Abstract

Serotonin (5-hydroxytryptamine, 5-HT) controls pyramidal cell activity in prefrontal cortex (PFC) through various receptors, in particular, 5-HT1A and 5-HT2A receptors. Here we report that the physiological stimulation of the raphe nuclei excites local, putatively GABAergic neurons in the prelimbic and cingulate areas of the rat PFC in vivo. These excitations had a latency of 36 ± 4 ms and a duration of 69 ± 9 ms and were blocked by the i.v. administration of the 5-HT3 receptor antagonists ondansetron and tropisetron. The latency and duration were shorter than those elicited through 5-HT2A receptors in pyramidal neurons of the same areas. Double in situ hybridization histochemistry showed the presence of GABAergic neurons expressing 5-HT3 receptor mRNA in PFC. These cells were more abundant in the cingulate, prelimbic and infralimbic areas, particularly in superficial layers. The percentages of GAD mRNA-positive neurons expressing 5-HT3 receptor mRNA in prelimbic cortex were 40, 18, 6 and 8% in layers I, II–III, V and VI, respectively, a distribution complementary to that of cells expressing 5-HT2A receptors. Overall, these results support an important role of 5-HT in the control of the excitability of apical dendrites of pyramidal neurons in the medial PFC through the activation of 5-HT3 receptors in GABAergic interneurons.

Introduction

The prefrontal cortex (PFC) plays a key role in higher brain functions (Fuster, 2001; Miller and Cohen, 2001) and controls, via the excitatory axons of pyramidal neurons, the activity of many subcortical motor and limbic areas (Groenewegen and Uylings, 2000). The activity of projection pyramidal neurons is controlled, among other areas, by the brainstem monoaminergic systems. In particular, the mesocortical dopaminergic system is involved in working memory and cognition through a complex control of the activity of pyramidal neurons (Williams and Goldman-Rakic, 1995; Goldman-Rakic, 1996; O’Donnell, 2003; Wang et al., 2003). There is also increasing evidence that the ascending serotonergic pathways originating in the dorsal and median raphe nuclei (DR and MnR, respectively) may play an important role in prefrontal function. Indeed, prefrontal neurons in various species express several 5-HT receptor subtypes (Pompeiano et al., 1992, 1994; Morales and Bloom, 1997; Hall et al., 2000; Talvik-Lotfi et al., 2000; Martinez et al., 2001; Arango et al., 2002; Amargós-Bosch et al., 2004), which suggests a role for 5-HT in the function of PFC. Hence, 5-HT2A receptors in dorsolateral PFC are involved in working memory (Williams et al., 2002) and recent work associates allelic variants of this receptor with memory capacity in humans (De Quervain et al., 2003). Hallucinogens such as LSD or DOI (1-[2,5]-dimethoxy-4-iodophenyl-2-aminopropane) are 5-HT2A receptor agonists, whereas atypical antipsychotics are 5-HT2A receptor antagonists (Kroeze and Roth, 1998; Meltzer, 1999). On the other hand, 5-HT1A receptor antagonists reverse drug-induced cognitive deficits (Harder and Ridley, 2000; Mello e Souza et al., 2001; Misane and Ögren, 2003).

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.

Materials and Methods

Animals and Tissue Preparation

Male albino Wistar rats weighing 250–320 g were used (Iffa Credo, Lyon, France). These were kept in a controlled environment (12 h light–dark 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 II–III, 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 0.5–1.7 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 4–10 MΩ. 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 10–30 KΩ. 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: 669–716, 1482–1520 and 1913–1960 of the rat 5-HT2A receptor mRNA (Pritchett et al., 1988); 728–772 and 1001–1045 of the rat 5-HT3A receptor subunit mRNA (GenBank Accession No. U59672); 159–213 and 514–558 of the GAD65 mRNA (GenBank Accession No. NM_012563); 191–235 and 1600–1653 of the GAD67 mRNA (GenBank Accession. N.o NM_017007); 127–172 and 1756–1800 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 (1× PBS: 8 mM Na2HPO4, 1.4 mM KH2PO4, 136 mM NaCl, 2.6 mM KCl), washed for 5 min in 3× PBS at room temperature twice for 5 min each in 1× 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 Tris–HCl pH 7.5, 5 mM EDTA. The enzymatic activity was stopped by immersion for 30 s in 2 mg/ml glycine in 1× PBS. Tissues were finally rinsed in 1× 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, 4× SSC (1× SSC: 150 mM NaCl, 15 mM sodium citrate), 1× Denhardt’s 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 Tris–HCl (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 Tris–HCl 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 Tris–HCl 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 Student’s 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.

Results

5-HT3-mediated Excitations of Local Neurons in mPFC

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 (I–III). 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 III–V 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).

We used the 5-HT3 receptor antagonists ondansetron and tropisetron to examine whether the DR-induced excitations were mediated by 5-HT3 receptors. Considering all cases (n = 11), 5-HT3 receptor blockade significantly blocked these excitations, reducing the success rate from 68 ± 11% to 26 ± 8% on average (P < 0.006). Ondansetron was used to reverse the excitations in seven cases (from 73 ± 16% to 29 ± 11%, P < 0.001). Typically, excitations were blocked by 0.5–2 mg/kg i.v. except in one unit which required 3 mg/kg. Tropisetron was used in four units (from 59 ± 12% to 20 ± 9% success rate, P < 0.003). Three of them were blocked at 0.5–1 mg/kg i.v., whereas one was blocked at 3 mg/kg i.v. Figure 3 shows the reversal of the excitations in two different units by ondansetron and tropisetron, respectively.

5-HT3 receptor-mediated responses have been shown to desensitize after exposure to 5-HT and 5-HT3 receptor agonists in vitro (Zhou and Hablitz, 1999). Here we examined whether physiological amounts of 5-HT released by raphe stimulation could elicit a similar desensitizing response in vivo. To this end, we calculated the success rate for 1 min intervals at different times after the onset of the stimulation. The corresponding values were 114 ± 14, 108 ± 16 and 101 ± 17 %, for the 2nd, 3rd and 4th minutes after beginning the raphe simulation, taking 100 % as the success rate during the first minute.

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 III–V 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).

The vast majority of 5-HT3 receptor-expressing cells also contained the transcript for GAD (Dig-labeled), as observed in double in situ experiments (Fig. 5). The estimated proportions of GAD + 5-HT3 receptor-expressing cells versus the total of GABAergic (GAD-expressing) cells in layers I–III were 16.1 ± 0.3, 23.9 ± 3.5 and 19.1 ± 3.6% in the cingulate, prelimbic and infralimbic areas, respectively (means of three rats; each value is the average of three sections; the average number of GAD-positive cells per field was between 29 and 33). However, the localization of these cells was not homogeneous in the various fields examined and sometimes appeared as clusters (several cells per field, as shown in Figs 4C and 5C, for instance). Unlike GAD-positive cells, the number of those double-labeled cells fell rapidly at a greater lateral coordinate. Table 2 shows the number of cells expressing GAD mRNA, 5-HT3 receptor mRNA and double-labeled cells in the various cortical layers of the prelimbic area. ANOVA showed a significant effect of the layer on the number of cells expressing GAD [F(3,8) = 88.3, P < 0.000005], 5-HT3 receptors [F(3,8) = 14.2, P < 0.0002] and the percentage of double-labeled cells [F(3,8) = 127.7, P < 0.000001]. Post hoc Tukey t-test revealed significant differences between layers. Thus, the number of double-labeled cells was significantly greater in layers II–III than in the rest of layers. However, due to the lower number of GABAergic neurons in layer I compared to other layers, the percentage of double-labeled cells was greater in this layer (40% in layer I versus 18% in layers II–III and 6–8% in deeper layers; Table 2).

We observed a minority of cells expressing the 5-HT3 receptor mRNA which apparently were not positive for GAD mRNA (Table 2). This suggests that the 5-HT3 receptor transcript may also be present in a few non-GABAergic neurons. A pilot double in situ hybridization experiment showed the presence of some 5-HT3 receptor-positive cells which also contained the vGluT1 mRNA, yet these results require further confirmation. Likewise, it cannot be excluded that those are GABAergic cells with a faint labeling (e.g. poor penetration of the oligonucleotides or the antibody against digoxygenin).

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 I–III. Shown in the same figure are the cells positive for vGluT1 (Fig. 6A) and GAD (Fig. 6B) mRNAs in layers I–VI 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 III–V, 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.

Discussion

The present study shows that (i) endogenous 5-HT excites putative GABAergic interneurons in the medial PFC through the activation of 5-HT3 receptors and (ii) GAD- and 5-HT3 receptor-expressing cells are mainly located in superficial layers of the PFC, a location different — but complementary — to that of GABAergic neurons expressing 5-HT2A receptors. These results add to previous studies in rat PFC indicating that 5-HT modulates the activity of cortical microcircuits in various ways, either directly through the activation excitatory and inhibitory receptors in pyramidal neurons or indirectly, through the activation of excitatory receptors in selected populations of GABAergic interneurons.

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 (0.2–0.5 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 II–III, 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 III–V of the PFC, with a conspicuous absence in layers I–II 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 I–III) 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.

Work supported by grants from SAF2001-2133 and La Marató de TV3. P.C. is recipient of a Ramón y Cajal contract from the Ministry of Science and Technology. N.S. and M.V.P. are recipients of predoctoral fellowships from CICYT and IDIBAPS, respectively. We thank VITA-INVEST for the generous supply of ondansetron.

Figure 1. (A) Section drawing taken from Swanson (1998) showing the localization of the units recorded (shaded rectangle in the cingulate and prelimbic areas of the mPFC). These units were not antidromically activated from DR or mediodorsal thalamus and showed orthodromic activation from the DR that was blocked by the 5-HT3 receptor antagonists. (B, C) Extracellular recordings of putative GABAergic neurons in mPFC. (B) Representative waveform (average of 10 sweeps) and firing pattern of a non-fast-spiking (non-FS) neuron whose orthodromic activation from the DR was blocked by 5-HT3 receptor antagonist administration. (C) Representative waveform (average of 10 sweeps) and firing pattern of a spontaneously fast-spiking (FS) neuron. Firing rates of these two units were 1.3 and 10.6 spikes/s, respectively.

Figure 1. (A) Section drawing taken from Swanson (1998) showing the localization of the units recorded (shaded rectangle in the cingulate and prelimbic areas of the mPFC). These units were not antidromically activated from DR or mediodorsal thalamus and showed orthodromic activation from the DR that was blocked by the 5-HT3 receptor antagonists. (B, C) Extracellular recordings of putative GABAergic neurons in mPFC. (B) Representative waveform (average of 10 sweeps) and firing pattern of a non-fast-spiking (non-FS) neuron whose orthodromic activation from the DR was blocked by 5-HT3 receptor antagonist administration. (C) Representative waveform (average of 10 sweeps) and firing pattern of a spontaneously fast-spiking (FS) neuron. Firing rates of these two units were 1.3 and 10.6 spikes/s, respectively.

Figure 2. Peristimulus time histograms showing the orthodromic excitations elicited by the electrical stimulation of the DR on (A) a putatively GABAergic, 5-HT3 receptor-containing neuron and (B) on a pyramidal neuron in the prelimbic PFC identified by antidromic stimulation. Both responses were selectively blocked by the administration of the respective antagonists ondansetron (A) and M100907 (B) (not shown). Note that, as many pyramidal neurons in mPFC, this unit had antidromic (arrowhead) and orthodromic responses to DR stimulation as a result of the reciprocal connectivity and functional interaction between the DR and mPFC (Puig et al., 2003). The latency and duration of the 5-HT3-mediated responses in putative GABAergic neurons was significantly lower than those evoked by 5-HT2A receptor activation in pyramidal neurons. The concordances of the units shown are 85% (A) and 43% (B). Each peristimulus consists of 200 triggers; bin size is 4 ms. The arrow at zero abcissa marks the stimulus artifact.

Figure 2. Peristimulus time histograms showing the orthodromic excitations elicited by the electrical stimulation of the DR on (A) a putatively GABAergic, 5-HT3 receptor-containing neuron and (B) on a pyramidal neuron in the prelimbic PFC identified by antidromic stimulation. Both responses were selectively blocked by the administration of the respective antagonists ondansetron (A) and M100907 (B) (not shown). Note that, as many pyramidal neurons in mPFC, this unit had antidromic (arrowhead) and orthodromic responses to DR stimulation as a result of the reciprocal connectivity and functional interaction between the DR and mPFC (Puig et al., 2003). The latency and duration of the 5-HT3-mediated responses in putative GABAergic neurons was significantly lower than those evoked by 5-HT2A receptor activation in pyramidal neurons. The concordances of the units shown are 85% (A) and 43% (B). Each peristimulus consists of 200 triggers; bin size is 4 ms. The arrow at zero abcissa marks the stimulus artifact.

Figure 3. Blockade of the 5-HT-induced excitations in putative GABAergic neurons by the 5-HT3 antagonists ondansetron and tropisetron. (A) Raster display and PSTHs of a neuron in basal conditions and after the administration of ondansetron (0.5–2 mg/kg i.v. cumulative doses; injection time shown by arrows in the raster display). Note the complete suppression of the DR-induced orthodromic excitation by ondansetron. Each PSTH corresponds to 200 triggers; bin size 4 ms. (B) Raster display and PSTHs of a neuron in basal conditions and after the administration of tropisetron (0.5–1 mg/kg i.v. cumulative doses; injection times shown by arrows in the raster display). Note the marked suppression of the DR-induced orthodromic excitation induced by tropisetron. Each PSTH corresponds to 100 triggers; bin size 4 ms. The brackets denote the times in the raster displays at which PSTH have been constructed. x-axis units are in seconds.

Figure 3. Blockade of the 5-HT-induced excitations in putative GABAergic neurons by the 5-HT3 antagonists ondansetron and tropisetron. (A) Raster display and PSTHs of a neuron in basal conditions and after the administration of ondansetron (0.5–2 mg/kg i.v. cumulative doses; injection time shown by arrows in the raster display). Note the complete suppression of the DR-induced orthodromic excitation by ondansetron. Each PSTH corresponds to 200 triggers; bin size 4 ms. (B) Raster display and PSTHs of a neuron in basal conditions and after the administration of tropisetron (0.5–1 mg/kg i.v. cumulative doses; injection times shown by arrows in the raster display). Note the marked suppression of the DR-induced orthodromic excitation induced by tropisetron. Each PSTH corresponds to 100 triggers; bin size 4 ms. The brackets denote the times in the raster displays at which PSTH have been constructed. x-axis units are in seconds.

Figure 4. Visualization of 5-HT3 receptor mRNA in the rat prefrontal cortex. (A) Nissl stained section consecutive to B used as anatomical reference for the areas where 5-HT3 receptors are expressed. (B) Macroscopic dark-field image from an emulsion-dipped coronal section showing the localization of cells containing 5-HT3 receptor mRNA. Note the preferential expression in superficial cortical layers in all cortical areas. Frame in B limits the approximate area shown at higher magnification in panel C. High magnification bright field (D1) and dark field (D2) photomicrographs of the areas marked in C, showing individual cells expressing 5-HT3 receptors. Note the abundance of silver grains. ACAd, anterior cingulate (dorsal); AId, agranular insular (dorsal); GU, gustatory area; ILA, infralimbic area; MOp, primary motor area; MOs, secondary motor area; layer VI; ORBI, orbital area (lateral); PIR, piriform area; PrL, prelimbic area; TT, taenia tecta. Scale bar = 1 mm (A, B); 250 µm (C); 30 µm (D1, D2).

Figure 4. Visualization of 5-HT3 receptor mRNA in the rat prefrontal cortex. (A) Nissl stained section consecutive to B used as anatomical reference for the areas where 5-HT3 receptors are expressed. (B) Macroscopic dark-field image from an emulsion-dipped coronal section showing the localization of cells containing 5-HT3 receptor mRNA. Note the preferential expression in superficial cortical layers in all cortical areas. Frame in B limits the approximate area shown at higher magnification in panel C. High magnification bright field (D1) and dark field (D2) photomicrographs of the areas marked in C, showing individual cells expressing 5-HT3 receptors. Note the abundance of silver grains. ACAd, anterior cingulate (dorsal); AId, agranular insular (dorsal); GU, gustatory area; ILA, infralimbic area; MOp, primary motor area; MOs, secondary motor area; layer VI; ORBI, orbital area (lateral); PIR, piriform area; PrL, prelimbic area; TT, taenia tecta. Scale bar = 1 mm (A, B); 250 µm (C); 30 µm (D1, D2).

Figure 5. High magnification photomicrographs showing the detection in layers II–III of prelimbic and cingulate areas of 5-HT3 receptor mRNA using 33P-labeled oligonucleotides (silver grains) in GABAergic cells, visualized by hybridization with Dig-labeled oligonucleotides complementary to GAD mRNA (dark precipitate). (A, C) Bright-field photomicrographs showing the presence of several double-labeled cells (red arrowheads) as well as GABAergic cells not expressing the 5-HT3 receptor (black arrowheads) in the infralimbic (A) and prelimbic (C) areas. (B) Dark-field photomicrograph showing three double-labeled cells with a very dense labeling of the 5-HT3 receptor transcript (silver grains are seen as yellowish dots) in the dorsal anterior cingulate area. Scale bar = 20 µm.

Figure 5. High magnification photomicrographs showing the detection in layers II–III of prelimbic and cingulate areas of 5-HT3 receptor mRNA using 33P-labeled oligonucleotides (silver grains) in GABAergic cells, visualized by hybridization with Dig-labeled oligonucleotides complementary to GAD mRNA (dark precipitate). (A, C) Bright-field photomicrographs showing the presence of several double-labeled cells (red arrowheads) as well as GABAergic cells not expressing the 5-HT3 receptor (black arrowheads) in the infralimbic (A) and prelimbic (C) areas. (B) Dark-field photomicrograph showing three double-labeled cells with a very dense labeling of the 5-HT3 receptor transcript (silver grains are seen as yellowish dots) in the dorsal anterior cingulate area. Scale bar = 20 µm.

Figure 6. Composite photomicrographs showing the localization of cells expressing vGluT1 (A), GAD (B), 5-HT3 (C) and 5-HT2A (D) mRNAs through layers I–VI at the level of the prelimbic area in the rat PFC. The continuous vertical line denotes the location of the midline whereas the dotted line shows the approximate border between layer I and II. Each panel (AD) corresponds to three consecutive microscopic fields. Pyramidal neurons (as visualized by vGluT1 mRNA) are present in layers II–VI whereas GAD mRNA-positive cells are present in all layers, including layer I. Note the different location of cells expressing 5-HT3 (C) and 5-HT2A receptors (D). 5-HT3 receptor transcript is expressed by a limited number of cells present in layers I–III, particularly in the border between layers I and II. However, they represent 40% of GABAergic neurons in layer I. On the other hand, cells in these locations, particularly in layer I, do not express 5-HT2A receptors (note that only a small proportion of the latter receptors are expressed by GABAergic neurons; Santana et al., 2004). The asterisk denotes an artifact of the emulsion, seen in the dark field. Scale bar = 150 µm.

Figure 6. Composite photomicrographs showing the localization of cells expressing vGluT1 (A), GAD (B), 5-HT3 (C) and 5-HT2A (D) mRNAs through layers I–VI at the level of the prelimbic area in the rat PFC. The continuous vertical line denotes the location of the midline whereas the dotted line shows the approximate border between layer I and II. Each panel (AD) corresponds to three consecutive microscopic fields. Pyramidal neurons (as visualized by vGluT1 mRNA) are present in layers II–VI whereas GAD mRNA-positive cells are present in all layers, including layer I. Note the different location of cells expressing 5-HT3 (C) and 5-HT2A receptors (D). 5-HT3 receptor transcript is expressed by a limited number of cells present in layers I–III, particularly in the border between layers I and II. However, they represent 40% of GABAergic neurons in layer I. On the other hand, cells in these locations, particularly in layer I, do not express 5-HT2A receptors (note that only a small proportion of the latter receptors are expressed by GABAergic neurons; Santana et al., 2004). The asterisk denotes an artifact of the emulsion, seen in the dark field. Scale bar = 150 µm.

Table 1


 Characteristics of 5-HT3 and 5-HT2A receptor-mediated excitations induced by stimulation of the DR

 5-HT3 5-HT2A 
Firing rate (spikes/s) 1.7 ± 0.3 1.1 ± 0.8 
Latency (ms) 36 ± 4* 71 ± 8 
Duration (ms) 69 ± 9* 101 ± 8 
Success rate (%) 68 ± 11* 38 ± 8 
Localization (DV, mm) 2.1 ± 0.3 2.2 ± 0.1 
n 11 10 
 5-HT3 5-HT2A 
Firing rate (spikes/s) 1.7 ± 0.3 1.1 ± 0.8 
Latency (ms) 36 ± 4* 71 ± 8 
Duration (ms) 69 ± 9* 101 ± 8 
Success rate (%) 68 ± 11* 38 ± 8 
Localization (DV, mm) 2.1 ± 0.3 2.2 ± 0.1 
n 11 10 

*P < 0.05, paired Student’s t-test.

Data from 5-HT2A receptor-mediated excitations calculated from Amargós-Bosch et al. (2004).

Table 2


 Expression of 5-HT3 receptors in prelimbic cortex

 Layer I Layers II–III Layer V Layer VI 
GAD mRNA 6.6 ± 0.4* 22.1 ± 1.2 26.7 ± 1.5 23.9 ± 0.1 
GAD + 5-HT3 receptor mRNAs 2.6 ± 0.2*** 4.1 ± 0.5* 1.5 ± 0.3 2.0 ± 0.1 
5-HT3 receptor mRNA alone 0.2 ± 0.1 0.4 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 
% double-labeled cells 40 ± 2* 18 ± 2** 6 ± 1 8.2 ± 0.4 
 Layer I Layers II–III Layer V Layer VI 
GAD mRNA 6.6 ± 0.4* 22.1 ± 1.2 26.7 ± 1.5 23.9 ± 0.1 
GAD + 5-HT3 receptor mRNAs 2.6 ± 0.2*** 4.1 ± 0.5* 1.5 ± 0.3 2.0 ± 0.1 
5-HT3 receptor mRNA alone 0.2 ± 0.1 0.4 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 
% double-labeled cells 40 ± 2* 18 ± 2** 6 ± 1 8.2 ± 0.4 

*P < 0.05 versus the rest of layers; **P < 0.05 versus deeper layers; *** P < 0.05 versus layers II–V (Tukey t-test).

Data are number of cells expressing the corresponding mRNAs in the various cortical layers in the prelimbic area (mean ± SEM of three rats). The values for each rat were calculated by averaging the number of cells in three consecutive fields per section (three sections per rat) as observed at ×40 magnification in a Nikon Eclipse E1000 microscope. Layer and area nomenclature are according to Swanson (1998).

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