Department of Neurochemistry, Institut dInvestigacions Biomèdiques de Barcelona (CSIC), IDIBAPS, 08036 Barcelona, Spain, \|[ast ]\| The first two authors contributed equally to this study
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Abstract |
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Key Words: GABA interneurons, 5-HT2A receptors, 5-HT3 receptors, medial prefrontal cortex, pyramidal neurons
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Introduction |
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5-HT3 receptors appear also to be involved in the cortical actions of 5-HT. Hence, 5-HT3 receptor antagonists display pro-cognitive actions (Staubli and Zu, 1995). These agents have been also reported to display anxiolytic and antipsychotic activity in animal models (Higgins and Kilpatrick, 1999
) and to improve the therapeutic action of antipsychotics in schizophrenic patients (Sirota et al., 2000
), perhaps through changes in dopamine release (Blandina et al., 1989
; Chen et al., 1992
; De Deurwaerdère et al., 1998
). Likewise, the atypical antipsychotic clozapine is an antagonist of 5-HT3 receptors (Watling et al., 1989
; Edwards et al., 1991
).
Early microiontophoretic studies showed that 5-HT and 5-HT3 receptor agonists suppressed pyramidal activity in rat PFC through the activation of 5-HT3 receptors by a direct action (Ashby et al., 1989, 1991, 1992). However, more recent in vitro studies indicate that 5-HT may increase IPSCs in cortical pyramidal neurons by activation of 5-HT3 receptors, likely as a result of a fast synaptic excitation of local GABAergic neurons (Zhou and Hablitz, 1999
; Férézou et al., 2002
). The latter observations are consistent with the presence of 5-HT3 receptors in GABAergic interneurons in the rat telencephalon (Morales et al., 1996
; Morales and Bloom, 1997
). Likewise, in macaque cortex, 5-HT3 receptors are expressed by a subpopulation of calbindin- and calretinin-positive interneurons (Jakab and Goldman-Rakic, 2000
). To gain further insight on the actions of 5-HT in PFC, we examined the localization of 5-HT3 receptors in GABA interneurons of the rat PFC and the effects of the physiological stimulation of the DR on the activity of such neurons recorded in vivo.
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Materials and Methods |
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Male albino Wistar rats weighing 250320 g were used (Iffa Credo, Lyon, France). These were kept in a controlled environment (12 h lightdark cycle and 22 ± 2°C room temperature) with food and water provided ad libitum. Animal care followed the European Union regulations (O.J. of E.C. L358/1 18/12/1986) and was approved by the local ethics committee. Stereotaxic coordinates were taken from bregma and duramater according to the atlas of Paxinos and Watson (1998). We used the brain maps of Swanson (1998
) for nomenclature of cortical areas.
Tissue Preparation
Rats used in electrophysiological experiments were killed by an anesthetic overdose. The location of stimulation electrodes was verified histologically (Neutral Red staining). The rats used for in situ hybridization histochemistry were killed by decapitation, the brains rapidly removed, frozen on dry ice and stored at 20°C. Tissue sections, 14 µm thick, were cut using a microtome-cryostat (HM500 OM; Microm, Walldorf, Germany), thaw-mounted onto APTS (3-aminopropyltriethoxysilane; Sigma, St Louis, MO)-coated slides and kept at 20°C until use.
Electrophysiological Recordings
We assessed the effects of the electrical stimulation of the DR at physiological rates on the activity of non-pyramidal neurons in the dorsal anterior cingulate and prelimbic areas of the rat PFC. Descents were carried out at AP +3.2 to +3.4, DV 1.1 to 3.6 below the brain surface. For the recording of 5-HT3-expressing GABAergic neurons, the lateral coordinate was adjusted between 0.2 and 0.5 mm in order to target cells in the border between layers I and IIIII, which show the greater abundance of cells expressing this receptor, as observed in in situ hybridization experiments (see below). To this end, the sinus was retracted to allow recording near the midline. As in previous studies, pyramidal neurons were identified by antidromic activation from projection areas of the medial prefrontal cortex (mPFC), such as the DR (at two different coordinates) or the mediodorsal thalamus (AP 2.8, L 0.5, DV 5.3), up to 2 mA and collision extinction with spontaneously occurring spikes (Fuller and Schlag, 1976). Non-projecting units which were spontaneously active with a slow firing rate were considered candidates for the examination of the in vivo effects of 5-HT through 5-HT3 receptors (see below). To this end, the DR (tip coordinates: AP 7.8, L 0, DV 6.5) was stimulated at 0.51.7 mA, 0.2 ms square pulses, 0.9 Hz. Peristimulus time histograms (PSTH) were constructed in baseline conditions and after the administration of the 5-HT3 receptor antagonists ondansetron (gift from VITA-INVEST, Sant Joan Despí, Spain) and tropisetron (Sigma).
Single-unit extracellular recordings were performed as follows. Rats were anesthetized (chloral hydrate 400 mg/kg i.p.) and positioned in an stereotaxic apparatus (David Kopf). Additional doses of chloral hydrate (80 mg/kg) were administered i.v. through the femoral vein. Body temperature was maintained at 37°C throughout the experiment with a heating pad. All wound margins and points of contact between the animal and the stereotaxic apparatus were infiltrated with lidocaine solution (5%). In order to minimize pulsation, the atlanto-occipital membrane was punctured to release some CSF. Putative GABAergic neurons were recorded extracellularly with glass micropipettes pulled from 2.0 mm capillary glass (WPI, Sarasota, FL) on a Narishige PE-2 pipette puller (Narishige Sci. Inst., Tokyo, Japan). Microelectrodes were filled with 2M NaCl. Typically, impedance was between 410 M. Bipolar stimulating electrodes consisted of two stainless steel enamel-coated wire (California Fine Wire, Grover Beach, CA) with a diameter of 150 µm and a tip of separation of
100 µm and in vitro impedance of 1030 K
. Constant current electrical stimuli were generated with a Grass stimulation unit S-48 connected to a Grass SIU 5 stimulus isolation unit. Single unit extracellular recordings were amplified with a Neurodata IR283 (Cygnus Technology Inc., Delaware Water Gap, PA), postamplified and filtered with a Cibertec amplifier (Madrid, Spain) and computed on-line using a DAT 1401plus interface system Spike2 software (Cambridge Electronic Design, Cambridge, UK).
Oligonucleotide Probes
The oligodeoxyribonucleotide probes used were complementary to the following bases: 669716, 14821520 and 19131960 of the rat 5-HT2A receptor mRNA (Pritchett et al., 1988); 728772 and 10011045 of the rat 5-HT3A receptor subunit mRNA (GenBank Accession No. U59672); 159213 and 514558 of the GAD65 mRNA (GenBank Accession No. NM_012563); 191235 and 16001653 of the GAD67 mRNA (GenBank Accession. N.o NM_017007); 127172 and 17561800 of the vGluT1 mRNA (GenBank Accession No. U07609). The probes for 5-HT2A receptor and GAD67 were synthesized on a 380 Applied Biosystem DNA synthesizer (Foster City Biosystem, Foster City, CA) and purified on a 20% polyacrylamide/8 M urea preparative sequencing gel. The rest of the probes were synthesized and HPLC purified by Isogen Bioscience BV (Maarsden, The Netherlands).
Oligonucleotides were individually labeled at the 3'-end either with [33P]-dATP (>2500 Ci/mmol; DuPont-NEN, Boston, MA) or with Dig-11-dUTP (Boehringer Mannheim) using terminal deoxynucleotidyltransferase (Roche Diagnostics GmbH, Mannheim, Germany), purified by centrifugation using QIAquick Nucleotide Removal Kit (QIAGEN GmbH, Hilden, Germany).
In Situ Hybridization Histochemistry Procedure
The protocols for single- and double-label in situ hybridization were based on previously described procedures (Tomiyama et al., 1997; Landry et al., 2000
) and have been already published (Serrats et al., 2003
). Frozen tissue sections were first brought to room temperature, fixed for 20 min at 4°C in 4% paraformaldehyde in phosphate-buffered saline (1x PBS: 8 mM Na2HPO4, 1.4 mM KH2PO4, 136 mM NaCl, 2.6 mM KCl), washed for 5 min in 3x PBS at room temperature twice for 5 min each in 1x PBS, and incubated for 2 min at 21°C in a solution of predigested pronase (Calbiochem, San Diego, CA) at a final concentration of 24 U/ml in 50 mM TrisHCl pH 7.5, 5 mM EDTA. The enzymatic activity was stopped by immersion for 30 s in 2 mg/ml glycine in 1x PBS. Tissues were finally rinsed in 1x PBS and dehydrated through a graded series of ethanol. For hybridization, the radioactively-labeled and the non-radioactively labeled probes were diluted in a solution containing 50% formamide, 4x SSC (1x SSC: 150 mM NaCl, 15 mM sodium citrate), 1x Denhardts solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 10% dextran sulfate, 1% sarkosyl, 20 mM phosphate buffer pH 7.0, 250 µg/ml yeast tRNA and 500 µg/ml salmon sperm DNA. The final concentrations of radioactive and Dig-labeled probes in the hybridization buffer were in the same range (
1.5 nM). Tissue sections were covered with hybridization solution containing the labeled probe/s, overlaid with Nescofilm coverslips (Bando Chemical Ind., Kobe, Japan) and incubated overnight at 42°C in humid boxes. Sections were then washed four times (45 min each) in a buffer containing 0.6 M NaCl and 10 mM TrisHCl (pH 7.5) at 60°C.
Development of Radioactive and Non-radioactive Hybridization Signal
Hybridized sections were treated as described by Landry et al. (2000). Briefly, after washing, the slides were immersed for 30 min in a buffer containing 0.1 M TrisHCl pH 7.5, 1 M NaCl, 2 mM MgCl2 and 0.5% bovine serum albumin (Sigma) and incubated overnight at 4°C in the same solution with alkaline-phosphatase-conjugated anti-digoxigenin-F(ab) fragments (1:5000; Boehringer Mannheim). Afterwards, they were washed three times (10 min each) in the same buffer (without antibody) and twice in an alkaline buffer containing 0.1 M TrisHCl pH 9.5, 0.1 M NaCl and 5 mM MgCl2. Alkaline phosphatase activity was developed by incubating the sections with 3.3 mg nitroblue tetrazolium and 1.65 mg bromochloroindolyl phosphate (Gibco BRL, Gaithersburg, MD) diluted in 10 ml of alkaline buffer. The enzymatic reaction was blocked by extensive rinsing in the alkaline buffer containing 1 mM EDTA. The sections were then briefly dipped in 70 and 100% ethanol, air-dried and dipped into Ilford K5 nuclear emulsion (Ilford, Mobberly, Chesire, UK) diluted 1:1 with distilled water. They were exposed in, USA) for 5 min, and fixed in Ilford Hypam fixer (Ilford).
Specificity of the Probes
The specificity of the hybridization signals has been previously established and published (Pompeiano et al., 1992, 1994; Serrats et al., 2003
). These controls included: (i) the thermal stability of the hybrids obtained was checked for every probe; (ii) for a given oligonucleotide probe, the hybridization signal was completely blocked by competition of the labeled probe in the presence of 50-fold excess of the same unlabeled oligonucleotide (iii) since we synthesized more than one probe for each mRNA analyzed, the hybridization signal obtained with each oligonucleotide for the same mRNA was identical at regional and cellular levels when used independently; and (iv) to assure the specificity of the non-radioactive hybridization signal, we compared the results obtained with the same probe radioactively labeled.
Analysis of the Results
The responses in putative GABAergic neurons evoked by DR stimulation were characterized by measuring the delay, magnitude and duration of excitatory responses from PSTH (4 ms bin width). Orthodromic excitations elicited spikes with short and variable latencies with a success rate greater than 10% (Celada et al., 2001). Success rate in PSTHs were corrected by the pre-stimulus firing. Drug changes were assessed with paired Students t-test.
Tissue sections were examined in bright- and dark-field in a Wild 420 macroscope (Leica, Heerbrugg, Germany) and in a Nikon Eclipse E1000 microscope (Nikon, Tokyo, Japan) equipped with bright- and dark-field condensers for transmitted light and with epi-illumination. Micrography was performed using a digital camera (DXM1200 3.0; Nikon) and analySIS Software (Soft Imaging System GmbH, Germany). Bright-field images were captured with transmitted light. Dark-field images were also captured with Darklite illuminator (Micro Video Instruments, Avon, MA). The figures were prepared for publication using Adobe Photoshop software (Adobe Software, Mountain View, CA).
Cell counting was performed manually at the microscope with the help of analySIS Software. Only cellular profiles showing great abundance of both transcripts were considered to co-express both mRNAs. Cells with a dense labeling of GAD mRNAs and occasional silver grains were not considered to co-express both receptors. P < 0.05 was considered statistically significant.
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Results |
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The present experiments were initiated in parallel to the study of the effect of DR/MnR stimulation on pyramidal neurons of mPFC mediated by 5-HT1A and 5-HT2A receptors (Puig et al., 2003, 2004; Amargós-Bosch et al., 2004
). Pyramidal neurons were recorded at a lateral coordinate typically between 0.5 and 1.0 mm. During these experiments, encompassing
230 neurons, we occasionally found cells that (i) were excited by DR/MnR stimulation but were not antidromically activated from the midbrain and thalamus and (ii) exhibited excitations with a latency and duration shorter than those typically elicited through the activation of 5-HT2A receptors. Because of the presence of 5-HT3 receptors in GABA interneurons in rat telencephalon (Morales and Bloom, 1997
), we hypothesized that these excitations might be due to the activation of 5-HT3 receptors. Five units were recorded at this location, whose excitations were reversed by 5-HT3 receptor antagonists. Based on these initial observations, we carried out in situ hybridization experiments to determine the location of cells expressing the 5-HT3 receptor mRNA. Once these results were available (see below), additional descents were systematically performed at a more central coordinate, between 0.2 and 0.5 mm, aiming at cells expressing 5-HT3 receptors in superficial layers (IIII). In all cases, only slow-spiking neurons, not antidromically activated from the DR or the mediodorsal thalamus were considered to be potential candidates to examine the effects of DR stimulation upon 5-HT3 receptors. A total of 14 excitations were considered to be potentially attributable to 5-HT3 receptor activation and blockade was successfully attempted in 11 cases with the 5-HT3 receptor antagonists ondansetron and tropisetron. Since other 5-HT receptors might potentially contribute to these excitations, here we report only the data of those cells whose excitations were reversed by these antagonists.
The electrical stimulation of the DR at a physiological rate (0.9 Hz, 0.2 ms square pulses) resulted in orthodromic excitations of slow-spiking putative GABAergic neurons. Fast-spiking neurons (>10 spikes/s) were not excited by DR stimulation (data not shown). The characteristics of the recorded neurons, as well as the latency and duration of the excitations are given in Figure 1 and Table 1. Unlike fast-spiking cells, these neurons exhibited a slow firing rate (<3 spikes/s), as recorded extracellularly, with a mean firing rate of 1.7 ± 0.3 spikes/s (n = 11, one neuron per rat). The latency and duration of these excitations were significantly lower than those elicited by DR stimulation, using the same parameters, in pyramidal neurons recorded in layers IIIV of cingulate and prelimbic areas (Table 1 and Fig. 2). The latter excitations were mediated by 5-HT2A receptor activation, since they were blocked by the i.v. administration of the 5-HT2A receptor antagonist M100907 (Puig et al., 2003; Amargós-Bosch et al., 2004
). Moreover, the success rate was significantly greater for the 5-HT3 receptor- than for 5-HT2A receptor-mediated excitations at the same current (68 ± 11% versus 38 ± 8, P < 0.04).
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Expression of 5-HT3 Receptors in GABA Interneurons
The presence of cells expressing the 5-HT3 receptor transcript in various areas of the rat PFC is illustrated in Figure 4. These cells were present in all cortical layers, although they had a preferential localization in superficial layers. In particular, they were more abundant in the cingulate, prelimbic and infralimbic areas as well as in primary and secondary motor areas (Fig. 4B,C). A smaller number of cells were also present in piriform cortex and adjacent olfactory areas. Some cells were also present in layer VI of medial and motor cortices whereas layers IIIV of these areas as well as the tenia tecta were almost without or with a much smaller population of neurons expressing 5-HT3 receptors. Most cells positive for the 5-HT3 receptor had a high level of expression, as judged from the large density of silver grains, corresponding to the 33P-labeled oligonucleotides used to hybridize with the mRNA (Figs 4D and 5). This was more marked than that of 5-HT2A receptors in GABAergic neurons in the same prefrontal areas observed using the same methodology (Santana et al., 2004). This difference may indicate a higher density of 5-HT3 receptors per cell although methodological reasons may also account (e.g. a higher hybridization of the oligonucleotides complementary to the 5-HT3 receptor mRNA).
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5-HT2A and 5-HT3 receptors mediate direct excitatory responses of 5-HT on the cells expressing these receptors (see Introduction). A previous immunohistochemical study suggested the localization of these two 5-HT receptors in different subpopulations of GABAergic interneurons in monkey neocortex (Jakab and Goldman-Rakic, 2000). We therefore examined the localization of cells expressing these receptors in the mPFC. As observed in the prelimbic area of PFC (Fig. 6), 5-HT3 receptor-expressing cells were located near the midline, in layers IIII. Shown in the same figure are the cells positive for vGluT1 (Fig. 6A) and GAD (Fig. 6B) mRNAs in layers IVI of this cortical area. GAD-positive cells were present in all layers, including layer I where, as expected, pyramidal cells (vGluT1-expressing) were absent. On the other hand, cells expressing 5-HT2A receptors were located mainly in layers IIIV, an area where the 5-HT3 receptor mRNA was much less abundant (Fig. 6C,D). The 5-HT2A receptor transcript is expressed by
60% of pyramidal (vGluT1-positive) cells and by
25% of GABAergic cells (GAD-positive; Santana et al., 2004
). However, the proportion between 5-HT2A receptors in GAD- and vGluT1-positive cells is similar in all areas of the PFC and, therefore, the total population of cells positive for the 5-HT2A receptor mRNA is representative of that in GABAergic neurons. The conspicuous absence of cells containing the 5-HT2A receptor mRNA in layer I indicates that the GABAergic neurons close to the midline express 5-HT3 but not 5-HT2A receptors.
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Discussion |
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Methodological Considerations
Previous studies examining the cellular phenotypes expressing 5-HT3 receptors used GABA immunoreactivity to label GABAergic interneurons (Morales et al., 1996; Morales and Bloom, 1997
). Here we identified GABAergic neurons by the presence of GAD67 or GAD65 mRNAs. Immunohistochemical and in situ hybridization histochemistry indicate that the majority of GABA-containing neurons in the brain co-express the genes encoding the two GAD isoforms (Erlander et al., 1991
; Esclapez et al., 1993
, 1994; Feldblum et al., 1993
). On the other hand, the cloning and characterization of glutamate vesicular transporters, vGluT1, vGluT2 and vGluT3, in rat brain (Takamori et al., 2000
, 2001; Gras et al., 2002
) has enabled the histological identification of a glutamatergic neuronal phenotype (Fremeau et al., 2001
; Takamori et al., 2001
; Gras et al., 2002
; Oliveira et al., 2003
). In particular, most rat cortical cells express very high levels of vGluT1 mRNA (Gras et al., 2002
; Ziegler et al., 2002
), which supports the use of vGluT1 to identify cortical glutamatergic pyramidal neurons.
Several classifications of GABAergic interneurons have been made, based on their morphology, chemical neuroanatomy and electrophysiological characteristics (De Felipe, 2002; Freund, 2003
). Considering their firing characteristics when recorded intracellularly, GABA interneurons have been classified as fast-spiking and non-fast-spiking (both regular and irregular) cells (Cauli et al., 1997
; Férézou et al., 2002
; Kawaguchi and Kondo, 2002
). Although extracellular recordings cannot discriminate between these cellular types, here we observed two main firing patterns of putative GABAergic interneurons, namely slow (non-fast-spiking, not firing in trains, discharge rate <3 spikes/s) and fast-spiking cells (firing in trains, discharge rate >10 spikes/s; Constantinidis and Goldman-Rakic, 2002
). Indeed, due to the inherent complexity of the in vivo recordings of putative GABAergic neurons, a limitation of the present study is that the recorded units were not neurochemically characterized. However, it is unlikely that these were pyramidal neurons, in view of the following reasons. First, they were not antidromically activated from the DR or the mediodorsal thalamus, which make up two main targets of the axons of mPFC pyramidal neurons, where recordings were made (Thierry et al., 1983
; Peyron et al., 1998
; Groenewegen and Uylings, 2000
). Secondly, more than half of the successful recordings were made close to the midline (0.20.5 mm lateral) to target the GAD- + 5-HT3-receptor-labelled cells observed in the parallel in situ hybridization studies. In close agreement with the present observations, Zhou and Hablitz (1999
) recorded 5-HT3 receptor-mediated responses in vitro in layer I of cortical slices. Thirdly, the DR-induced excitations were unequivocally mediated by 5-HT3 receptor activation since they were reversed by the selective antagonists ondansetron and tropisetron. Thus, although there may be a very small proportion of 5-HT3 receptors in non-GABAergic neurons (Morales and Bloom, 1997
; this study) it is unlikely that these were recorded.
Effect of DR Stimulation on Putative GABAergic Neurons in mPFC
Cortical microcircuits consist of principal (pyramidal) neurons and local (mainly GABAergic) interneurons that modulate pyramidal activity (Somogyi et al., 1998). 5-HT can modulate the activity of these microcircuits in various ways. Direct inhibitory and excitatory actions on pyramidal neurons are mediated by 5-HT1A and 5-HT2A receptors respectively (Araneda and Andrade, 1991
; Aghajanian and Marek, 1997
, 1999; Zhou and Hablitz, 1999
). Indirect actions are mediated by the activation of 5-HT receptors present on GABAergic interneurons and afferent terminals (heteroceptors; e.g. 5-HT1B) (Ashby et al., 1990
; Tanaka and North, 1993
; Zhou and Hablitz, 1999
). The electrical stimulation of the DR and MnR in the anesthetized rat can excite or inhibit pyramidal neurons in the cingulate and prelimbic PFC through the activation of 5-HT2A and 5-HT1A receptors, respectively (Puig et al., 2003
; Amargós-Bosch et al., 2004
). In this manner, 5-HT may influence the descending excitatory input into limbic and motor structures where the prefrontal cortex projects (Groenewegen and Uylings, 2000
).
However, the role of 5-HT3 receptors in the control of cortical neurons is less well understood. These receptors have been reported to be present in axons and in the somatodendritic region of cortical neurons (Miquel et al., 2002). The microiontophoretic application of 5-HT and selective 5-HT3 agonists in the rat mPFC suppressed the firing of cells in layers IIIII, an effect blocked by 5-HT3 receptor antagonists (Ashby et al., 1989
, 1991, 1992). Likewise, the stimulation of ascending serotonergic fibers at high frequency (15 Hz) evoked a suppression of cortical, possibly pyramidal, cells which was also blocked by 5-HT3 antagonists (Ashby et al., 1991
, 1992). Based on the inability of the microiontophoretic application of the GABAA receptor antagonist SR 95103 to block these effects, it was concluded that cortical neurons were directly inhibited through 5-HT3 receptor activation (Ashby et al., 1989
, 1991, 1992). In contrast to these reports, whole cell recordings in rat sensorimotor cortex revealed that 5-HT induces a fast synaptic excitation in a subpopulation of regular or irregular slow-spiking (but not fast-spiking) VIP- and CCK-containing GABAergic interneurons in layer II (Férézou et al., 2002
). These effects were mimicked by the 5-HT3 receptor agonist m-phenylbiguanide and blocked by tropisetron, indicating the involvement of 5-HT3 receptors, whose presence in the recorded neurons was determined by single cell RT-PCR (Férézou et al., 2002
). Moreover, 5-HT and the 5-HT3 agonist 1-(m-chlorophenyl)-biguanide increased a TTX-independent inward current in layer I interneurons (Zhou and Hablitz, 1999
). This cortical effect is consistent with the ionic characteristics of the 5-HT3 receptor (Maricq et al., 1991
) and agrees with earlier data in hippocampus showing that 5-HT can excite GABA interneurons through 5-HT3 receptors (Ropert and Guy, 1991
; Kawa, 1994
; McMahon and Kauer, 1997
). Thus, these observations suggest that the 5-HT3 receptor-mediated inhibitory action of 5-HT on cortical pyramidal neurons is indirect, involving an increase of local GABA inputs.
Our in vivo data in mPFC accord with the above in vitro observations (Zhou and Hablitz, 1999; Férézou et al., 2002
) and indicate that 5-HT, released in the PFC by the physiological stimulation of the DR, can excite slow-spiking GABAergic neurons through the activation of 5-HT3 receptors. However, unlike to the exogenous in vitro application of 5-HT (Zhou and Hablitz, 1999
), the response to endogenous 5-HT does not appear to desensitize, at least during the observation period used herein (4 min). The inability of the DR stimulation to evoke a similar excitation in spontaneous fast-spiking interneurons agrees with the fact that 5-HT3 receptors are only expressed by a subpopulation of GABAergic neurons (Morales and Bloom, 1997
; Férézou et al., 2002
; this study). We cannot give an estimate of the proportion of cells responding to DR stimulation with 5-HT3 receptor-mediated responses, but indeed this is very low, consistent with the low proportion of neurons expressing 5-HT3 receptor observed in the parallel histological study. Systematic descents in the recording area enabled to record few cells that (i) were spontaneously firing, (ii) were not antidromically activated from the DR or the mediodorsal thalamus and (iii) responded to DR stimulation with an excitation that (iv) was blocked by 5-HT3 receptor antagonists. Hence, although the total number of cells reported here may appear low (n = 11), a much larger number were recorded to obtain such data. Similarly, Férézou et al. (2002
) reported that only 19 out of a total of 107 attempted neurons were excited in vitro by 5-HT through 5-HT3 receptors in slices of sensorimotor cortex.
The latency and duration of the 5-HT receptor-mediated excitations in putative GABAergic neurons were shorter than those observed in pyramidal neurons in the same areas of the PFC after the stimulation of the DR at the same rate (the latter are 5-HT2A receptor-mediated; Puig et al., 2003; Amargós-Bosch et al., 2004
). This difference may indicate a higher conduction velocity of the 5-HT fibers targeting 5-HT3 receptors. Indeed, two main types of serotonergic axons have been reported that differ in their morphology (Kosofsky and Molliver, 1987
). On the other hand, this difference could also be attributed to the ionic nature of the 5-HT3 response which results in fast synaptic actions of 5-HT on these neurons (Maricq et al., 1991
; Férézou et al., 2002
). In contrast, the actions of 5-HT2A receptors on neuronal excitability are mediated by metabotropic mechanisms (Aghajanian, 1995
). The short latency and duration 5-HT3 receptor-mediated activation of GABAergic inputs onto pyramidal neurons may perhaps contribute to a short-latency, 5-HT1A receptor-independent inhibition observed in pyramidal neurons after the stimulation of the DR (Amargós-Bosch et al., 2004
).
Localization of GABAergic Neurons Expressing 5-HT3 Receptors
Consistent with previous data in various telencephalic areas in rat (Morales and Bloom, 1997) and mouse brain (Hermann et al., 2002
), here we found that a very large proportion of 5-HT3 receptor is expressed by GABAergic neurons in PFC. Few non-GABAergic cells exhibited the presence of the 5-HT3 receptor transcript. Given the larger proportion of pyramidal versus GABAergic cells in neocortex (the latter represent a 15% of total; Beaulieu, 1993
) we cannot exclude that a minority of the 5-HT3 receptor-positive cells are pyramidal neurons.
5-HT3 receptor-immunoreactive cells were found through all layers in frontal, temporal and parietal cortex in monkeys (Jakab and Goldman-Rakic, 2000). In contrast, these appear to be located preferentially in superficial layers in the rat, as judged from histological and functional studies (Morales and Bloom, 1997
; Zhou and Hablitz, 1999
; Férézou et al., 2002
; this study). In particular, we show an enrichment of these cells in superficial layers of the cingulate, prelimbic and infralimbic areas of the rat PFC. This localization suggests that 5-HT3 receptors may be the target of the dense plexus of serotonergic fibers in superficial cortical layers (Blue et al., 1988
). Indeed, the expression of other cortical 5-HT receptors, such as 5-HT1A, 5-HT2A, or 5-HT2C is more marked in intermediate and deep layers (Pompeiano et al., 1992
, 1994; Amargós-Bosch et al., 2004
; Santana et al., 2004
; see also Fig. 6). Interestingly, the distribution of cells expressing 5-HT3 and 5-HT2A receptors in PFC seems complementary. The latter were expressed in glutamatergic and GABAergic neurons in layers IIIV of the PFC, with a conspicuous absence in layers III and a low expression in layer VI (Amargós-Bosch et al., 2004
; Santana et al., 2004
; this study). Only a small proportion of all 5-HT2A receptor-expressing cells is GABAergic (Santana et al., 2004
), although their distribution follows the pattern of all 5-HT2A receptor-containing cells. In contrast, 5-HT3 receptor-expressing cells were found near the midline (particularly layers IIII) and to a much lesser extent in layer VI. 5-HT3 receptors have been localized to calbindin- and calretinin-containing, small size GABAergic interneurons, whereas 5-HT2A receptors are expressed by parvalbumin-containing large size interneurons (e.g. basket cells) (Morales and Bloom, 1997
; Jakab and Goldman-Rakic, 1998
, 2000). The presence of 5-HT3 receptors in layer I GABAergic neurons, a cortical level devoid of pyramidal cell bodies (see, for instance, Fig. 6), suggests that 5-HT can modulate the inputs onto the apical dendrites of pyramidal neurons in PFC via 5-HT3 receptors located in GABAergic interneurons. In this manner, 5-HT might modulate the cortico-cortical and thalamo-cortical inputs into superficial layers through an enhancement of synaptic GABAergic inputs (Krettek and Price, 1977
; Linke and Schwegler, 2000
; Mitchell and Cauler, 2001
). On the other hand, 5-HT2A receptors are involved in the feed-forward inhibition of pyramidal neurons through large, perisomatic parvalbumin-containing GABAergic neurons (Jakab and Goldman-Rakic, 2000
). Thus, although the present study did not characterize the subtype(s) of GABAergic interneurons expressing 5-HT3 and 5-HT2A receptors, the distinct localization of cells expressing one or other receptor strongly supports an anatomical and functional segregation of both receptors in cortical microcircuits in the rat PFC, as observed in macaque cortex (Jakab and Goldman-Rakic, 2000
). Moreover, 5-HT3 receptor-mediated excitations are faster and last less than those induced by the activation of 5-HT2A receptors, which indicates that 5-HT2A and 5-HT3 receptor-mediated responses are also temporally segregated.
In summary, the present study adds to previous in vivo data indicating that endogenous 5-HT, released by the physiological stimulation of the DR, is able to control the activity of neurons in the cingulate and prelimbic areas of the PFC through various cortical receptors, in particular the 5-HT1A, 5-HT2A and 5-HT3 subtypes (Puig et al., 2003; Amargós-Bosch et al., 2004
). The distinct temporal patterns of activation and the different cellular localizations of these receptors suggest a complex regulation of the cortical activity by 5-HT which deserves further investigation.
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Notes |
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Address correspondence to Francesc Artigas, Department of Neurochemistry, Institut dInvestigacions Biomèdiques de Barcelona (CSIC), IDIBAPS, Rosselló 161, 6th floor, 08036 Barcelona, Spain. Email: fapnqi{at}iibb.csic.es.
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