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The Journal of Neuroscience, September 1, 1998, 18(17):6963-6976
Noradrenergic Excitation and Inhibition of GABAergic Cell Types in Rat Frontal Cortex
Yasuo Kawaguchi and Tomomi Shindou Laboratory for Neural Circuits, Bio-Mimetic Control Research Center, The Institute of Physical and Chemical Research (RIKEN), Shimoshidami, Moriyama, Nagoya 463-0003, Japan
ABSTRACT
Top
Abstract
Introduction
Noradrenaline (NA) from the locus coeruleus and GABA from intracortical nonpyramidal cells exert strong influences on cortical activity. To assess possible interaction between the two, the effects of noradrenergic agonists on spontaneous GABAergic IPSCs as well as on the activity of identified GABAergic cell types were investigated by in vitro whole-cell recordings from the frontal cortex of 18- to 22-d-old rats. NA (3-50 µM) and an
-adrenergic agonist, 6-fluoronorepinephrine (FNE; 30-50 µM), induced an increase of IPSC frequency in pyramidal cells, but a
-adrenergic agonist did not. This increase was reduced by tetrodotoxin, bicuculline, and
Key words: noradrenaline; frontal cortex; nonpyramidal cell; cholecystokinin; somatostatin; GABA;
INTRODUCTION
In the neocortex, noradrenaline (NA) and acetylcholine (ACh) are released, respectively, from afferent fibers originating in noradrenergic cells in the locus coeruleus and cholinergic cells in the nucleus basalis of the basal forebrain. NA and ACh may diffuse through the extracellular space and mediate their effects at sites beyond the synaptic cleft (Séguéla et al., 1990
; Umbriaco et al., 1994
Application of NA usually increases the excitability of pyramidal cells, with reduction of spike frequency adaptation and slow depolarization (Haas and Konnerth, 1983
The activity of cortical GABAergic nonpyramidal cells is also modulated by NA or adrenaline; both cause increases in the frequency of IPSPs or IPSCs in pyramidal cells of the hippocampus, piriform cortex, and somatosensory cortex (Madison and Nicoll, 1988
Like NA, ACh also induces IPSPs or IPSCs in pyramidal cells indirectly through excitation of cortical GABAergic cells in the neocortex (McCormick and Prince, 1986
MATERIALS AND METHODS
Slice preparation and whole-cell recording. The experiments were performed on young Wistar rats (18-22 d postnatal). Animals were deeply anesthetized with ether and decapitated. The brains were quickly removed and submerged in ice-cold physiological Ringer's solution. Sections of frontal cortex (200 µm thick) were cut, immersed in a buffered solution containing (in mM) NaCl 124.0, KCl 3.0, CaCl2 2.4, MgCl2 1.2, NaHCO3 26.0, NaH2PO4 1.0, glucose 10.0, and aerated with a mixture of 95% O2 and 5% CO2. Cells in the frontal cortex (medial agranular cortex and anterior cingulate cortex) were recorded in a whole-cell mode at 30°C using a 40× water immersion objective.
Voltage-clamp recordings from pyramidal cells. The electrode solution for the voltage-clamp recording consisted of (in mM): cesium methanesulfonate 120, KCl 5.0, EGTA 10.0, CaCl2 1.0, MgCl2 2.0, ATP 4.0, GTP 0.3, HEPES 8, QX314 5.0, and biocytin 20. The quaternary lidocaine derivative QX314 (Astra, Westborough, MA) was included to suppress fast sodium currents. Voltage-clamp recordings were made with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Spontaneous postsynaptic currents were collected and analyzed using Spike 2 software (Cambridge Electronic Design, Cambridge, UK).
Current-clamp recordings from nonpyramidal cells. The electrode solution for the current-clamp recording consisted of (in mM): potassium methylsulfate 115, KCl 5.0, EGTA 0.5, MgCl2 1.7, ATP 4.0, GTP 0.3, HEPES 8.5, and biocytin 17. Current-clamp recordings were made in the bridge mode with an Axoclamp-2B amplifier (Axon Instruments). Voltage responses to current pulses were collected using Axodata software (Axon Instruments) and analyzed with Axograph software (Axon Instruments). Continuous voltage signals were collected and analyzed with Spike 2 software. Resting potentials were measured just after the patched membranes were ruptured by suction. Input resistances of cells were determined by passing hyperpolarizing current pulses (duration, 500-600 msec), which induced voltage shifts of 6-15 mV negative to rest. The generation of two or more spikes on slow humps from hyperpolarization (burst-spiking) was investigated by depolarizing current pulses of threshold strength from
75 to
Drugs were applied by replacing the solution superfusing the slice with one containing a set concentration. D-2-amino-5-phosphonovaleric acid (APV; 50 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10-20 µM) were obtained from Tocris (Bristol, UK); tetrodotoxin (TTX; 0.3-1 µM) was from Sankyo (Tokyo, Japan); NA (3-50 µM) was obtained from Wako (Osaka, Japan); (
Antibodies. A monoclonal antibody raised against gastrin/CCK (28.2 MoAb) was provided by CURE/UCLA/DDC Antibody/RIA Core. For control it was preabsorbed with an excess (10
6 M) of sulfated CCK-octapeptide (Peptide Inst. Inc., Japan), resulting in no staining in the rat frontal cortex. A rat monoclonal antibody against somatostatin (MAB354) was purchased from Chemicon (Temecula, CA). Preabsorption with an excess (10
It was confirmed by double immunofluorescence that the cellular distributions of neurochemical markers (parvalbumin, somatostatin, CCK, and VIP) of nonpyramidal cells in frontal cortex of 19- to 21-d-old rats were similar to those of adult rats. In the 19- to 21-d-old animal, parvalbumin-positive cells (n = 2279) and somatostatin-positive cells (n = 1218) were always singly stained with no cross-immunoreactivity. Similarly parvalbumin-positive (n = 2237) and CCK-positive cells (n = 284) were clearly separable, as were parvalbumin-positive (n = 1485) and VIP-positive cells (n = 469), somatostatin-positive (n = 1642) and CCK-positive cells (n = 311), and somatostatin-positive (n = 780) and VIP-positive cells (n = 595). CCK-positive cells (n = 146 in layers II/III, 107 in layer V, and 100 in layer VI) were sometimes positive for VIP (58% in layers II/III, 64% in layer V, and 40% in layer VI). Similarly VIP-positive cells (n = 412 in layers II/III, 277 in layer V, and 205 in layer VI) were occasionally positive for CCK (21% in layers II/III, 25% in layer V, and 20% in layer VI). These observations were in line with the literature and thus showed these chemical markers to be distributed similarly in the frontal cortex of both 19- to 21-d-old and adult rats (Kubota et al., 1994
Histological procedures for immersion-fixed slices. Tissue slices containing biocytin-loaded cells were fixed by immersion in 4% paraformaldehyde and 0.2% picric acid in 0.1 M sodium phosphate buffer (PB), pH 7.4, overnight at 4°C, and incubated in PB containing 10% sucrose for 30 min and 20% sucrose for 1 hr. The tissue was next frozen on dry ice, thawed twice, and incubated in PB containing 0.5% H2O2 for 30 min to suppress endogenous peroxidase activity. The slices, without resectioning, were then washed with 0.05 M Tris-HCl-buffered saline (TBS), pH 7.6, for 1 hr. Each slice was further treated by one of the following two procedures.
(1) After they were washed in TBS, some slices were incubated with avidin-biotin-peroxidase complex (1:100; Vector, Burlingame, CA) in TBS containing 0.1% Triton X-100 (TX) for 4 hr at room temperature. After they were washed in TBS, the slices were reacted with 3,3'-diaminobenzidine tetrahydrochloride (DAB) (0.05%) and H2O2 (0.003%) in Tris-HCl buffer.
(2) The other slices were processed for double-fluorescence immunohistochemistry. All staining procedures were performed at room temperature. The slices were incubated overnight with the mouse monoclonal antibody against gastrin/CCK (diluted 1:2000) and the rat monoclonal antibody against somatostatin (1:500) in TBS containing 2% bovine serum albumin (BSA), 10% normal goat serum (NGS), and 0.5% TX. After the slices were washed in TBS, they were incubated in a mixture of dichlorotriazinyl amino fluorescein (DTAF)-conjugated donkey anti-mouse IgG (1:100; 192F, Chemicon) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rat IgG (1:100; 183R, Chemicon) in TBS containing BSA, NGS, and TX for 4 hr, followed by incubation with a sulfonated derivative of 7-amino-4-metylcoumarin-3-acetic acid (AMCA-S)-conjugated streptavidin (1:600; S-6364, Molecular Probes, Eugene, OR) in TBS for 2 hr. After they were washed in TBS, the sections were coverslipped in 50% glycerin in TBS, observed, and photographed with a fluorescence microscope (BX-50, Olympus) using filters (Olympus): U-MNUA (360-370 nm exciter and 420-460 nm emitter) for AMCA-S to identify biocytin-labeled cells, U-MNIBA (470-490 nm exciter and 515-550 nm emitter) for DTAF, and U-MWIG (520-550 nm exciter and >580 nm emitter) for TRITC. Only one color was visualized with each filter combination. Cross-reactivity of the secondary antibodies was not observed. After examination for fluorescence, the slices were incubated with avidin-biotin-peroxidase complex in TBS and reacted with DAB and H2O2 in Tris-HCl buffer.
In both cases, the slices were washed in TBS, mounted on gelatin-coated glass slides, and dried. They were then immersed in PB containing 0.1% osmium tetroxide for 10 min, dehydrated, and embedded in Epon (TAAB, Berkshire, UK). The dendrites, axonal processes, and somata of the biocytin-labeled neurons were drawn using a camera lucida with a 60× or 100× oil-immersion lens. Terminal boutons were observed with differential interference contrast. The cross-sectional area and the longest extent of somata were measured in the 200 µm sections containing whole stained cells using a computer-based image analysis system (National Institutes of Health Image).
Statistics. Paired t tests were performed to compare the raw values of the control with the responses in the presence of adrenergic agonists applied to the same cells. Responses to agonists were also expressed as a percentage of the control obtained before the addition of the agonists. The effects of agonists, expressed as percentages, in the presence or absence of antagonists (two different groups of cells) were compared using Mann-Whitney U tests. The effects of agonists in the presence or absence of TTX (two different groups of cells) were also compared using Mann-Whitney U tests. The tests determined whether the decrease in the effect of the agonist was significant.
RESULTS
NA-induced increase of IPSC frequency in pyramidal cells
To monitor the activity of GABAergic cells in the frontal cortex, spontaneous outward-going currents were recorded at holding potentials of ~0 mV in pyramidal cells in a solution containing blockers of excitatory transmission (10-20 µM CNQX and 50 µM APV) (Fig. 1). Because these outward currents were reversibly suppressed by the GABAA-receptor antagonist bicuculline (10 µM), they were considered to be GABAergic IPSCs (Salin and Prince, 1996
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Figure 1. Spontaneous GABAergic postsynaptic currents in layer V pyramidal cells. Outward currents were recorded in pyramidal cells at a holding potential (Vh) of 0 mV in a solution containing antagonists for non-NMDA and NMDA receptors (20 µM CNQX and 50 µM APV) (A, B). These outward currents were suppressed by an antagonist for GABAA receptors (10 µM bicuculline) (A, C) and recovered after washing (A, D).
NA (3-50 µM; n = 25) induced a reversible increase in the frequency and amplitude of GABAergic currents in a solution containing CNQX and APV (CNQX/APV) (Fig. 2). The NA-induced currents in a solution containing CNQX/APV were suppressed by TTX (Fig. 3A). GABAergic IPSCs larger than 30 pA were counted for measurement of the frequency over a period of 30-120 sec. The frequency of spontaneous IPSCs in layer V pyramidal cells (n = 7) was 2.89 ± 1.26/sec (mean ± SD) before the application, and 5.73 ± 2.06/sec after the application of 10 µM NA [p < 0.01, paired t test; 212 ± 82% (SD) after application]. The frequency increase of IPSCs was also significant after application of 30 and 50 µM NA (p < 0.05) (Fig. 3B). Prior application of TTX (0.5 µM; n = 4) reduced the NA (10 µM)-induced increase of IPSC frequency to 126 ± 12% (p < 0.05, Mann-Whitney U test) (Fig. 3B).
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Figure 2. Noradrenaline (NA)-induced increase of spontaneous IPSCs. A, Spontaneous IPSCs in a layer V pyramidal cell under control conditions (a) and in the presence of 10 µM NA (b). B, The amplitude and frequency of spontaneous IPSCs were increased by application of NA in a solution containing 10 µM CNQX and 50 µM APV (CNQX/APV). The currents at the black bars (a) and (b) are shown in A. Each bin in the histogram represents the frequency (Hz) of the spontaneous IPSCs for 10 sec.
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Figure 3. Reduction of NA-induced synaptic currents by tetrodotoxin (TTX). A, NA-induced increase of GABAergic currents was not observed in a solution containing TTX (0.5 µM) and CNQX/APV. B, Dose dependency of NA (3-50 µM)-induced increase of IPSC frequency and its reduction by TTX. The effects of NA are normalized as a percentage of control values. The error bars represent SEM. The numbers of cells investigated are given in parentheses. The effects of NA (10 µM) in the presence or absence of TTX were compared using Mann-Whitney U test. *p < 0.05.
Involvement of
The above results suggest that NA-induced IPSCs are caused by firing of GABAergic cells to a certain extent. Adrenoceptors are pharmacologically divided into
The IPSC frequency of pyramidal cells in a solution containing CNQX/APV increased after application of a selective
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Figure 4. Increase of spontaneous IPSCs induced by an
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Figure 5. Reduction of FNE-induced currents by TTX and bicuculline. A, FNE (50 µM)-induced synaptic currents were reduced by TTX (1 µM) application. The solution always contained CNQX/APV. B, Both spontaneously occurring and FNE (50 µM)-induced outward currents were completely suppressed by simultaneous application of the GABAA receptor antagonist bicuculline (100 µM). The solution always contained CNQX/APV. C, Dose dependency of FNE (10-50 µM)-induced increase of IPSC frequency and its reduction by TTX. The data are percentages of the control value. The error bars represent SEM. The numbers of cells examined are given in parentheses. The effects of FNE (50 µM) in the presence or absence of TTX (0.3 µM) were compared using Mann-Whitney U test. **p < 0.01.
The FNE-induced IPSCs were also blocked immediately by bicuculline (Fig. 5B). In addition, bicuculline abolished discrete GABAergic outward currents and induced inward currents (Fig. 1) (Salin and Prince, 1996
The frequency of spontaneous IPSCs in layer V pyramidal cells (n = 11) was increased from 4.82 ± 2.57/sec (SD) before the application to 8.8 ± 5.12/sec after the application of 50 µM FNE [p < 0.01, paired t test; 188 ± 51% (SD) after application]. A significant frequency increase was also observed with application of 30 µM FNE (p < 0.01) (Fig. 5C). Prior application of TTX (0.3 µM; n = 5) reduced the FNE (50 µM)-induced increase to 115 ± 26% (p < 0.01, Mann-Whitney U test) (Fig. 5C).
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Figure 6. Pharmacological characterization of NA-induced GABAergic currents. A, An
An
Effects of NA and an
The above results indicated that some GABAergic cells were excited by NA via
GABAergic nonpyramidal cells in the rat frontal cortex can be divided into three main physiological classes with regard to their firing patterns in response to depolarizing current pulses (Kawaguchi and Kubota, 1996
In the following experiments, each cell was physiologically identified as one of the above physiological classes before application of NA or FNE. NA or FNE was applied in solutions containing 10-20 µM CNQX and 50 µM APV (CNQX/APV), 0.5 µM TTX, or both. Furthermore, RS/BSNP cells were neurochemically identified as positive for somatostatin or CCK by double-fluorescence immunohistochemistry after fixation (Fig. 7). We also investigated morphological characteristics of the FS, LS, somatostatin RS/BSNP, and CCK RS/BSNP cells, whose responses to NA or FNE had been determined. Three types of membrane potential change were observed in GABAergic cells by NA or FNE application: (1) depolarization, (2) hyperpolarization followed by depolarization, and (3) hyperpolarization.
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Figure 7. Immunohistochemical identification of regular/burst-spiking nonpyramidal cells, in which the effects of NA or FNE were investigated. A, B, A biocytin-injected cell (A) immunoreactive for somatostatin (SOM) (arrow in B). C, D, A biocytin-injected cell (C) immunoreactive for cholecystokinin (CCK) (arrow in D). Scale bar, 50 µm.
FS cells
In response to depolarizing current pulses, FS cells started nonadapting repetitive discharges abruptly above some current intensity, and continuous repetitive firing was easily achieved when combined with constant depolarizations (Fig. 8A).
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Figure 8. Depolarization of fast-spiking (FS) cells by application of NA or FNE. A, Spike discharges of an FS cell induced by current pulses. Resting potentials,
FS cells (n = 25 in layers II/III, n = 11 in layer V) were all depolarized by NA or FNE application (Fig. 8B,C, Table 1). In a CNQX/APV-containing solution, 28 FS cells were depolarized by 10 µM NA [5.8 ± 2.5 mV (SD); n = 4], 50 µM NA (6.0 ± 1.8 mV; n = 5), or 50 µM FNE (5.5 ± 1.5 mV; n = 19), but no spike firing was noted (Table 1). In a TTX-containing solution, FNE (10-50 µM) also depolarized FS cells (n = 8) by 3-7 mV (Table 2). The FS cells depolarized by NA or FNE were morphologically common multipolar cells with extended axonal arborizations (see Fig. 10A-C) and chandelier cells with vertical rows of axonal boutons (see Fig. 10D). Some terminals of common multipolar FS cells were apposed to other somata by multiple boutons. Axonal arborizations of the common FS cells were dense just above or around their somata (see Fig. 10A,C). The axon collaterals of some FS cells did not branch so frequently close to the somata as the above FS cells and ran more widely (see Fig. 10B).
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Table 1. Actions of noradrenaline (NA) or 6-fluoronorepinephrine (FNE) on identified GABAergic cell types
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Table 2. Amplitude of responses induced by 6-fluoronorepinephrine (FNE) in identified GABAergic cell types LS cells
LS cells were identified by their ramp-like depolarizing response to a square-wave current injection of 0.5-1.0 sec duration, which induced a spike at the end of a pulse (Fig. 9A).
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Figure 9. Noradrenergic depolarization of a late-spiking (LS) neurogliaform cell. A1,2, Voltage responses of an LS cell induced by current pulses. Note the ramp-like depolarizing response to a square-wave current injection (A1). Resting potential,
Like FS cells, LS cells (n = 7 in layers II/III) were all depolarized by NA or FNE application (Fig. 9B, Table 1). In a CNQX/APV-containing solution, six LS cells were depolarized by 10 µM NA [6.2 ± 2.9 mV (SD); n = 3] or 50 µM NA (6.0 ± 1.0 mV; n = 3), but none fired spikes on the induced depolarizations (Table 1). An LS cell was depolarized by 50 µM FNE in a TTX-containing solution (Table 2). The LS cells depolarized by NA or FNE included morphologically neurogliaform cells (Fig. 10E).
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Figure 10. Four FS cells and an LS cell that were all depolarized by NA or FNE application in a solution containing CNQX/APV, TTX, or both. The somata and dendrites are shown in black and the axons in gray. Roman numerals correspond to the cortical layers. A, FS cell with dense innervation mainly above the cell body. B, FS cell with wide innervation. C, FS cell with dense innervation mainly at the level of the cell body. D, FS chandelier cell. E, LS neurogliaform cell.
Somatostatin RS/BSNP cells
Somatostatin or CCK cells were of BSNP or RSNP type. BSNP cells fired two or more spikes on slow depolarizing humps from hyperpolarized potentials (Fig. 11A). RSNP cells did not show fast-spiking characteristics, spike bursts on depolarization from negative membrane potentials, or ramp-like depolarizing responses before spike generation (Fig. 11B). Nonpyramidal cells not categorized into one of the FS, LS, and BSNP groups were categorized as RSNP cells.
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Figure 11. Burst-spiking nonpyramidal (BSNP) cells and regular-spiking nonpyramidal (RSNP) cells affected by application of NA or FNE. A, Voltage responses of a BSNP cell immunoreactive for CCK. Four action potentials on an all-or-none slow depolarizing potential were induced by a depolarizing current pulse from a hyperpolarized potential (
NA or FNE was applied to 21 somatostatin RSNP cells (n = 20 in layers II/III, n = 1 in layer V) and 3 somatostatin BSNP cells (n = 1 in layers II/III, n = 2 in layer V) (Table 1).
Most somatostatin cells (19 RSNP and 2 BSNP cells) were depolarized by NA or FNE application (Fig. 12A,B, Table 1). In a CNQX/APV-containing solution, 12 somatostatin cells were depolarized by 10 µM NA (n = 3), 50 µM NA (n = 1), 10 µM FNE (n = 4), or 30 µM FNE (n = 4), and 11 cells fired spikes on the induced depolarization (Fig. 12A). In a TTX-containing solution, nine somatostatin cells were also depolarized by 10-30 µM FNE (Fig. 12B, Table 2).
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Figure 12. Noradrenergic effects on somatostatin-immunoreactive cells. A, A somatostatin cell was depolarized with spike discharges by NA (10 µM) application in a solution containing CNQX/APV. B, A somatostatin cell was depolarized by FNE (10 µM) application in a solution containing TTX and CNQX/APV. C, A somatostatin cell was minimally hyperpolarized (arrow) followed by depolarization with spike discharges by NA (10 µM) application in a solution containing CNQX/APV.
A few somatostatin cells (two RSNP and one BSNP) exhibited NA- or FNE-induced hyperpolarization followed by depolarization (Table 1). By application of 10 µM NA in a CNQX/APV-containing solution, a somatostatin cell elicited spikes on the depolarization after weak hyperpolarization (
By application of NA or FNE in a solution containing CNQX/APV, but not TTX, 92% of somatostatin RSNP cells elicited spikes (Table 1).
These somatostatin cells morphologically included multipolar or bitufted cells with mainly ascending axonal arbors (Martinotti cells; see Fig. 14A) or with axonal arbors extending in all orientations (wide arbor cells; see Fig. 14B).
CCK RS/BSNP cells
NA or FNE was applied to 22 CCK RSNP cells (n = 21 in layers II/III, n = 1 in layer V) and 4 CCK BSNP cells (n = 4 in layers II/III) (Table 1). From the somatic size, these cells appeared to belong to the large CCK cell category (Kubota and Kawaguchi, 1997
Seventeen CCK cells (13 RSNP and 4 BSNP cells) were depolarized by NA or FNE application (Fig. 13A, Table 1). In a CNQX/APV-containing solution, 14 CCK cells were depolarized by 10 µM NA (n = 9), 50 µM NA (n = 2), 10 µM FNE (n = 1), or 30 µM FNE (n = 2), accompanied by spike firing in six cases (Fig. 13A). In a TTX-containing solution, three CCK cells were also depolarized by 10 µM FNE (n = 2) or 30 µM FNE (n = 1) (Table 2).
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Figure 13. Noradrenergic effects on CCK-immunoreactive cells. A, A CCK cell was depolarized with spike discharges by NA (10 µM) application in a solution containing CNQX/APV. B, A CCK cell demonstrated hyperpolarization followed by depolarization with spike discharges after NA (10 µM) application in a solution containing CNQX/APV. C, A CCK cell was hyperpolarized and then depolarized by FNE (10 µM) application in a solution containing TTX and CNQX/APV. D, A CCK cell was hyperpolarized by FNE (10 µM) application in a solution containing TTX and CNQX/APV.
Seven CCK RSNP cells exhibited hyperpolarization followed by depolarization by application of NA or FNE (Fig. 13B,C, Table 1). In a CNQX/APV-containing solution, five CCK cells were hyperpolarized and later depolarized by NA or FNE. The early hyperpolarization was
Two CCK RSNP cells were only hyperpolarized by application of 10 or 50 µM FNE in a TTX-containing solution (Fig. 13D, Tables 1, 2).
By application of NA or FNE in a solution containing CNQX/APV, but not TTX, 40% of CCK RSNP cells and 50% of CCK BSNP cells elicited spikes (Table 1).
These CCK cells morphologically included large multipolar or bitufted cells making boutons apposed to other cell bodies (large basket cells) (Fig. 14C). Morphological differences among the three types of CCK cells with different adrenergic responses could not be found.
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Figure 14. Two somatostatin cells and a CCK cell affected by NA or FNE application. The somata and dendrites are shown in black and the axons in gray. A, A somatostatin cell with ascending axonal arbors. This neuron was depolarized by FNE (10 µM) application in a solution containing TTX. B, A somatostatin cell with wide axonal arbors. This neuron was depolarized with spike discharges by FNE (30 µM) application in a solution containing CNQX/APV. C, A large CCK cell with axonal arbors making multiple boutons on other cell bodies. This neuron was hyperpolarized and then demonstrated depolarization with spike discharges on FNE (10 µM) application in a solution containing CNQX/APV.
DISCUSSION
The major findings of the present study in the frontal cortex of young rats can be summarized as follows. (1) NA or a selective
Noradrenergic increase of GABAergic IPSCs
Adrenergic induction of IPSCs has been observed in several regions. The frequency of spontaneous IPSCs is increased by adrenaline in the sensorimotor cortex (Bennett et al., 1998
Iontophoretically applied NA in vivo decreases or enhances the spontaneous activity and the activity evoked by sensory stimuli of cortical cells (Bevan et al., 1977
Excitation and inhibition of GABAergic cells via
In the present study, most GABAergic cells were depolarized by NA or FNE application, but some CCK-containing cells and a few somatostatin-containing cells exhibited hyperpolarization preceding depolarization. A few CCK cells were only hyperpolarized. Responses of small VIP cells to NA application were not investigated. Because most somatostatin cells fired spikes on the NA-induced depolarization, these GABAergic cells are considered to contribute to NA-induced increase of spontaneous IPSC in vitro slice preparations. Some CCK cells may also contribute to NA-induced induction of IPSCs in pyramidal cells.
Because these induced depolarizations or hyperpolarizations were observed in both TTX- and CNQX/APV-containing solutions, the responses are most likely direct actions of NA or FNE on GABAergic cells. However, NA or FNE may affect the membrane potentials of GABAergic cells through the change of tonic inhibitions attributable to the action potential-independent release of GABA because bicuculline was not included in the solutions. The TTX-insensitive tonic GABAergic currents were observed in pyramidal cells (Figs. 1, 3A, 5A,B).
Activation of
NA is considered to be released diffusely to the extracellular space from the noradrenergic axons (Séguéla et al., 1990
Adrenergic modulation of GABAergic inhibition on pyramidal cells
In the present study, application of NA increased spontaneous GABAA receptor-mediated IPSCs through excitation of GABAergic cells, which may cause tonic shunting inhibition in pyramidal cells. Different from spontaneous IPSCs, evoked IPSCs in pyramidal cells by electrical stimulation of the sensorimotor cortex are depressed, enhanced, or not affected by adrenoceptor activation, although spontaneous IPSC frequency is increased in all cells (Bennett et al., 1997
Differential monoaminergic and cholinergic modulation among GABAergic cell types
Like NA action through
In addition to NA, there are other monoaminergic ascending systems such as serotonin (5-HT), histamine, and dopamine, which are also considered to control the pattern of activity and level of excitability in the cortex (Nicoll et al., 1990
Conclusions
GABAergic cell types have been identified in the rat frontal cortex on the basis of their firing responses to a depolarizing current, axon arborization patterns, synaptic connections, and coexpression of neuroactive substances (Kawaguchi and Kubota, 1997
FOOTNOTES Received March 3, 1998; revised June 5, 1998; accepted June 9, 1998.
This work was supported by the Frontier Research Program, RIKEN, and Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture. The authors thank N. Wada and S. Kato for technical assistance. We are grateful to Dr. Robert Benoit for an antiserum against somatostatin 28. Antibody 28.2 MoAb raised against gastrin/CCK was provided by CURE/UCLA/DDC Antibody/RIA Core.
Correspondence should be addressed to Dr. Yasuo Kawaguchi, Bio-Mimetic Control Research Center, RIKEN, 2271-130 Anagahora, Shimoshidami, Moriyama, Nagoya 463-0003, Japan.
Dr. Shindou's present address: Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo, Sunto-gun, Shizuoka, 411-0943, Japan.
REFERENCES
- Alreja M, Liu W (1996) Noradrenaline induces IPSCs in rat medial septal/diagonal band neurons: involvement of septohippocampal GABAergic neurons. J Physiol (Lond) 494:201-215[ISI][Medline].
- Armstrong-James M, Fox K (1983) Effects of ionophoresed noradrenaline on the spontaneous activity of neurones in rat primary somatosensory cortex. J Physiol (Lond) 335:427-447
[Abstract/Free Full Text] .- Aston-Jones G, Chiang C, Alexinsky T (1991) Discharge of noradrenergic locus coeruleus neurons in behaving rats and monkeys suggests a role in vigilance. In: Progress in brain research, Vol 88, Neurobiology of the locus coeruleus (Barnes CD, Pompeiano O, eds), pp 501-520. Amsterdam: Elsevier.
- Bassant MH, Ennouri K, Lamour Y (1990) Effects of iontophoretically applied monoamines on somatosensory cortical neurons of unanesthetized rats. Neuroscience 39:431-439[ISI][Medline].
- Behrends JC, ten Bruggencate G (1993) Cholinergic modulation of synaptic inhibition in the guinea pig hippocampus in vitro: excitation of GABAergic interneurons and inhibition of GABA-release. J Neurophysiol 69:626-629
[Abstract/Free Full Text] .- Benardo LS (1993) Characterization of cholinergic and noradrenergic slow excitatory postsynaptic potentials from rat cerebral cortical neurons. Neuroscience 53:11-22[ISI][Medline].
- Bennett BD, Huguenard JR, Prince DA (1997) Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABAB receptors in rat sensorimotor cortex. J Neurophysiol 78:561-566
[Abstract/Free Full Text] .- Bennett BD, Huguenard JR, Prince DA (1998) Adrenergic modulation of GABAA receptor-mediated inhibition in rat sensorimotor cortex. J Neurophysiol 79:937-946
[Abstract/Free Full Text] .- Bergles DE, Doze VA, Madison DV, Smith SJ (1996) Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurons. J Neurosci 16:572-585
[Abstract/Free Full Text] .- Berridge CW, Foote SL (1991) Effects of locus coeruleus activation on electroencephalographic activity in neocortex and hippocampus. J Neurosci 11:3135-3145[Abstract].
- Bevan P, Bradshaw CM, Szabadi E (1977) The pharmacology of adrenergic neuronal responses in the cerebral cortex: evidence for excitatory alpha- and inhibitory beta-receptors. Br J Pharmacol 59:635-641[ISI][Medline].
- DeFelipe J, Hendry SHC, Hashikawa T, Jones EG (1991) Synaptic relationships of serotonin-immunoreactive terminal baskets on GABA neurons in the cat auditory cortex. Cereb Cortex 1:117-133
[Abstract/Free Full Text] .- Demeulemeester H, Vandesande F, Orban GA, Brandon C, Vanderhaeghen JJ (1988) Heterogeneity of GABAergic cells in cat visual cortex. J Neurosci 8:988-1000[Abstract].
- Dodt HU, Pawelzik H, Zieglansberger W (1991) Actions of noradrenaline on neocortical neurons in vitro. Brain Res 545:307-311[ISI][Medline].
- Doze VA, Cohen GA, Madison DV (1991) Synaptic localization of adrenergic disinhibition in the rat hippocampus. Neuron 6:889-900[ISI][Medline].
- Foehring RC, Schwindt PC, Crill WE (1989) Norepinephrine selectively reduces slow Ca2+- and Na+-mediated K+ currents in cat neocortical neurons. J Neurophysiol 61:245-256
[Abstract/Free Full Text] .- Foote SL, Berridge CW, Adams LM, Pineda JA (1991) Electrophysiological evidence for the involvement of the locus coeruleus in alerting, orienting, and attending. In: Progress in brain research, Vol 88, Neurobiology of the locus coeruleus (Barnes CD, Pompeiano O, eds), pp 521-532. Amsterdam: Elsevier.
- Gellman RL, Aghajanian GK (1993) Pyramidal cells in piriform cortex receive a convergence of inputs from monoamine activated GABAergic interneurons. Brain Res 600:63-73[ISI][Medline].
- Gereau RW, Conn PJ (1994) Presynaptic enhancement of excitatory synaptic transmission by beta-adrenergic receptor activation. J Neurophysiol 72:1438-1442
[Abstract/Free Full Text] .- Haas HL, Konnerth A (1983) Histamine and noradrenaline decrease calcium-activated potassium conductance in hippocampal pyramidal cells. Nature 302:432-434
