3) cells in response to
5-HT2 agonists.
DOI (10-30 µM) depolarized four of seven layers II-III
pyramidal cells (5 ± 2 mV). Two cells did not respond, and one
cell was hyperpolarized (data not shown). Ketanserin prevented the depolarization in three of four pyramidal cells (data not shown). Ketanserin alone had no effect on membrane potential (n = 4). Together, these data suggest that at least some of the
depolarizations in both pyramidal and nonpyramidal cells were mediated
by 5-HT2A or 5-HT2C
receptors (Davies et al., 1987; cf. Araneda and Andrade, 1991). Further
study with more specific agonists and antagonists is required to test
for potential roles of other receptor subtypes (e.g.,
5-HT3, 5-HT4,
5-HT7) (Beique and Andrade, 2001).
Ionic basis
Hyperpolarization
In layer V pyramidal neurons, the
5-HT1A-mediated hyperpolarization was found to be
caused by an increase in K+ conductance
(Spain 1994). On average, we found that the 5-HT-mediated hyperpolarization in interneurons (measured at the original resting potential) was associated with no significant change in input resistance (control: 231 ± 38 M; 5-HT: 277 ± 40 M;
n = 10). Input resistance was calculated from Ohm's
law (RN = V/I). The current injection (500 msec
step) was adjusted to elicit a 10-20 mV voltage response.
We tested the reversal potential of the hyperpolarization in six
interneurons (Fig. 3) (in the presence of
1 µM TTX). With 3 mM extracellular
K+, the reversal potential was 86 ± 4 mV (Fig. 3A). At 32°C and with 140 mM internal K+, the
Nernst potential for 3 mM extracellular
K+ is approximately 100 mV. For 9 mM extracellular K+,
the Nernst potential is 71.5 mV (predicted shift with change from 3 to 9 mM extracellular
K+ = 28.5 mV). The 5-HT-mediated
hyperpolarization reversed at 62 ± 4 mV in 9 mM K+ (average
shift = 24 ± 3 mV) (Fig. 3B). In pyramidal cells
with 3 mM extracellular
K+, the hyperpolarization induced by 5-HT
reversed at 86 ± 2 mV (n = 8; data not shown).
This reversal potential shifted to 65 ± 4 mV (n = 5) in 9 mM extracellular
K+ (average shift = 21 ± 3 mV;
data not shown). There was also no significant change in pyramidal cell
input resistance with 5-HT (control: 224 ± 66 M; 5-HT:
227 ± 67 M; n = 4). These data suggest that
the 5-HT1A-mediated hyperpolarization in both
interneurons and pyramidal neurons involved a small increase in
K+ conductance (as evidenced by the
decrease in slope of the voltage response to current ramps) (Fig.
3).
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Figure 3.
Ionic basis for 5-HT-induced hyperpolarizations.
A, Response of a layer II nonpyramidal neuron to bath
application of 30 µM 5-HT (3 mM extracellular
K+). The recording was done in the presence
(extracellular) of 1 µM TTX. The control
(Ctl) and 5-HT traces intersect at approximately
91 mV (calculated Nernst potential for K+ = 100 mV). Calibration also applies to B. Current ramp
used as stimulus shown above. Bottom, Box plot
illustrating population data (n = 10 cells) for
reversal potential in 3 mM extracellular
K+. B, Same cell showing response to
5-HT in 9 mM extracellular K+. The
reversal potential shifted positive (by 28 mV) to 63 mV (expected
shift if Nernstian = 28.5 mV). Bottom, Box plot
illustrating population data (n = 4 cells) for
reversal potential in 9 mM extracellular
K+. Current ramp used as stimulus shown above.
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Depolarization
For the depolarization of nonpyramidal cells, there was also no
significant change in average input resistance (control: 289 ± 40 M; 5-HT: 339 ± 54 M; n = 16;
p = 0.06). We tested the reversal potential of the
depolarization in six nonpyramidal cells, but no consistent pattern
emerged. On average, the reversal potential was 73 ± 6 mV with
3 mM extracellular
K+ (data not shown). In two cells the
reversal potential approached EK
(95, 86 mV), but in the remaining four cells the reversal potential
was more positive (73, 65, 61, 58 mV). Similarly, in pyramidal
neurons, the reversals were 95 and 65 mV (in 3 mM K+). The
5-HT2A-mediated depolarization in pyramidal
neurons has been attributed to a decrease in
K+ conductance (Davies et al., 1987;
Araneda and Andrade, 1991). Depolarization in cat pyramidal
neurons was found to be caused by an increased
IH (Spain, 1994). It has been
suggested that the 5-HT2-mediated depolarization
in layer V pyramidal cells from medial prefrontal cortex is caused by
activation of a nonspecific cation current (Haj-Dahmane and Andrade,
1998). Our data do not differentiate between these
possibilities. In pyramidal cells, there was no significant change in
input resistance with hyperpolarization by 5-HT (control: 232 ± 27 M; 5-HT: 216 ± 25 M; n = 8).
Cell types
We next tested whether the variability in interneuronal responses
to 5-HT was caused by cell type-specific effects.
Layer I
Our morphological findings for layer I neurons were similar to
previous investigations (Hestrin and Armstrong, 1996; Zhou and Hablitz,
1996a). We identified four groups of cells in layer I (Fig.
4): Cajal Retzius cells (CR), small cells
at the pia surface, vertical axon cells, and horizontal axon cells. We
identified seven vertical axon cells (Fig. 4A). These
cells varied in soma size and sent an axonal projection to layers
deeper than layer I. The horizontal extent of both the axonal and
dendritic arbors was restricted (typically <200 µm) (Fig.
4A). Our vertical axon cells may include both the
"vertical cell" and the "cells sending axon collaterals to deeper
layers" groups of Zhou and Hablitz (1996a).
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Figure 4.
Camera lucida reconstruction of biocytin-filled
(Horikawa and Armstrong, 1988) layer I interneurons. A,
Vertical axon cell (soma, axon, dendrites). The soma and dendrites (no
axon) of this cell are shown separately on the right.
The pia (solid line) and layers I-II border
(broken line) are indicated on the left.
The dendritic tree branches throughout the entire thickness of layer I,
whereas most axonal branches are confined to layers II-III and do not
enter layer I. Although this cell was located at the I-II border,
vertical axon cells were located at all depths of layer I. B, Horizontal axon cell. The axons originated from two
opposite poles of the cell body from a short trunk. The precise
transition between dendrites and axons was difficult to discern. Note
that the axonal and dendritic branches are primarily confined to the
top half of layer I. C, Cajal
Retzius neuron (P9 animal). The soma and dendrites of this cell
are shown at higher magnification (2×) on the right.
Note the thick dendrites and that the axonal branches were mostly
confined to the top half of layer I. The cells in
B and C were drawn with a 100×
objective, whereas the cell in A was drawn with a 40×
objective. Scale bars, 100 µm (and apply to the reconstruction of
axons). The drawings of the dendritic trees on the right
were enlarged 2× from the drawings on the left.
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Sixteen horizontal axon cells (Fig. 4B) were recorded
from, two of which were clearly neurogliaform (cf. Hestrin and
Armstrong, 1996). The axonal and dendritic arbors of the horizontal
axon cells extended horizontally in layer I for up to 300-400 µm
(Fig. 4B). These cells did not send axons deeper than
the border between layers I-II. Zhou and Hablitz (1996a) found this
cell type to be the most common in layer I. Layer I neurogliaform cells
(Hestrin and Armstrong, 1996) had a very dense local axonal projection, and we considered them to be a subpopulation of horizontal axon cells.
We recorded from four CR cells in slices from animals aged P9-13 (Fig.
4C). These cells were characterized by their large size,
horizontal orientation, and thick dendrites (Fairen et al., 1984;
Hestrin and Armstrong, 1996; Zhou and Hablitz, 1996a). The pia surface
cells (n = 4) were small, with fine, sparse dendrites that remained local (data not shown). A subpial cell type was described
by Zhou and Hablitz (1996a, their Fig. 14).
Interneurons could also be classified on the basis of their physiology
(Kawaguchi and Kubota 1996, 1997, 1998; Cauli et al., 1997). FS
interneurons have brief spikes followed by a large fast afterhyperpolarization (fAHP), show little spike frequency adaptation or sAHP, and exhibit irregular, interrupted firing in response to near
threshold current injections. A variant of the FS pattern is exhibited
by LS cells, which fire after a prolonged voltage ramp (Kawaguchi and
Kubota, 1996, 1997, 1998). In contrast, RS interneurons exhibit broad
spikes, repetitive firing with spike frequency adaptation, and sAHPs
(Kawaguchi and Kubota 1996, 1997, 1998; Cauli et al., 1997). Zhou and
Hablitz (1996b) reported that layer I neurons had FS physiology.
Hestrin and Armstrong (1996) found layer I cells to be more
heterogeneous. We found that the firing pattern of layer I interneurons
(other than CR cells) varied from regular spiking (RS) to fast spiking.
The distribution of AP half-widths formed a continuum in this layer
(data not shown), and there was no clear relationship between firing
patterns and cell morphology or response to 5-HT. Some (but not all)
horizontal axon, vertical axon, and pia surface interneuron types had
properties resembling those of FS cells (Hestrin and Armstrong, 1996;
Zhou and Hablitz, 1996b), i.e., brief spikes, large fAHPs, and
interrupted firing to near threshold stimuli.
Layer II
Seven of the layer II cells could be classified on the basis of
axonal morphology as basket-like (extended plexus) cells (Feldman and
Peters, 1978; White 1989; Somogyi et al., 1998) (Fig.
5A). These cells had smooth
dendrites with few or no spines. Pericellular varicosities were
observed throughout the axonal arbor. In some cases, one or more
collaterals descended to deeper layers (Fig. 5A). All of the
basket-like cells exhibited physiological characteristics of LS or FS
cells: brief action potentials, lack of an sAHP or spike frequency
adaptation, and interruptions in the firing pattern in response to near
rheobasic current injections (McCormick et al., 1985; Kawaguchi and
Kubota, 1996, 1997, 1998). We also sampled cells that were bipolar and
multipolar (data not shown), and we recorded from one chandelier cell
with characteristic vertical axonal cassettes (Fig. 5C).
This cell had FS physiology and did not respond to 5-HT. One cell was
classified as neurogliaform (Fig. 5B). Two layer II
interneurons (both bipolar) had RS physiology, and 21 had FS or LS
physiology.
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Figure 5.
Morphological types of interneurons in layers II
and III. All cells were filled with biocytin during recording and
processed with modifications of Horikawa and Armstrong (1988). The
cells were then drawn with the aid of a camera lucida, copied onto
acetate, and digitized with a scanner. A, Layer II
basket-like cell. Note long vertical axonal projection as well as the
horizontal one. Multipolar soma and dendrites are shown without the
axon at right. B, Neurogliaform cell in
layer II. Note extensive local processes. C, A
chandelier cell in layer II. This cell did not respond to 5-HT. Note
extensive vertical cassettes. Soma and dendrites shown in isolation
below. All of the cells were drawn with a 100× objective. Scale bars:
A, left, 100 µm; A,
dendritic inset, 25 µm; B, 50 µm;
C, 100 µm; C, dendritic
inset, 25 µm.
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5-HT effects by cell type/layer
There was no clear relationship between the firing type or cell
morphology of layer II interneurons and their response to 5-HT (Tables
1, 3) (Para et al., 1998). A few cells were inadequately filled for
classification by the axonal arbor, but were clearly not pyramidal (no
prominent apical dendrite) and showed physiological characteristics of
FS or LS cells. The basket-like and chandelier cells also had FS
physiology, so we combined them with the morphologically unclassified
cells as an FS/LS group. 5-HT depolarized 14 of these 21 cells (67%),
hyperpolarized 3 (14%); 1 cell showed both responses (5%), and 4 cells did not respond to 5-HT (19%) (Fig. 1, Table 3).
In layer I, three of four CR cells were hyperpolarized, and one did not
respond (Tables 1, 3). All four pia surface cells that we recorded from
were depolarized by 5-HT (Table 1). In response to 5-HT, four or seven
vertical axon cells (57%) were strongly depolarized (Table 3). One
cell did not respond to 5-HT, and two of seven cells were
hyperpolarized (29%; both 2 mV) (Tables 1, 3). Bath application of
5-HT led to depolarization in 4 of 16 (25%) of horizontal axon cells
(including neurogliaform) (Table 3). Two cells (12.5%) were not
affected by 5-HT, and 10 (62.5%) were hyperpolarized (Tables 1,
3).
Statistics
We used Fisher's exact test to test the hypothesis that responses
to 5-HT differed by layer and cell type. We found that the distribution
of cells (Table 3) responding with depolarization versus
hyperpolarization (vs no change) was statistically different (p = 0.03) for layer I versus II. When the
amplitude and sign of 5-HT responses were compared, Student's
t test also revealed a statistical difference between layers
I and II (p < 0.001; two-tailed test). On
average, layer I cell membrane potential depolarized by 0.5 ± 1 mV (n = 31) and layers II-III interneurons were
depolarized by 4.3 ± 1 mV (n = 23). Because
horizontal and vertical axon cells in layer I appeared to respond
differently to 5-HT, we also tested whether these two populations of
cells were different. The distributions (Table 3) were not
statistically different (Fisher's exact test; p = 0.1), but amplitude of the response did differ between the two cell
types (t test; p < 0.001). On average,
vertical axon cells were depolarized (5 ± 2 mV; n = 7), and horizontal axon cells were hyperpolarized (3 ± 1 mV;
n = 16) by 5-HT.
The responses of layer I cells whose axons projected to layers II-III
(vertical cell) and those of interneurons whose somas (and axons) were
found in layers II-III were similar, suggesting that they form a
functional group. We tested this hypothesis by comparing the
combination of layer I vertical cells plus layers II-III interneurons
with layer I horizontal cells (Table 3). CR cells were not included in
this analysis because they may not be GABAergic. Pia surface cells were
also not included because their axonal projections were not well
filled. The distribution of responses was statistically different
(p < 0.002; Fisher's exact test), and response
amplitude also differed significantly (t test). Cells whose
projection remained in layer I were on average hyperpolarized by 3 ± 1 mV (n = 16), and cells projecting to layers II-II
were depolarized (5 ± 1 mV; n = 30). Responses of
layer I projecting (horizontal axon cells) versus layers II-III
projecting cells (layer I vertical axon cells plus layers II-III
interneurons) are summarized graphically in Fig.
6. These data suggest that interneuronal
responses to 5-HT can be functionally differentiated by axonal targets.
That is, axonal projection pattern is a better predictor of response to
5-HT than soma location or physiological type.
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Figure 6.
Box plot (Tukey, 1977) illustrating the
distribution of membrane potential responses of interneurons whose
axonal projection was restricted to layer I (layer I horizontal axon
cells) to the responses of interneurons with axonal projections within
layers II-III (layer I vertical axon cells plus layers II-III
interneurons). The horizontal dashed line indicates no
response to 5-HT. Hyperpolarizing responses fall below the
line; depolarizations are above the
line.
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5-HT effects on sIPSCs
We have described spontaneous synaptic currents in whole-cell
recordings of pyramidal cells (van Brederode et al., 2001). Most of
these currents are GABAA-mediated IPSCs (van
Brederode et al., 2001).
To test whether 5-HT2-mediated depolarization of
supragranular interneurons results in altered tonic inhibition of
pyramidal cells, we examined the effects of the selective
5-HT2 agonist DOI (10 µM, bath
applied) on sIPSCs in pyramidal cells from layers II and III (Fig.
7). We used whole-cell recordings with a
CsCH3SO4-based internal
solution (to block effects of postsynaptic
K+ conductances) and applied CNQX (10 µM) and AP5 (50 µM) to block AMPA receptor-
and NMDA receptor-mediated responses, respectively. This selects for
GABAA receptor-mediated events (van Brederode et
al., 2001).
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Figure 7.
5-HT2A receptor activation increased
the frequency of spontaneous IPSCs recorded in layer II pyramidal
neurons. A, Histogram of the inter-IPSC interval for a
single pyramidal cell as a function of time before, during, and after
bath application of 30 µM of the 5-HT2
agonist DOI (means ± SEM). DOI was present for the time indicated
by the black bar. The interevent interval reversibly
decreased in DOI (increased frequency of IPSCs). B,
Representative traces showing IPSCs from the initial
control period, in the presence of DOI, and control wash for the same
cell as in A. C, Cumulative frequency
plots for IPSC amplitude (left) or interval
(right) showing that in DOI the intervals decrease
(frequency increase) with no change in IPSC amplitude.
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We tested 14 cells with DOI (Fig. 7) at a holding potential of 60 mV.
Mean sIPSC amplitude did not change in DOI (Fig. 7C). In 9 of 14 cells, we found an increase in mean frequency of at least 5%
(range, 6-40%); there was a decrease in 1 cell, and no change in 4 cells. The initial control sIPSC frequency was 36 ± 4 Hz (17-73
Hz). In DOI, the mean frequency was significantly increased to 39 ± 4 Hz (19-73 Hz; p < 0.05; paired t
test). After a 10 min wash in control media, the mean frequency was
35 ± 11 Hz (13-75 Hz). We also calculated the percentage change
in frequency for each cell. For all 14 cells combined, the average
response was an increase in frequency of 11 ± 3%. In the nine
cells showing an increase, the average increase was 16 ± 4%.
Thus 5-HT2 receptor activation of interneurons
results in an increased frequency of tonic inhibition in pyramidal
neurons [see also Zhou and Hablitz (1999b)].
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DISCUSSION |
We determined the dominant effect of 5-HT on membrane potential in
different types of neocortical neurons. Previous work on cellular
effects of 5-HT concentrated on layer V pyramidal cells, which were
depolarized, hyperpolarized, or showed mixed responses to bath-applied
5-HT (Davies et al., 1987; Araneda and Andrade, 1991; Spain, 1994;
Marek and Aghajanian, 1998). Responses of supragranular pyramidal cells
to 5-HT had not been studied previously. We found that approximately
half of the superficial layers II-III pyramidal cells were depolarized
and approximately half were hyperpolarized by bath-applied 5-HT. Mixed
responses were not observed. In deep layers II-III, nearly all
pyramidal cells (90%) were depolarized. Additionally, the sAHP was
reduced and firing behavior was altered. Overall, these data indicate
that 5-HT variably affects pyramidal cell membrane potential and
facilitates responses to large suprathreshold depolarizations.
To obtain a broader perspective of the action of 5-HT on cortical
circuitry, we applied 5-HT to nonpyramidal cells. As with pyramidal
neurons, interneuronal responses to 5-HT were variable. Our main
finding was that the direction and amplitude of interneuron responses
were related to the laminar pattern of their axonal projections.
Hyperpolarizations
Our data suggest that interneuronal hyperpolarizations were caused
by activation of 5-HT1A receptors, on the basis
of mimicry of the hyperpolarization by 8-OHDPAT (specific
5-HT1A and 5-HT7 agonist)
(Middlemiss et al., 1986). Hyperpolarizations were not prevented by
5-HT2/5-HT7 antagonists
(Hoyer et al., 1994). This is consistent with findings in other cell
types (Andrade and Nicoll, 1987; Collino and Halliwell, 1987; Araneda
and Andrade, 1991; Schmitz et al., 1998). Immunocytochemistry and
in situ hybridization indicate that
5-HT1A receptors are expressed by interneurons
and pyramidal cells in supragranular layers of sensorimotor cortex (Pazos and Palacios, 1985; Morilak et al., 1993; Wright et al., 1995).
Reversal potentials and ion substitution experiments clearly implicate
K+ channels in the 5-HT-induced
hyperpolarizations of both interneurons and pyramidal neurons,
consistent with findings on layer V pyramidal neurons (Spain, 1994) and
other cell types (Collino and Halliwell, 1987). The lack of measurable
change in input conductance suggests that conductance changes are
small, obscured by other effects, or occur in a location remote from
the soma. In CA1 pyramidal neurons, 5-HT1A
receptors activate G-proteins
(Gi/Go subclass), which
interact with inwardly rectifying K+
channels (Andrade and Nicoll, 1987).
Depolarizations
With bath-applied 5-HT, depolarizations are at least partly
5-HT2 receptor mediated. This conclusion is based
on mimicry of the depolarization by a specific
5-HT2 agonist (DOI) and block of 5-HT-induced
depolarizations by specific 5-HT2 antagonists (ketanserin, ritanserin). The agents that we used do not allow us to
distinguish between 5-HT2A and
5-HT2C receptors. Both receptor types are present
in cortex (Pompeiano et al., 1994; Wright et al., 1995). In prefrontal
cortex, layer III was intensely stained for
5-HT2A receptors (Jakab and Goldman-Rakic, 1998).
5-HT2C receptors are sparsely distributed in
cortex (Molineaux et al., 1989). A role for other receptors
(cf. Gustafson et al., 1996; Beique and Andrade, 2001) requires further study.
In primate (Jakab and Goldman-Rakic, 1998) and rat (Willins et al.,
1997; Hamada et al., 1998) prefrontal cortex,
5-HT2A receptors are found on proximal apical
dendrites of pyramidal neurons. This region is hypothesized to be a
"hot spot" for 5-HT2A-mediated physiological
actions relevant to normal and "psychotic" states (Jakab and
Goldman-Rakic, 1998). 5-HT2A receptors are also
present on terminals and in a subset of interneurons (Jakab and
Goldman-Rakic, 1998; Vysokanov et al., 1998). In piriform cortex,
5-HT2A receptors are preferentially located on
nonpyramidal interneurons, which are depolarized by
5-HT2 receptor activation (Sheldon and
Aghajanian, 1991; Gellman and Aghajanian, 1994).
In hippocampal interneurons, fast depolarizations were induced by
activation of 5-HT3 receptors (Kawa, 1994;
McMahon and Kauer, 1997). In neocortex, 5-HT3
receptors are reportedly primarily expressed on a subset of
nonpyramidal cells [but see Edwards et al. (1990) and Morales and
Bloom (1997)]. 5-HT3 induced fast inward currents in a small percentage of layer I interneurons (Zhou and Hablitz, 1999b) and cultured neocortical neurons (Matsuoka et al.,
1997). 5-HT3 receptor activation may depolarize a
subset of interneurons in layer V (Williams et al., 1999; Xiang et al., 1999). We observed one positive response to a limited number of direct
(n = 1 of 13) or bath applications (n = 3) of a 5-HT3 agonist. Because we did not
systematically test direct applications of higher doses of
5-HT3 agonists, and bath application is likely to
cause significant desensitization of 5-HT3
receptors [Jackson and Yakel (1995); but see Roerig et al. (1997)],
we do not consider the role of 5-HT3 receptors to
be adequately tested.
Laminar and cell type comparisons
In layer I, 5-HT depolarized or hyperpolarized cells with similar
frequency. An interesting pattern emerged from the most commonly
recorded neuron types: horizontal axon cells and vertical axon cells.
The principal effect of 5-HT on horizontal axon cells was
hyperpolarization. This could reduce tonic inhibition and selectively disinhibit responses of pyramidal cells to distal inputs. The horizontal extent of the axonal arbors suggests that this effect would
influence a large area (e.g., multiple columns). Such disinhibition could also reduce the independence of Ca2+
dynamics in distal dendrites (e.g., apical tuft) of layer V pyramidal cells versus the soma/proximal dendrite (Yuste et al., 1994; Callaway and Ross, 1995; Schiller et al., 1997). Alternatively, the distal dendritic responses to GABAA activation may be
depolarizing (Owens et al., 1996; Cerne and Spain, 1997; Mienville,
1998). Serotonergic reduction in such inputs would then decrease
pyramidal cell excitability.
Most layer I vertical axon cells were depolarized by 5-HT. Axonal
anatomy suggests that these cells could inhibit pyramidal cell
dendrites proximally, within a more local area than horizontal axon
cells (e.g., within column), suggesting possible differences between
serotonergic effects on inter-column and intra-column inhibition. 5-HT
would thus increase tonic inhibition of nearby pyramidal cells.
Most layers II-III nonpyramidal cells, including RS and FS cells, are
depolarized by 5-HT. Our major finding is that interneurons whose axons
terminate in layers II-III (layers II-III interneurons, layer I
vertical axon) form a functional group. Depolarization of these cells
would increase tonic inhibition of pyramidal cells, thereby reducing
repetitive firing (Kim et al., 1995; van Brederode and Spain, 1995),
responses to excitatory inputs, and backpropagation of APs
(Tsubokawa and Ross, 1996). This could bias responsiveness of
pyramidal neurons to strong versus weak stimuli. Somatic and proximal
dendritic inputs have also been proposed to control rhythmicity of
local circuitry in cortex (Somogyi et al., 1998; Whittington et al.,
2000). Serotonergic depolarization of interneurons providing proximal
inputs to pyramidal cells (e.g., basket and chandelier cells) (Somogyi
et al., 1998) may therefore play a more complicated role than direct
control of membrane excitability.
Synaptic transmission
DOI application increases GABA release in cortex, suggesting
excitation of GABAergic interneurons via 5-HT2
receptors (Abi-Saab et al., 1999). We confirmed that the
frequency of sIPSCs on pyramidal neurons was increased by
5HT2 activation (Zhou and Hablitz, 1999b). Zhou
and Hablitz (1999b) further suggest that the effects of 5-HT are
dependent on AP-induced GABA release, because miniature IPSCs (in the
presence of TTX) were not modulated by 5-HT. The
5-HT2-induced increase in spontaneous IPSCs on
pyramidal neurons should result in increased tonic inhibition, reducing
overall network excitability. Williams et al. (2002) suggested that
increased feedforward inhibition in response to activation of
5HT2 receptors on FS interneurons might enhance
spatial tuning of working memory in primate prefrontal cortex.
5-HT increased the frequency of sEPSCs recorded in neocortical
pyramidal cells (layer V >> II/) (Zhou and Hablitz, 1999b; Aghajanian and Marek, 2000; Lambe et al., 2000; Beique and Andrade, 2001) [but see Newberry et al. (1999)] and hippocampus (Ropert and
Guy, 1991; Shen and Andrade, 1998). Zhou and Hablitz (1999b) also suggest a temporal variable to 5-HT modulation of cortical circuitry: 5-HT2A activation initially causes
increase in the frequency of sIPSCs, which then desensitizes. The
enhancement of sEPSCs is weaker but longer lasting. Thus, short epochs
of 5-HT release would lead to increased inhibition of cortical
circuitry, but prolonged 5-HT presence would facilitate this circuitry.
We did not observe biphasic responses, but we did not systematically examine responses to long applications of DOI.
Function
5-HT2A receptors on apical dendrites of
pyramidal neurons may act to gate incoming signals (Jakab and
Goldman-Rakic, 1998). A proper balance of 5-HT activation would be
required in prefrontal cortex to facilitate working memory and latent
inhibition. Too much activation of these receptors would prevent
pyramidal cells from shutting down, possibly leading to the
schizophrenic state (Abi-Dargham et al., 1997). The present results
suggest that this balance must also include the differential laminar
modulation of GABAergic inhibition by 5-HT. Interestingly, increased
levels of GABAA receptors are reported in
superficial layers of cingulate cortex in schizophrenic patients (Benes
et al., 1992).
5-HT2A receptors may be the site of action of
hallucinogens and antipsychotic drugs (Jakab and Goldman-Rakic, 1998;
Aghajanian and Marek, 1999). The preponderance of serotonergic
excitation of interneurons and consequent tonic inhibition of pyramidal
cells would also suppress seizure generation [genetically
epilepsy-prone rats have a low density of serotonergic projections to
forebrain (Jobe, 1973; Statnick et al., 1996)].
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FOOTNOTES |
Received April 12, 2002; revised June 4, 2002; accepted June 13, 2002.
This work was supported by a Veterans Affairs Merit Award
(W.J.S), National Institutes of Health Grant NS34769 (J.F.M.v.B.), and
National Institute of Neurological Disorders and Stroke Grant NS 33579 (R.C.F.). We thank Richard Lee for excellent technical assistance and
Dr. W. Armstrong for reading an earlier version of this manuscript.
Correspondence should be addressed to Dr. Robert C. Foehring,
Department of Anatomy and Neurobiology, University of Tennessee, Memphis, 855 Monroe Avenue, Memphis, TN 38163. E-mail:
foehring{at}nb.utmem.edu |
TOP
ABSTRACT
INTRODUCTION
