Cerebral Cortex Advance Access originally published online on December 7, 2005
Cerebral Cortex 2006 16(10):1440-1452; doi:10.1093/cercor/bhj081
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Cortical Sources of CRF, NKB, and CCK and Their Effects on Pyramidal Cells in the Neocortex
Laboratoire de Neurobiologie et Diversité Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7637, École Supérieure de Physique et de Chimie Industrielles, 10 rue Vauquelin, 75005 Paris, France
Address correspondence to Dr Bertrand Lambolez, Neurobiologie Processus Adaptatifs, CNRS UMR 7102, Université Pierre et Marie Curie 9 quai St Bernard, 75005 Paris, France. Email: bertrand.lambolez{at}snv.jussieu.fr.
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Abstract |
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In order to investigate how neuropeptide transmission can modulate the neocortical network, we mapped the expression of neurokinin (NK) B, cholecystokinin (CCK), and corticotropin-releasing factor (CRF) and their receptors to neuronal types using patch-clamp and single-cell reverse transcription–polymerase chain reaction in acute slices of rat neocortex. Classification of neurons by unsupervised clustering based on the analysis of multiple electrophysiological and molecular properties disclosed 3 GABAergic interneuron clusters and 1 pyramidal cell cluster. The 3 neuropeptides were expressed in a cluster of interneurons characteristically expressing vasoactive intestinal peptide. CRF was additionally found in a cluster containing almost exclusively somatostatin-expressing interneurons, whereas CCK was present in all clusters. The respective receptors of these peptides, NK-3, CCK-B, and CRF-1, were essentially expressed in pyramidal cells. At –60 mV, pyramidal cells were weakly depolarized by each of these peptides. When pyramidal neurons were maintained to about 5 mV below spike threshold, depolarization induced by each peptide resulted in a long-lasting action potential discharge. Neuropeptide effects were prevented by selective antagonists of NK-3, CCK-B, and CRF-1 receptors. These results suggest that pyramidal neurons are the primary target of NKB, CCK, and CRF in the neocortex. They further indicate that specific interneuron types coordinate the release of these peptides and can induce a long-lasting increase of the excitability of the neocortical network.
Key Words: cholecystokinin • corticotropin-releasing factor • GABAergic interneuron • neurokinin B • pyramidal cells
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Introduction |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialReferences |
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Neurons of the neocortex are broadly classified as excitatory glutamatergic pyramidal cells and inhibitory GABAergic interneurons (Peters and Jones 1984
; Connors and Gutnick 1990; DeFelipe and Farinas 1992; Thomson and Deuchars 1994; Somogyi and others 1998). As compared with pyramidal cells, interneurons are highly diverse in terms of morphological and firing properties as well as gene expression patterns (DeFelipe 1993; Kubota and Kawaguchi 1994; Cauli and others 1997, 2000; Kawaguchi and Kubota 1997; Gupta and others 2000; Wang and others 2004). Furthermore, interneurons are key coordinators of intercellular communication via GABAergic and electrical connections and through the release of neuropeptides. GABAergic and electrical connections play a critical role in gating rapid responses to afferent inputs and in synchronizing neuronal populations (Galarreta and Hestrin 1999, 2001a, 2001b; Gibson and others 1999; Beierlein and others 2000; Amitai and others 2002; Meyer and others 2002; Traub and others 2003; Galarreta and others 2004). Neuropeptides differ from classical neurotransmitters in size, synthesis, and mechanism of action (Zupanc 1996; Hokfelt and others 2000; Baraban and Tallent 2004), and their explicit contribution to neocortical network activities remains largely unknown.
Psychopharmacological studies indicate that several neuropeptides expressed in the neocortex are involved in emotional and cognitive processes (Freund 2003). Among these peptides, cholecystokinin (CCK) and corticotropin-releasing factor (CRF) participate in anxiety-like behaviors (van Megen and others 1996; Hernandez-Gomez and others 2002; Reul and Holsboer 2002), whereas neurokinin B (NKB)–mediated transmission is a target of antipsychotic drugs (Kamali 2001). It is known that these peptides are expressed by subsets of GABAergic interneurons. In contrast to CCK that has been extensively used as a marker of neuronal-type diversity (Somogyi and others 1984; Demeulemeester and others 1988; Cauli and others 1997, 2000; Kubota and Kawaguchi 1997), the distribution of CRF and NKB has been the subject of a limited number of studies (Kaneko and others 1998; Taki and others 2000; Hioki and others 2004). In particular, the respective localization of these peptides and their distribution relative to neuronal types defined physiologically or neurochemically is still unclear.
These three peptides activate distinct G-protein–coupled receptors whose expression patterns and functional effects in neocortical neurons are largely uncharacterized. The actions of CCK are mediated by CCK-A and CCK-B receptors. CCK-B is expressed throughout the brain (Noble and Roques 1999; Mercer and others 2000), whereas CCK-A, detected only in a few brain areas, is absent from the neocortex (Carlberg and others 1992). CRF is selective for the CRF-1 and CRF-2 receptors. CRF-1 is widely distributed in the brain, whereas CRF-2 is virtually restricted to subcortical structures (Chalmers and others 1995; Lovenberg and others 1995; Bittencourt and Sawchenko 2000; Van Pett and others 2000). NKB belongs to the family of tachykinins that additionally comprise substance P and NKA and act at NK-1, NK-2, and NK-3 receptors. NK-3 is the most selective for NKB (Shigemoto and others 1990; Guard and others 1991) and is widely distributed in the brain including the neocortex (Ding and others 1996; Shughrue and others 1996; Langlois and others 2001). Postsynaptic activation of this receptor increases the excitability of neocortical pyramidal neurons (Stacey and others 2002; Rekling 2004).
The aim of the present study was to identify neocortical neuronal types involved in the peptidergic transmission mediated by NKB, CRF, and CCK. To this end, the expression of these peptides and their receptors was investigated by patch-clamp and single-cell reverse transcription–polymerase chain reaction (scPCR) (Lambolez and others 1992) in neocortical neurons classified by unsupervised clustering analysis based on both their electrophysiological and molecular phenotypes (Cauli and others 2000; Monyer and Markram 2004). We found that NKB and CRF were selectively expressed in interneurons, whereas CCK distributed to all neuronal classes. The receptors for these peptides were primarily found in pyramidal cells. Activation of these receptors increased the excitability of pyramidal neurons.
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Materials and Methods |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialReferences |
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Slice Preparation
Young Wistar rats (postnatal days 14–18) were decapitated, and the brains were quickly removed and placed into cold (
4 °C) oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 20 glucose, 5 pyruvate, and 1 kynurenic acid. Parasagittal sections (300-µm thick) of cerebral sensorimotor cortex were prepared as described previously (Cauli and others 1997). Slices were collected and subsequently transferred to a holding chamber containing ACSF saturated with 95% O2/5% CO2 and held at room temperature.
Whole-Cell Recordings
Individual slices were then transferred to a recording chamber placed under a microscope (Axioscop FS Zeiss, Oberkochen, Germany). Slices were maintained immersed, continuously superfused at 1–2 ml/min with oxygenated ACSF at room temperature, and allowed to equilibrate for at least 1 h before recordings. Patch micropipettes (3–5 M
) were pulled from borosilicate glass capillaries (1.5-mm outer diameter, 0.86-mm inner diameter, Harvard Apparatus, Les Ulis, France) on a Brown–Flaming micropipette puller (Model P-97, Sutter Instrument, Novato, California). Electrodes were filled with 8 µl of internal solution containing (in mM) 123 K-gluconate, 21 KCl, 3 MgCl2, 0.5 EGTA, and 10 HEPES plus 2 mg/ml biocytin (Sigma, St Louis, Missouri). The pH was adjusted to 7.2 and osmolarity to 285/295 mOsm. Whole-cell recordings were made from neocortical neurons identified under infrared video microscopy (Stuart and others 1993) and with a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, California) connected to a Digidata 1200B interface board (Axon Instruments). Signals were amplified and collected using the data acquisition software P-Clamp-8.02 (Axon Instruments). Resting membrane potential was measured just after passing in whole-cell configuration, and only cells with a resting membrane potential more hyperpolarized than –50 mV were analyzed. Membrane potentials were not corrected for junction potential. Cells were maintained at a holding potential of –60 mV by continuous current injection, and their firing behavior was tested by applying depolarizing current pulses. Action potential discharges were recorded by using the I-clamp fast mode of the amplifier.
The signals were filtered at 5 kHz, digitized at 10 kHz, saved to a personal computer, and analyzed off-line with Clampfit 9 software (Axon Instruments).
Drugs
The drugs used were NKB (1 µM, Bachem, Weil am Rhein, Germany), senktide (500 µM, Tocris, Bristol, UK), CCK-8s (1 µM, Bachem), CRF (250 nM, Tocris), LY 225910 (CCK-B receptor antagonist, 200 nM, Tocris), SB 222200 (NK-3 receptor antagonist, 1 µM, Tocris), and NBI 27914 hydrochloride (CRF-1 receptor antagonist, 200 nM, Tocris). Drug stock solutions were prepared in distilled water or in 0.1% dimethyl sulfoxide (LY 225910 and NBI 27914 hydrochloride), and stored at –80 °C. On the day of use, drugs were diluted in ACSF to their working concentration. Agonists were applied for 60 s, whereas their specific antagonists were applied for longer periods (
10 min) to examine their effects alone and in the presence of the agonist. All recordings were performed in the presence of bicuculline methiodide (GABA-A receptor antagonist, 10 µM, Sigma) and kynurenic acid (nonspecific glutamate receptors antagonist, 1 mM, Sigma) to prevent glutamatergic and GABAergic transmission
Single-Cell Reverse Transcription–Polymerase Chain reaction
At the end of the recording, the cell's cytoplasm was aspirated into the recording pipette while maintaining the tight seal. Then the pipette was removed delicately to allow outside-out patch formation. Next, the content of the pipette was expelled into a test tube, and reverse transcription (RT) was performed in a final volume of 10 µl as described previously (Lambolez and others 1992). Next, 2 steps of polymerase chain reaction (PCR) were performed essentially as described previously (Ruano and others 1995). The cDNAs present in 10 µl of the RT reaction first were amplified simultaneously by using all the primer pairs described in Table 1 (for each primer pair the sense and antisense primers were positioned on 2 different exons). Taq polymerase (2.5 U; Qiagen GmbH, Hilden, Germany) and 20 pmol of each primer were added to the buffer supplied by the manufacturer (final volume, 100 µl), and 21 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 35 s) of PCR were run. Second rounds of PCR were performed using 2 µl of the first PCR product as template. In this second round, each cDNA was amplified individually with a second set of primer pair internal to the primer pair used in the first PCR (nested primers, see Table 1) and positioned on 2 different exons. Thirty-five PCR cycles were performed (as described earlier). Then 10 µl of each individual PCR were run on a 2% agarose gel, with 
X174 digested by HaeIII as a molecular weight marker and stained with ethidium bromide. The RT-PCR protocol was tested on 500 pg of total RNA purified from rat neocortex. All the transcripts were detected from 500 pg of neocortical RNA except for the CRF-2 and CCK-A mRNAs, which were detected from 500 pg of whole-brain RNA. The sizes of the PCR-generated fragments were as predicted by the mRNA sequences (see Table 1). A control for mRNA contamination from surrounding tissue was performed by placing a patch pipette in the slice without establishing a seal. Positive pressure was then interrupted, and following the removal of the pipette, its content was processed as described. No PCR product was obtained using this protocol (n = 20).
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Intracellular Labeling
Identification of some of the recorded neurons was confirmed by histochemical revelation of intracellular biocytin, performed using the ABC elite kit (Vector Laboratories, Burlingame, California). All pyramidal neurons (n = 38) exhibited triangular somata with a prominent apical dendrite extending to layer I, whereas interneurons (n = 7) showed bipolar/bitufted or multipolar dendritic morphology (not shown).
Electrophysiological Analysis
Electrophysiological parameters were measured as previously described (Cauli and others 1997, 2000). The parameters are input resistance determined from the application of an 800-ms hyperpolarizing current step (–50 pA), amplitude of the first and the second spike, spike amplitude reduction, duration of the first and the second spike measured at half amplitude, and spike duration increase. The amplitude of the afterhyperpolarizations (AHPs) of the first and the second spike was measured between the spike threshold and the peak of the AHP. Accommodation parameters were also measured on discharges elicited by the application of 800-ms depolarizing current pulses. Thus, the frequency adaptation occurring during the first 200 ms of discharge (early adaptation) and the frequency adaptation occurring after the first 200 ms of discharge (late adaptation) were measured. In some neurons, a pronounced reduction of the amplitude of the action potentials was followed by an increase of the spike amplitude during an 800-ms depolarization protocol. This firing property was measured as the difference between the peak of the smallest action potential and the peak of the following biggest action potential and was termed accommodative hump (Cauli and others 2000). Two additional parameters, the minimal discharge frequency observed during 800 ms of depolarization for a current step eliciting an initial firing frequency of 50 Hz and the time at which this minimal frequency occurred, took into account irregularities in the discharge. Three groups of interneurons were subjectively defined according to their intrinsic electrophysiological properties and their action potential firing behavior. Fast-spiking (FS) cells were identified based on their thinner action potential and the high amplitude of the AHP (Kawaguchi 1993, 1995; Kawaguchi and Kubota 1993). FS cells also exhibited higher firing rates with little spike frequency adaptation in response to depolarizing current pulses. In contrast, regular-spiking nonpyramidal cells (RSNP) exhibited broad action potentials and were unable to sustain high frequencies of repetitive discharges (Kawaguchi 1995). Finally, interneurons displaying an irregular firing pattern were classified as Irregular-Spiking (IS) cells (Cauli and others 1997, 2000).
Statistical Analysis
All data are presented as mean ± standard error of mean unless otherwise stated. Analysis by Student's t-test was performed for paired and unpaired observations. Mann–Whitney U-test was employed to compare electrophysiological properties between cell types. P values of 
0.05 were considered statistically significant. The multidimensional data set (13 molecular variables) was explored using principal components analysis (PCA) to examine relationships between the expressions of each variable. PCA determines a set of principal components as linear combinations of original dimensions, such that the first principal component is in the direction of highest variance of the distribution, the next principal component is in the direction of highest of the remaining variance, and so on (Mardia and others 1979). Spearman rank correlation test was employed to determine the degree of association between the molecular markers expression. P values of 0.01 were considered statistically significant. The cluster analysis was used to classify the neocortical neurons sampled without a priori knowledge (Cauli and others 2000) of the number of groups by combining the molecular and 14 electrophysiological variables described earlier. After standardizing the data, cluster analysis was performed using squared Euclidean distances and Ward's method linkage rules (Ward 1963). A Thorndike analysis of the critical threshold was conducted to suggest the likely number of different clusters in the data set (Thorndike 1953). PCA, descriptive statistics, and cluster analysis were calculated with Statistica v 6.0 (StatSoft France, Paris, France).
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Results |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialReferences |
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Neocortical neurons (n = 157) from layers II/III and V were electrophysiologically characterized and subsequently analyzed by scPCR. Pyramidal cells and interneurons were selected according to the shape of their soma and proximal dendrites, as seen with infrared video microscopy, and 14 electrophysiological parameters were determined for each neuron (see Materials and Methods and Table 3). The scPCR protocol was designed to detect the expression of mRNAs encoding NKB, CCK, and CRF and their respective receptors (NK-3, CCK-A and CCK-B, CRF-1 and CRF-2) together with 8 molecular markers commonly used to define subtypes of neocortical neurons: GABA-synthesizing enzymes, Glutamic acid decarboxylase (GAD65 and GAD67); vesicular glutamate transporter (vGlut1); calcium-binding proteins calretinin (CR) and calbindin (CB); and neuropeptides vasoactive intestinal peptide (VIP), somatostatin (SOM), and neuropeptide Y (NPY). The identity of some of the recorded neurons was confirmed by their morphology following histochemical revelation (see Materials and Methods). In this report, cells positive for GAD65 and/or GAD67 are denoted as GAD positive, and these mRNAs are thus considered as a single molecular variable. CCK-A and CRF-2 mRNAs were never detected in our sample of neocortical neurons, as expected from their expression being restricted to subcortical structures (Moran and others 1986
; Chalmers and others 1995; Lovenberg and others 1995; Mercer and Beart 1997; Van Pett and others 2000), and are not further considered.
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Correlated Expressions of Molecular Markers
On the basis of coexpression of the different mRNAs at the single-cell level, a PCA was performed such that mRNAs with closely related expression pattern will appear close to each other and mRNAs with unrelated expression are farther apart (Fig. 1). Coexpression between each pair of markers (see cross-expression in Supplementary Table 1) was further analyzed using a Spearman rank correlation test to determine significant correlation (Table 2).
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The PCA clearly separated GAD and vGlut1 (Fig. 1 and Table 2), as expected from their differential distribution in interneurons and pyramidal cells, respectively. The PCA further disclosed 3 groups of markers (Fig. 1). In 1 group, the peptide receptors NK-3, CCK-B, CRF-1 were found close to vGlut1. Indeed, their expression presented a positive correlation with that of vGlut1 and an inverse correlation with GAD (Table 2), suggesting that these receptors were preferentially expressed by glutamatergic pyramidal cells. On the other hand, interneuron markers distributed in 2 other groups close to GAD. One group comprised VIP, CR, NKB, and CRF and the other group NPY, CB, and SOM. Indeed, it is well established that VIP and CR are frequently coexpressed and define interneuron populations distinct from those expressing SOM, NPY, or CB (Kubota and Kawaguchi 1994
; Kubota and others 1994; Cauli and others 1997; Kawaguchi and Kubota 1997; Porter and others 1998). Consistent with the PCA, NKB and CRF were preferentially coexpressed, and each presented a positive correlation with VIP and GAD and a negative correlation with vGlut1 (Table 2). These results suggest that NKB and CRF are preferentially expressed in VIP-containing GABAergic interneurons. In contrast, CCK did not correlate with any marker of neocortical neuronal types (Fig. 1 and Table 2). These results suggest that peptidergic transmission involving NKB, CRF, and CCK and their receptors may map to neuronal types previously defined using other parameters.
Cluster Analysis of Neocortical Neurons
A classification of the recorded neocortical neurons was then performed using unsupervised cluster analyses based on the combination of the 13 scPCR variables used in the PCA and the 14 electrophysiological parameters (see Materials and Methods and Table 3). This multifactorial analysis perfectly segregated pyramidal cells (black boxes, n = 77) and interneurons (gray boxes, n = 80) into 2 clusters of cells separated by a large aggregation distance (Fig. 2A). The histogram in Figure 2B shows the percentage of pyramidal cells and interneurons expressing each molecular marker. A representative example of electrophysiological and molecular characterization of pyramidal cells is shown in Figure 4A. Electrophysiological and molecular properties of each neuron of the pyramidal cell cluster are listed in Supplementary Tables 2 and 3. All pyramidal cells expressed vGlut1 but not GAD, confirming the reliability of their visual identification and glutamatergic nature (Fremeau and others 2004). As expected from the PCA results, the vast majority of NK-3, CRF-1, and CCK-B mRNAs were found in pyramidal cells (95%, 76%, and 78%, respectively, Fig. 2B). In contrast, NKB and CRF mRNAs were only expressed by interneurons except for 3 pyramidal cells expressing CRF. CCK was detected in both interneurons (44.6%) and pyramidal cells (55.4%). SOM and NPY were predominantly expressed by interneurons (67% and 72%, respectively) but were also found, to a lesser extent, in pyramidal cells as previously described in the case of SOM (Ong and others 1994). VIP was only found in interneurons.
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These results suggest that pyramidal cells selectively express NK-3, CRF-1, and CCK-B and thus would be the primary neuronal target of NKB, CRF, and CCK in the neocortex. They further suggest that in this structure, NKB, CRF, and CCK originate from interneurons, although the last is also present in pyramidal cells. Considering the diversity of neocortical interneurons, we next examined the distribution of these peptides with respect to interneuron subtypes.
Cluster Analysis of Interneurons
To characterize the type of interneurons expressing NKB, CRF, and CCK, our sample of 80 interneurons was classified using the same cluster analysis as described earlier. The Thorndike threshold (dotted line) revealed 3 main groups of cells corresponding to branches a, b, and c in the tree diagram (Fig. 3). These clusters were named according to their prominent characteristics: VIP-cluster, SOM-cluster, and FS-cluster. Electrophysiological and molecular properties of each interneuron of these clusters are listed in Supplementary Tables 2 and 3.
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The properties of the interneurons assigned to these clusters (Fig. 3 and Table 3) were remarkably similar to those obtained in a previous classification based on the same analysis, but that took into account neither the neuropeptide receptors nor NKB and CRF (Cauli and others 2000
). Strikingly, when these latter molecular parameters were removed from the analysis of the present sample, the only modification observed was that 2 cells moved from the FS-cluster, 1 falling into the VIP-cluster and 1 into the SOM-cluster (not shown). This confirms the robustness of the present multifactorial classification and indicates that NKB, CRF, and CCK distribute to interneuron subtypes defined otherwise.
The major molecular characteristics of neurons in the VIP-cluster were the high occurrence of VIP (n = 27/28, 96%) and CR (68%). This cluster comprised RSNP cells (57%) and IS cells (43%) that segregated selectively to this cluster. The electrophysiological hallmarks of the VIP-cluster were the large amplitude reduction, duration increase of their spikes, and the large early adaptation and low minimal frequency of their discharge that presented a marked accommodative hump (Table 3). A representative example of a VIP-cluster neuron is shown in Figure 4B. The SOM-cluster was exclusively composed of RSNP cells (n = 23/23) that were characterized by the large amplitudes of their 2 first action potentials (Table 3). In addition to the predominant occurrence of the SOM mRNA (96%), these neurons expressed to a lower extent CB and CR (52% and 43%, respectively, Fig. 3). A representative example of a SOM-cluster neuron is shown in Figure 4C. Most neurons of the FS-cluster (n = 22/29, 76%) did correspond to the electrophysiologically defined FS cell type characterized by high firing rates with little spike frequency adaptation (Kawaguchi 1993, 1995; Cauli and others 1997). Other neurons in the FS-cluster were RSNP cells. As a whole, neurons of the FS-cluster characteristically exhibited a small input resistance, short spike durations, large AHPs, and a high frequency of discharge (Table 3). These cells frequently expressed NPY (48%) and to a lower extent CB and SOM (38%). A representative example of an FS-cluster neuron is shown in Figure 4D.
The interneuron classes demarcated in this study showed differential expression patterns of NKB, CRF, and CCK (Fig. 3). The VIP-cluster was the only class where expression of the 3 peptides was found (57%, 50%, and 57% of VIP-cluster neurons expressed NKB, CRF, and CCK, respectively). Indeed, NKB was almost exclusively found in neurons of the VIP-cluster where 37.5% of NKB-containing cells additionally expressed both CCK and CRF. In the SOM-cluster, CRF was frequently detected (48%), CCK was only detected in 17% of the neurons, and NKB was not found. Among the 3 peptides, only CCK frequently occurred in the FS-cluster (45%). Although expression of neuropeptides overlapped onto different interneuron clusters, neurons from the VIP-cluster appeared to be a main source for the coordinated actions of NKB, CRF, and CCK in the neocortex.
Expression of neuropeptide receptors was only found at low frequency in interneurons and did not appear to be specific of a given cluster (Fig. 3). Indeed, NK-3, CRF-1, and CCK-B were only detected in 1, 6, and 7 interneurons, respectively. Hence, NKB, CRF, and CCK are expected to act primarily on pyramidal neurons in the neocortex.
NKB, CCK, and CRF Increase the Excitability of Pyramidal Cells
Whole-cell patch-clamp recordings were then carried out in current-clamp mode to assess the effects of NKB, CRF, and CCK on the membrane potential and the firing rate of pyramidal cells. For all these experiments, exogenous peptides were bath applied for 60 s in the presence of glutamate and GABA transmission blockers (kynurenic acid, 1 mM; bicuculline, 10 µM).
In a first series of experiments, membrane potential was set at –60 mV, and consecutive hyperpolarizing and depolarizing current pulses (1 s) were applied every 20 s. Bath application of senktide (a specific NK-3 agonist, 0.5 µM), NKB (1 µM), CCK (1 µM), and CRF (0.25 µM) produced small membrane depolarization (n = 10/14, n = 3/5, n = 4/6, and n = 6/10, respectively; see Fig. 5A–C and Table 4). When action potential firing was blocked by tetrodotoxin (TTX, 1µM), depolarization induced by senktide (n = 9), CCK (n = 2), and CRF (n = 3) persisted, indicating that these effects depended on postsynaptic receptors expressed by pyramidal cells (Table 4). The membrane resistance was not significantly modified by CRF or CCK application, as measured by responses to hyperpolarizing current steps. However, the senktide-induced depolarization was associated with a small but significant increase in membrane resistance of +4.3 ± 1.3% in the absence of TTX and +3.5 ± 1.4% in the presence of TTX (P < 0.05). During these experiments, depolarizing current pulses (100 pA) elicited a discharge presenting the marked early accommodation distinctive of pyramidal cells (Fig. 5). The number of spikes in the discharge was significantly increased during the membrane depolarization induced by senktide (+118 ± 25.8%, Fig. 5A), CCK (+50.9 ± 15.2, Fig. 5B), and CRF (+33 ± 17.5%, Fig. 5C), as compared with that obtained in control conditions. The firing rates recovered their control values at the end of the membrane depolarization, except with CRF for which the number of spikes was significantly different from that measured in control conditions (Fig. 5A–C). The effects of NKB (not shown) were qualitatively similar to those of senktide in these experiments.
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In another set of experiments, the effects of these peptides were studied when pyramidal cells were maintained to about 5 mV below spike threshold (Fig. 5D). Under these conditions, NKB (n = 14/14), CCK (n = 9/9), and CRF (n = 13/13) consistently induced membrane depolarization (6 ± 0.4 mV, 3.9 ± 0.7 mV, and 5 ± 0.48 mV, respectively). The depolarization was always accompanied by a long-lasting discharge of action potentials that lasted for 14.7 ± 2.4 min (NKB), 6.4 ± 0.45 min (CCK), and 20.5 ± 4.4 min (CRF). The duration of this discharge exceeded by far that of depolarization.
It is noteworthy that using the above protocol, all pyramidal cells tested responded to NKB, CCK, or CRF, although mRNA expression of their cognate receptors was only detected in part of the pyramidal cells analyzed by scPCR (see Fig. 2). The involvement of these receptors in the responses of pyramidal neurons to NKB, CCK, and CRF was thus assessed using their selective antagonists (Fig. 6). Both the membrane depolarization and the discharge of action potentials were prevented when NKB, CCK, and CRF were applied in the presence of a selective NK-3 antagonist (SB 222200, 1 µM, n = 5), CCK-B antagonist (LY 225910, 0.2 µM, n = 6), and CRF-1 antagonist (NBI 27914, 0.2 µM, n = 4), respectively. Responses to CCK and CRF recovered after washout of the antagonist. In contrast, recovery of the NKB-induced response was not observed even after more than 20 min of SB 222200 washout. These results indicate that NK-3, CCK-B, and CRF-1 are expressed by the vast majority of neocortical pyramidal neurons. Activation of any of these receptors by its natural agonist induced a depolarization of pyramidal cells and a long-lasting increase of their excitability that resulted in an action potential discharge when neurons were sufficiently depolarized.
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NKB, CCK, and CRF Act Primarily on Pyramidal Cells
We next verified that NK-3, CCK-B, and CRF-1 are only expressed in a small proportion of neocortical interneurons as suggested by their mRNA expression profiles. Interneurons were recorded in current-clamp mode at resting membrane potential (range, –59 to –65 mV), and exogenous peptides were bath applied for 60 s in the presence of TTX (1 µM). No effect of senktide (0.5 µM) was observed on RSNP (n = 10 out of 10) or FS (n = 3 out of 3) neurons. Upon CCK application (1 µM), no response was detected in 16 out of 17 RSNP and 5 out of 6 FS cells tested. A depolarizing effect of CCK was observed in the remaining RSNP (+3.8 mV) and FS (+10.2 mV) neurons. Finally, no effect of CRF (0.25 µM) was observed in FS neurons (n = 3 out of 3) and in 12 out of 13 RSNP cells tested. The remaining RSNP cell was depolarized by CRF (+15.2 mV). These results confirm that NK-3, CCK-B, and CRF-1 are predominantly expressed by pyramidal neurons in the neocortex.
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Discussion |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialReferences |
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The expression of NKB, CRF, and CCK and their receptors was investigated by patch-clamp and scPCR in neocortical neurons classified by unsupervised clustering analysis based on both molecular and electrophysiological properties. NKB was almost exclusively found in VIP-cluster interneurons, CRF expression was restricted to VIP- and SOM-cluster interneurons, whereas CCK was found in all clusters demarcated in the present study. NK-3, CRF-1, and CCK-B were predominantly expressed in pyramidal cells, and their activation increased the excitability of these neurons. This suggests that peptidergic transmission mediated by NKB, CRF, and CCK allows GABAergic interneurons to increase the excitability of the neocortical network.
Expression Patterns of NKB, CCK, and CRF
Our results are in overall good agreement with previous histochemical studies of their distribution. Indeed, our findings that all NKB-positive cells expressed GAD and that NKB was detected in 53% of VIP-, 29% of CR-, and 36% of CRF-expressing interneurons (not shown) are consistent with immunohistochemical reports (Marksteiner and others 1992; Kaneko and others 1998). Similarly, immunohistochemical evidence suggesting that CRF predominantly colocalizes with VIP and to a lesser extent with SOM (Demeulemeester and others 1988; Taki and others 2000; Karube and others 2004) supports our findings that 46.5% of VIP- and 31.5% of SOM-positive interneurons expressed CRF (not shown).
The present observation that the CCK mRNA is expressed in both interneurons (44.6%) and pyramidal cells (55.4%) is consistent with previous scPCR (Cauli and others 1997, 2000; Toledo-Rodriguez and others 2005) and in situ hybridization studies (Burgunder and Young 1990; Schiffmann and Vanderhaeghen 1991; Senatorov and others 1995). CCK is by far the most abundant neuropeptide expressed in the cerebral cortex where it is found at a concentration of 5 nmole/g protein as measured by radioimmunoassay (Schneider and others 1979; Beinfeld and others 1981; Crawley 1985). For comparison (Crawley 1985), it is about seven times more abundant than VIP (below 0.7 nmole/g protein) and about 50 times more abundant than CRF (below 0.1 nmole/g protein). However, immunohistochemistry reveals fewer cells positive for CCK than for VIP or CRF (Taki and others 2000; Karube and others 2004), suggesting that a large proportion of neocortical CCK immunoreactivity is not detected by immunohistochemical staining. Indeed, colchicine pretreatment allows CCK immunoreactivity to become detectable in pyramidal cells (Morino and others 1994). Hence, we propose that CCK is expressed at low levels in multiple neocortical neuronal types but can nonetheless be released because large CCK efflux is observed by in vivo microdialysis (Becker and others 2001). Furthermore, the present results showing the high occurrence of the CCK mRNA in VIP-cluster neurons and its low frequency in SOM-cluster neurons are in good agreement with immunohistochemical colocalization of these markers (Somogyi and others 1984; Kubota and others 1994; Kubota and Kawaguchi 1997).
The different sensitivities of scPCR and immunohistochemistry may also account for the presently found coexpression patterns (e.g., CCK and SOM, CCK and NPY) that are rarely observed by immunohistochemistry (Kubota and others 1994). The detection of the vGlut1 mRNA in 20% of GABAergic interneurons of the present study is consistent with its reported expression in several GABAergic cell types throughout the brain (Danik and others 2005). Nonetheless, the absence of vGlut1 immunoreactivity in symmetrical synapses (Alonso-Nanclares and others 2004) and the clear GABAergic phenotype of interneuron output in the neocortex suggest a low level of vGlut1 expression in these interneurons.
Expression Patterns of NK-3, CCK-B, and CRF-1
Very few studies have investigated the expression of these receptors in the neocortex, either at the histochemical or at the functional level. Here, their expression was only found at low frequency in interneurons and did not appear to be specific of a given interneuron cluster. Our results thus indicate that in the neocortex, these receptors are expressed predominantly by pyramidal neurons, consistent with previous histochemical reports of NK-3 and CRF-1 distribution in this structure (Ding and others 1996; Shughrue and others 1996; Chen and others 2000; Langlois and others 2001). Although the presence of CCK-B in the neocortex has been demonstrated (Mercer and others 2000), we first describe its distribution with cellular resolution. In the present report, responses to NKB, CRF, and CCK persisted in the presence of glutamate and GABA transmission blockers and TTX and were blocked by selective antagonists of NK-3, CRF-1, and CCK-B. This clearly demonstrates the functional expression of these receptors in pyramidal cells. Conversely, the effects of these peptides appeared to be mediated exclusively by these receptors, with no contribution from other receptors of their respective families. Indeed, CCK-A and CRF-2 were not detected in this study, as expected from their selective expression in subcortical structures (Moran and others 1986; Chalmers and others 1995; Lovenberg and others 1995; Mercer and Beart 1997; Van Pett and others 2000). Furthermore, NK-1 is absent from pyramidal cells, and NK-2 is not expressed in the neocortex (Ding and others 1996; Vruwink and others 2001; and our unpublished PCR observations). Consistent with this, NKB effects were very similar to those of the selective NK-3 agonist, senktide.
When pyramidal neurons were sufficiently depolarized, all showed a response to activation of NK-3, CRF-1, and CCK-B, although corresponding mRNAs were detected in only a fraction of pyramidal neurons. In a previous study where glutamate AMPA receptor mRNAs were quantified at the single-cell level on cultured hippocampal pyramidal cells, the detection limit by scPCR was estimated to be around 25 molecules of mRNA harvested in the patch pipette, due to low RT efficiency. Furthermore, only one-third of the cellular mRNA was harvested by somatic patching, the other two-third being presumably localized in the dendritic tree (Tsuzuki and others 2001). Assuming a similar overall efficiency for peptide receptors in the present study, this would indicate that the mean number of peptide receptor mRNA per pyramidal cell was around 75 and would thus belong to the class of low-abundance mRNA (Velculescu and others 1995). Nonetheless, the functional expression of these receptors observed in the present study clearly indicates that the vast majority of neocortical pyramidal cells do coexpress NK-3, CCK-B, and CRF-1.
Neuronal Types Involved in Peptidergic Transmission Mediated by NKB, CCK, and CRF
NKB, CCK, and CRF and their receptors distributed relatively selectively to neuronal clusters defined by the multifactorial analysis. This indicates that local transmission using these peptides operates in the frame of the neocortical functional architecture as defined by its neuronal constituents. The present unsupervised clustering disclosed 4 main classes of neurons comprising pyramidal cells and 3 populations of interneurons, as found in most multifactorial studies of neocortical neuronal diversity so far (Kawaguchi and Kubota 1993, 1997; Kubota and Kawaguchi 1994; Kubota and others 1994; Kawaguchi 1995; Cauli and others 2000; Toledo-Rodriguez and others 2004). The present classes correspond to a probabilistic definition based upon the distribution of a large set of electrophysiological and molecular properties. Hence, these classes cannot perfectly match with neuronal types defined on the basis of only a few discriminant essential properties (Tyner 1975). Nonetheless, the present clusters show a good overall correspondence with previously defined neuronal types. Indeed, it is accepted that pyramidal cells and GABAergic interneurons form 2 distinct types of neurons with distinct function. Furthermore, our interneuron clusters showed distinctive hallmarks that define VIP-containing (Kubota and Kawaguchi 1994; Gabbott and Bacon 1997; Bayraktar and others 1997; Cauli and others 1997, 2000; Porter and others 1998, 1999; Ferezou and others 2002), SOM-containing (Hendry and others 1984; Schmechel and others 1984; Somogyi and others 1984; Lin and others 1986; Demeulemeester and others 1988; Kubota and Kawaguchi 1994; Kubota and others 1994; Gonchar and Burkhalter 1997; Wang and others 2004), and FS interneurons (Connors and Gutnick 1990; Kawaguchi 1993; Kawaguchi and Kubota 1993). In the present study, although expression of NKB, CRF, and CCK and their receptors overlapped onto different neuronal types, neurons from the VIP-cluster appear to be the coordinating center and pyramidal neurons the main target of neurotransmission using these peptides.
NKB, CRF, and CCK Increase the Excitability of the Neocortical Network
We found that senktide, NKB, CRF, and CCK induced a TTX-resistant depolarization of pyramidal neurons and elicited a long-lasting discharge of action potentials when neurons were sufficiently depolarized. An increase in membrane resistance was additionally observed with senktide and NKB. These functional data and the almost selective expression of NK-3 in pyramidal neurons observed in the present study are consistent with the reported effects of senktide and NKB in the neocortex (Stacey and others 2002; Rekling 2004). These previous studies additionally revealed that NK-3 activation induced an increase in the frequency of excitatory postsynaptic currents that was blocked by TTX. This suggests that these receptors distribute selectively to the somatodendritic compartment of pyramidal cells. Direct somatodendritic effects of CRF and CCK on neocortical pyramidal neurons have not been reported so far. However, it is known that CRF (Aldenhoff and others 1983; Haug and Storm 2000; Blank and others 2003) and CCK (Dodd and Kelly 1981; Boden and Hill 1988; Bohme and others 1988; Hughes and others 1990; Shinohara and Kawasaki 1997) increase the excitability of pyramidal neurons in the hippocampus.
Taken together, these 3 peptides originate mostly from interneurons and only colocalize in neurons of the VIP-cluster. Their coordinated effects converge primarily onto pyramidal cells where they likely modulate multiple intracellular effectors through activation of adenylate cyclase (CRF) and phospholipase C (NKB and CCK) signaling pathways (Chen and others 1986; Battaglia and others 1987; Pihoker and others 1992; Shinohara and Kawasaki 1994; Shigeri and others 1996; Haug and Storm 2000; Yang and others 2003). Neuropeptide release requires a high level of activity and may depend upon a sustained elevation of intracellular calcium (Zupanc 1996; Ludwig and Pittman 2003; Baraban and Tallent 2004). Our results suggest that NKB, CRF, and CCK originating from inhibitory interneurons induce a long-lasting increase of the excitability of the neocortical network of pyramidal cells while the properties of the interneuron network would remain essentially unaffected. This may directly affect pyramidal neurons outputs to other structures. It is noteworthy that NK-3 is involved in several cognitive disorders including schizophrenia (Kamali 2001), whereas CRF-1 and CCK-B participate in anxiety and other emotional disorders (van Megen and others 1996; Hernandez-Gomez and others 2002; Reul and Holsboer 2002).
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialReferences |
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Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialReferences |
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Funding to pay the Open Access publication charges for this article was provided by the Centre National de la Recherche Scientifique.
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We thank Paul Schweitzer, Bruno Cauli, Nathalie Gibelin, Isabelle Férézou, and Elisa Hill for their valuable help.
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