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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Eur J Neurosci. Author manuscript; available in PMC Jun 12, 2008.
Published in final edited form as:
PMCID: PMC2426826

Projections from basal forebrain to prefrontal cortex comprise cholinergic, GABAergic and glutamatergic inputs to pyramidal cells or interneurons


The present study was undertaken to characterize the pre- and postsynaptic constituents of the basal forebrain (BF) projection to the prefrontal cortex in the rat, and determine whether it includes glutamatergic in addition to established γ-aminobutyric acid (GABA)ergic and cholinergic elements. BF fibres were labelled by anterograde transport using biotin dextran amine (BDA) and dual-stained for the vesicular transporter proteins (VTPs) for glutamate (VGluT), GABA (VGAT) or acetylcholine (VAChT). Viewed by fluorescence microscopy and estimated by stereology, proportions of BDA-labelled varicosities were found to be stained for VGluT2 (and not VGluT1 or 3), VGAT or VAChT (representing, respectively, ~15%, ~52% and ~19% within the infralimbic cortex). Each type was present in all, though commonly most densely in deep, cortical layers. Material was triple-stained for postsynaptic proteins to examine whether BDA+VTP+ varicosities might form excitatory or inhibitory synapses, respectively, labelled by postsynaptic density-95 kDA (PSD-95) or gephyrin (Geph). Viewed by confocal microscopy, a majority of BDA+/VGluT2+ varicosities were found to be apposed to PSD-95+ elements, and a majority of BDA+/VGAT+ varicosities to be apposed to Geph+ elements. Other series were triple-stained for cell marker proteins to assess whether the varicosities contacted interneurons or pyramidal cells. Viewed by confocal microscopy, BDA-labelled VGluT2+, VGAT+ and VAChT+ BF terminals were all found in contact with calbindin+ interneurons, whereas VGAT+ BF terminals were also seen in contact with parvalbumin+ interneurons and non-phosphorylated neurofilament+ pyramidal cells. Through distinct glutamatergic, GABAergic and cholinergic projections, the BF can thus influence cortical activity in a diverse manner.

Keywords: gephyrin, PSD-95, VAChT, VGAT, VGluT


Through widespread projections to the cerebral cortex, including dense projections to the prefrontal cortex (PFC), the basal forebrain (BF) plays important and diverse roles in the modulation of cortical activity in association with different behavioural states (Everitt & Robbins, 1997; Sarter et al., 2003; Weinberger, 2003; Jones, 2004). Although cholinergic neurons were thought to be of prime importance in the basalocortical system, non-cholinergic neurons subsequently appeared to be of equal though possibly different importance in mediating the influence of the BF upon cortical activity and behaviour (Dunnett et al., 1991; Wenk, 1997).

Based on the presence of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), retrograde tracing studies established that cortically projecting BF neurons comprise both acetylcholine- (ACh) and γ-aminobutyric acid (GABA)-synthesizing cells (Zaborszky et al., 1986; Fisher et al., 1988; Gritti et al., 1997). Yet the proportions of ChAT+ and GAD+ neurons only accounted for ~2/3 of the cortically projecting neurons (Gritti et al., 1997). The remaining contingent was proposed to utilize glutamate (Glu) as a neurotransmitter, since a major proportion of cortically projecting BF neurons contain phosphate-activated glutaminase (PAG; Manns et al., 2001), the synthetic enzyme for neurotransmitter Glu (Kaneko & Mizuno, 1988). However, PAG is also present in significant proportions of ChAT+ and GAD+ neurons, suggesting that Glu might be synthesized by cholinergic and GABAergic neurons in addition to putative glutamatergic BF neurons (Manns et al., 2001; Gritti et al., 2006).

To determine whether BF fibres innervating the cortex utilize Glu as a neurotransmitter and how they relate to those that utilize ACh or GABA, we examined immunostaining of their varicosities for the vesicular transporter proteins (VTPs) for Glu (VGluT1, 2 and 3; Bellocchio et al., 2000; Fremeau et al., 2001, 2002; Fujiyama et al., 2001), for ACh (VAChT; Gilmor et al., 1996; Arvidsson et al., 1997) and for GABA (VGAT; McIntire et al., 1997; Chaudhry et al., 1998), which serve as specific markers for neurotransmitter uptake and associated release (Takamori et al., 2000, 2001; Fremeau et al., 2004). Following iontophoretic application of biotin dextran amine (BDA, 10 000 MW) in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) in rats, BDA-labelled fibres and axon terminals were examined for VTP immunostaining in the PFC, where they densely innervate medial (infralimbic, IL) and lateral (ventrolateral orbital and lateral orbital, VLO/LO) regions (Saper, 1984; Luiten et al., 1987; Grove, 1988). Additional series were processed for double- or triple-staining of multiple VTPs with BDA to assess colocalization of VGluT, VAChT and/or VGAT. Stereological analysis was carried out to determine the proportions of VGluT, VAChT and VGAT terminals. To assess whether these terminals might form synapses, sections were triple-stained for BDA, VTPs and the postsynaptic proteins (PSPs) postsynaptic density-95 kDA (PSD-95), a marker for excitatory synapses (Sheng, 2001; Sassoe-Pognetto et al., 2003), or gephyrin (Geph), a marker for inhibitory synapses (Sassoe-Pognetto & Fritschy, 2000; Sassoe-Pognetto et al., 2000). Finally, sections triple-stained for BDA, VTPs and the cell marker proteins, non-phosphorylated neurofilament (NPNF; Campbell & Morrison, 1989; Kirkcaldie et al., 2002) for pyramidal cells or the calcium-binding proteins, parvalbumin (PV) or calbindin (CB) for interneurons (Kubota et al., 1994; DeFelipe, 1997; Gabbott et al., 1997), were examined to assess whether glutamatergic, cholinergic and GABAergic terminals contact principal and/or interneurons in the PFC.

Materials and methods

Animals and surgery

All procedures were approved by the McGill University Animal Care committee, and conform to the guidelines of the Canadian Council on Animal Care and the US NIH.

As described previously (Henny & Jones, 2006a,b), Long–Evans rats (200–250 g, Charles River Canada, St Constant, Quebec, Canada) were anaesthetized with ketamine/xylazine/acepromazine (65/5/1 mg/kg, i.p.) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) for surgery. Glass micropipettes (tip diameter 15–25 μm) were back-filled with a 0.5 M NaCl solution containing 2% 10 000 MW BDA (BDA-10 000, Molecular Probes, Eugene, OR, USA). Following trephination over the BF on each side, the micropipette was lowered into the region of the MCPO (from bregma: anterior–posterior (A–P), −0.5 mm; lateral, ± 2.5 mm; vertical, −8.5 mm) with the aid of a micropositioner (Model 660, David Kopf Instruments). A holding current of −300 nA was maintained [using a Microiontophoresis Dual Current Generator 260, World Precision Instruments (WPI), Sarasota, FL, USA] during the descent to avoid leakage of the solution. Microinjection of BDA was performed by iontophoresis applying positive current pulses (5–10 μA) in a duty cycle of 1 s (0.5 s on, 0.5 s off) for a period of 25–30 min through a stimulator (Pulsemaster A300, WPI) and stimulus isolation unit (Iso-Flex, A.M.P.I., Israel). After the injection, the micropipette was held in place for 10 min before being removed with renewed application of the holding current.

Rats were maintained for 5 or 6 days to allow anterograde transport of the tracer from BF to PFC (~4 mm). During survival, the rats were maintained with food and water ad libitum. They were subsequently perfused transcardially under deep sodium pentobarbital anaesthesia (100 mg/kg, i.p.) with ~500 mL 4% paraformaldehyde fixative solution. The brains were removed and put in a 30% sucrose solution for 2–3 days or until they sunk, after which they were frozen at −50 °C and stored at −80 °C for subsequent processing.


Forebrains, including cortex and BF, were cut in sections of 25 μm thickness, which corresponded to the maximal thickness permissible for complete immunostaining (see below). The sections were collected in eight series, using intervals of 200 μm between sections. The first series was processed for evaluation of the BDA injection site and fibre distribution in the forebrain under light microscopy. It was stained for BDA using the Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine intensified with nickel (DAB-Ni) and counterstained for Nissl substance with Neutral Red.

Adjacent series of sections containing the PFC were processed for double- or triple-fluorescent staining of BDA, VTPs, PSPs and/or cell marker proteins (Tables 1 and and2).2). As assessed under epifluorescent and confocal microscopy, it was first established that full penetration of antibodies through the 25-μm-thick sections occurred during double- and triple-fluorescent staining with respective use of 0.1% and 0.3% Triton X-100 (TX). Free-floating sections from each series were rinsed for 30 min in Trizma saline buffer (TS, 0.1M, pH 7.4) followed by incubation for 30 min with a blocking solution of normal donkey serum (NDS, 6% in TS) containing TX. Sections were subsequently incubated overnight at room temperature with one or two primary antibodies in TS containing 1% NDS and TX. The sections were then rinsed for 30 min and incubated for 3 h in appropriate secondary antibodies in NDS and TX. With the exception of double-staining for two VTPs (see below), sections were then rinsed for 30 min and incubated for 3 h in streptavidin for BDA revelation.

Table 1
Primary antibodies used for immunostaining of VTPs, PSPs and cell marker proteins
Table 2
Combination and sequential processing of primary and secondary antibodies along with streptavidin used for double- or triple-fluorescent staining of VTPs, PSPs, cell marker proteins and/or BDA

For double-labelling of BDA and one of the VTPs, sections were incubated first with primary antibodies against VAChT, VGATor VGluT (1, 2 or 3) and then with indocarbocyanine (Cy3)-conjugated secondary antibodies (from donkey, Dky) followed by cyanine (Cy2)-conjugated streptavidin for revelation of BDA (see Table 2, Double BDA/VTP).

For double-labelling of two VTPs, sections were incubated with two primary antibodies against VAChT, VGAT or VGluT2 (from different host species), and then with corresponding Cy2- and Cy3-conjugated secondary antibodies (from Dky; see Table 2, Double VTP/VTP).

For triple-labelling of BDA, VAChT and VGAT, sections were incubated with primary antibodies (from different host species) against VAChT and VGAT, and then with corresponding Cy3 or indodicar-bocyanine (Cy5) secondary antibodies (from Dky). The sections were subsequently incubated with Cy2-conjugated streptavidin for revelation of BDA (see Table 2, Triple BDA/VTP/VTP).

For triple-labelling of BDA, the VTPs and PSPs, sections were incubated with two primary antibodies (from different host species) against VAChT, VGAT or VGluT2 and Geph or PSD-95, and then with corresponding Cy3 or Cy5 secondary antibodies (from Dky). Then, sections were subsequently incubated with Cy2-conjugated streptavidin for revelation of BDA (see Table 2, Triple BDA/VTP/PSP).

For triple-labelling of BDA, the VTPs and the cell marker proteins, sections were incubated with two primary antibodies against VAChT, VGAT or VGluT2 and NPNF, PV or CB, respectively (from different host species), and then with appropriate Cy3 or Cy5 secondary antibodies (from Dky). They were subsequently incubated with Cy2-conjugated streptavidin for revelation of BDA (see Table 2, Triple BDA/VTP/cell marker protein).

All sections were mounted out of Trizma water, and the mounted sections dehydrated through alcohols, cleared in xylene and covers-lipped with Permount. According to z-axis measurements (below), the thickness of the mounted sections (~10 μm) was approximately 40% of the cut sections (25 μm).

Fluorescent microscopy and stereological analysis

Sections were examined for single- or double-labelling under light and epifluorescent microscopy with a Leica DMLB microscope or Nikon Eclipse E800, which were equipped with xyz motorized stages, video or digital camera and filters appropriate for FITC (or Cy2) and rhodamine (or Cy3) fluorescence. Single and composite images were acquired using Neurolucida software (MicroBrightField, MBF, Williston, VT, USA). Labelled varicosities or cells were randomly sampled and counted to obtain unbiased estimates of numbers or proportions using the Optical Fractionator probe of StereoInvestigator software (MBF).

Injection sites from eight rats (BDA 14, 15, 16, 18, 19, 20, 21 and 22) were examined under brightfield illumination in DAB-Ni/Neutral Red-stained material. In 14 injection sites (referred to as ‘cases’ on left or right sides), the labelled cells were centred in the MCPO-SI (on the left and/or right sides), and were used for analysis with double- and triple-fluorescent staining for BDA, VTPs, PSPs or cell marker proteins in the PFC. From these, six cases from three rats (BDA 16, 18 and 19) were selected for systematic examination and quantitative estimate of the proportions of BDA/VTP varicosities within the IL and VLO/LO regions of the PFC.

For stereological sampling and analysis, the IL was delimited according to cytoarchitectonic criteria (Krettek & Price, 1977; Hurley et al., 1991) by contours positioned at three antero-posterior levels at 400-μm intervals (11.8, 11.4 and 11.0 anterior to interaural zero with an average sampling area of ~1.00 mm2 per level; see Fig. 1). Contours of molecular (I), intermediate (II–III) and deep (V–VI) layers were also delineated within the IL for subsequent estimation of the proportions of sampled varicosities across layers within this region. Estimates of BDA/VTP varicosities were also obtained for the VLO/LO region for which a 900 μm × 900 μm square box was used at the same three levels, with a sampling area of ~0.80 mm2 per level (not shown).

Fig. 1
Injection site in BF and projection areas in PFC. (A) Atlas section through the BF where BDA 10 000 MW was placed by microiontophoretic application into the magnocellular preoptic nucleus (MCPO)-substantia innominata (SI) region (A ~8.2, mm anterior to ...

Having found double-labelling of BDA-positive (+) varicosities with VAChT, VGAT and VGluT2 (see Results), unbiased estimates of the total numbers of single BDA+ and double-labelled BDA+/VAChT+, BDA+/VGAT+ or BDA+/VGluT2+ varicosities were performed by stereology in five cases within the IL and VLO/LO. Counts were performed under a 100 × oil objective (with 1.40 numerical aperture) using the Optical Fractionator probe of the Stereo Investigator software (MBF) on the Leica microscope. Varicosities were sampled in the IL or VLO/LO at three levels (above). The grid size (150 μm × 150 μm) was set to be larger than the counting frame (75 μm × 75 μm) so as to sample 25% of the area in each of the regions. Counting was performed through ~8.5 μm of the section thickness (starting 1 μm from the surface of the mounted sections, which had an average thickness of ~10 μm). In each counting block, all BDA+ varicosities (in green, Cy2) were counted, including those which were or were not double-labelled for VTPs (in red, Cy3) to allow an estimate of the proportion of double-labelled varicosities for each VTP.

Double-labelling for VAChT or VGAT with VGluT2 and also VAChT with VGAT was assessed systematically in the IL and VLO/LO under epifluorescent illumination in three cases (Table 2).

Confocal microscopy and image processing

Confocal laser-scanning microscopy was applied to triple-labelled material for assessment of the presence of VGAT in BDA+/VAChT+ varicosities and for examination of the association of BDA+/VTP+ varicosities with postsynaptic elements or proximity to cortical neurons. Triple-stained sections for BDA/VAChT/VGAT, BDA/VTP/PSP or BDA/VTP/cell marker proteins were analysed with a Zeiss LSM 510 laser-scanning microscope equipped with Argon 488 nm, helium–neon 543 nm and helium–neon 633 nm lasers, respectively, for Cy2, Cy3 and Cy5 excitation, as well as with appropriate filters for detection of Cy2 (bandpass 500–530 nm, green), Cy3 (bandpass 565–615 nm, red) and Cy5 (bandpass 697–719 nm, infrared). Scanning was performed through a Plan-Apochromat 100 × (with 1.40 numerical aperture) objective and pinhole size setting of 1 Airy Unit for the three channels. Images were acquired for the three chromogens using the resident LSM 510 software, and consisted of stacks taken through the z-axis in optical slices of ~0.33 μm for PSP series or ~0.50 μm for cell marker protein series.

Rendered three-dimensional views of the image stacks were obtained using the image software Volocity 3.5.1 (Improvision, Lexington, MA, USA, http://www.improvision.com), which allowed interactive visualization, magnification and rotation of the three-dimensional images in order to determine the relative location of each of the elements from the three channels.

Figures of image stacks were composed using Volocity, and of single sections from the three orthogonal planes using LSM software. Adjustments for brightness and contrast in brightfield images and tonal range for each individual RGB channel (‘Adjust/levels’ command) in fluorescent images were performed with Adobe Photoshop Creative Suite (Adobe System, San Jose, CA, USA). Figures were composed in Adobe Illustrator Creative Suite (Adobe).

Statistical analysis of results (one- or two-way ANOVA, and Bonferroni-corrected post hoc paired comparisons) was performed using Systat (v10.2, San Jose, CA, USA).


BDA injection site and axonal labelling in the cortex

As described previously (Henny & Jones, 2006b), iontophoretic application of BDA-10 000 into the region of the MCPO-SI (Fig. 1A) produced a well restricted, spherical injection site (Fig. 1B) containing labelled cell bodies and dendrites (Fig. 1C). The average number of BDA-labelled neurons per injection was ~1400 (mean ± SEM, 1430.6 ± 315, n = 5), of which ~90% were located in the MCPO and ~6% in the SI. Although some cell bodies were labelled in the surrounding regions (olfactory tubercle, lateral olfactory tract nucleus or anterior amygdaloid area, representing ~3.5% of cells) and isolated cells were labelled in other adjacent structures in some cases (fundus of the striatum or piriform cortex), no cell bodies were labelled in distant regions, including the PFC, indicating minimal to no retrograde transport of the BDA-10 000 (Henny & Jones, 2006b).

BDA was found in fibres within regions known to receive abundant projections from MCPO-SI, including both cortical and subcortical areas (Saper, 1984; Luiten et al., 1987; Grove, 1988; Gritti et al., 1994, 1997; Henny & Jones, 2006b). BDA-labelled axons extended into medial and lateral cortical regions at rostral as well as caudal levels, as previously described by others (Saper, 1984; Gaykema et al., 1990). In the PFC, medially coursing fibres densely innervated the medial PFC, most prominently the IL region along with the tenia tecta and less prominently the prelimbic area. Fibres arriving along a lateral course densely innervated the lateral PFC, most prominently the VLO/LO region. In both IL and VLO/LO cortices, BDA-labelled fibres were often orientated in a perpendicular manner to the surface, yet were also orientated in a diagonal or tangential manner. The fibres were morphologically diverse and bore both boutons en passant, as well as boutons terminaux, which were collectively considered as varicosities. Varicosities were distributed across all cortical layers, though were most densely distributed in the deep layers.

VAChT, VGAT or VGluT within BDA-labelled varicosities in the PFC

Series that were double-stained for BDA and VTPs were systematically examined to determine if BF axonal varicosities in the PFC (IL and VLO/LO) were immunopositive (+) for VGluT1, 2 or 3, VAChT or VGAT.

In series processed for BDA and VAChT, a portion of the BDA-labelled varicosities was double-labelled for VAChT (Fig. 2A and B). The BDA+/VAChT+ varicosities were relatively small and most often appeared as oblong swellings of labelled axons (Fig. 2A), though they were also seen occasionally at the end of axons ostensibly as boutons terminaux (Fig. 2B). Although the intensity of VAChT staining varied, it was detectable in most swellings along any single VAChT+, BDA-labelled fibre.

Fig. 2Fig. 2
Biotin dextran amine (BDA)-labelled axons containing vesicular acetylcholine transporter (VAChT), vesicular GABA transporter (VGAT) or vesicular glutamate transporter (VGluT) in the PFC. Images from epifluorescent microscopy showing BDA-labelled axonal ...

In series stained for BDA and VGAT, VGAT was found in a large number of BDA+ axon varicosities, in amongst a larger number of surrounding VGAT+ terminals in the cortex (Fig. 2C–E). The BDA+/VGAT+ varicosities appeared somewhat larger and rounder than BDA+/VAChT+ ones, and were not infrequently seen at the end of axons as boutons terminaux (Fig. 2C and D), though they were most frequently seen along the axons as boutons en passant (Fig. 2D and E). Along single BDA-labelled fibres bearing VGAT+ varicosities, the VGAT staining was detectable in the majority of the BDA-labelled varicosities.

In series processed for BDA and VGluT1, 2 or 3, VGluT2 (Fig. 2F and G), and not VGluT1 or VGluT3 (not shown), was found to be present in BDA-labelled varicosities. The BDA+/VGluT2+ varicosities were most frequently seen as boutons en passant (Fig. 2F and G) though also seen as boutons terminaux, and appeared to be somewhat larger and rounder than the VAChT+ varicosities. In single BDA-labelled fibres bearing VGluT2+ varicosities, the VGluT staining was visible in virtually all varicosities along the axon.

To examine the possibility that VGluT2 was present in VAChT and/or VGAT containing terminals, material dual-stained for VTPs was subsequently examined in fluorescence microscopy (Fig. 3). In material stained for VAChT and VGluT2, no evidence for colocalization of the two proteins was found (Fig. 3A). Similarly, in material stained for VGAT and VGluT2, there were no instances of colocalization between these two proteins (Fig. 3B). Series stained for VAChT and VGAT were also examined. Most of VAChT+ varicosities were found to be negative for VGAT and, reciprocally, the vast majority of VGAT+ varicosities were found to be negative for VAChT (Fig. 3C). Yet, some varicosities were found to be double-labelled for VAChT and VGAT. To assess whether this small number could correspond to BDA-labelled cholinergic fibres coming from MCPO-SI neurons (and not cortical interneurons), triple-staining was performed for BDA, VAChT and VGAT, and analysed by confocal microscopy. Of > 290 BDA+/VAChT+ varicosities from several axons from three cases analysed, less than 4% were double-labelled for VGAT (not shown).

Fig. 3
Virtual absence of vesicular acetylcholine transporter (VAChT), vesicular glutamate transporter (VGluT2) or vesicular GABA transporter (VGAT) colocalization in terminals within the PFC. Merged epifluorescent images from dual immunostained material stained ...

Proportion and laminar distribution of VAChT+, VGAT+ and VGluT2+, BDA-labelled varicosities

Having established an absence of colocalization of VGluT2 with VAChT or VGAT, and rare colocalization of VAChT with VGAT in BF axonal varicosities (above), the proportions of cholinergic, GABAergic and glutamatergic BF axon terminals were determined by stereological estimates of the total numbers of BDA+ and of BDA+/VTP+ varicosities in the IL and VLO/LO at three levels (~A11.8, A11.4 and A11.0, Fig. 1). In IL (n = 5), ~19% of BDA+ varicosities in the VAChT series were found to be BDA+/VAChT+~52% of the BDA+ varicosities in the VGAT series were BDA+/VGAT+ and ~15% of the BDA+ varicosities in the VGluT2 series were BDA+/VGluT2+ (Table 3). In the VLO/LO (n = 6), ~23% of BDA+ varicosities in the VAChT series were BDA+/−VAChT+ ~49% of the BDA+ varicosities in the VGAT series were BDA+/VGAT+ and ~8% of the BDA+ varicosities in the VGluT2 series were BDA+/VGluT2+. The proportions were similarly different for the three VTPs between the two areas (with two-way ANOVA for VTP and region, F = 18.042, P < 0.001 for VTP; F = 0.078, P > 0.05 for region; and F = 0.291, P > 0.05 for VTP × region).

Table 3
Proportions and laminar distribution of BDA-labelled BF axonal varicosities, which were VAChT+, VGAT+ or VGluT2+ in the IL

To examine the laminar distribution of BF fibres, the proportions of BDA+ and BDA+/VTP+ varicosities located in layers I, II–III or V–VI of IL were determined. Of the total number of BDA+ varicosities sampled stereologically in the three series (see Materials and methods), ~31% were located in layer I, ~11% in layers II–III and ~58% in layers V–VI. Of the total number of BDA+/VTP+ varicosities sampled in each series, proportions for each BDA+/VTP+ group were higher in layers V–VI (~77%, ~71% and ~75% for BDA+/VAChT+, BDA+/VGAT+ and BDA+/VGluT2+, respectively) when compared with layer I (~14%, ~27% and ~11%) and with layers II–III (~9%, ~2% and ~15%; Table 3).

Presence of PSPs PSD-95 or Geph in relation to VAChT+, VGAT+ or VGluT2+, BDA-labelled terminals

To assess whether BF axon terminals might form synapses with postsynaptic elements in the PFC, series were processed for triple-staining of BDA, the VTPs and the PSPs, PSD-95 or Geph (see Tables 1 and and2).2). Material was examined and z-stacks of images acquired by confocal microscopy, to be subsequently analysed as single sections, image stacks and three-dimensional-rendered reconstructions of image stacks (Fig. 4). In each case, possible associations of labelled varicosities with PSP profiles were assessed by full rotation of the images and judged as indicative of a potential synaptic association if no separation of the pre- and postsynaptic elements was apparent in orthogonal planes.

Fig. 4
PSPs postsynaptic density-95 kD (PSD-95) or gephyrin (Geph) in relation to vesicular acetylcholine transporter (VAChT)+, vesicular GABA transporter (VGAT)+ or vesicular glutamate transporter (VGluT)2+ BF terminals in the PFC. Projected views of merged ...

In series stained for BDA, VAChT and PSD-95 (Fig. 4A), only a small number of BDA+/VAChT+ varicosities was found associated with PSD-95+ profiles (five out of 36 varicosities examined or ~14%). In series stained for BDA, VAChT and Geph (Fig. 4B), a larger number of BDA+/VAChT+ profiles was found in association with Geph+ profiles (32 out of 108 varicosities examined or ~30%).

In series stained for BDA, VGAT and PSD-95 (Fig. 4C), a very small number of BDA+/VGAT+ varicosities was found in association with PSD-95 (two out of 15 varicosities examined or ~13%). In series stained for BDA, VGAT and Geph (Fig. 4D), many BDA+/VGAT+ varicosities were found to be associated with Geph+ profiles (57 out of 79 varicosities examined or ~72%).

In series stained for BDA, VGluT2 and PSD-95 (Fig. 4E), many BDA+/VGluT2+ varicosities were associated with PSD-95+ profiles (16 out of 30 varicosities examined or ~53%). In contrast, only a small number of BDA+/VGluT2+ varicosities could be found that were associated with Geph profiles (two out of 12 varicosities examined or ~17%) in series stained for BDA, VGluT2 and Geph (Fig. 4F).

VAChT+, VGAT+ and VGluT2+ BDA-labelled terminals in relation to NPNF+ pyramidal cells and PV+ or CB+ interneurons

Series that were triple-stained for BDA, the VTPs and cell marker proteins (n = 3 for each combination) were examined, and images acquired by confocal microscopy to determine if different BF axonal varicosities were located close to or in apposition to NPNF+ pyramidal cells or PV+ or CB+ interneurons in the PFC (Table 2, Fig. 5). Appositions between varicosities and cells were examined in single optical slices, image stacks and three-dimensional-rendered image stacks, and judged to be contacts if no separation was evident between the elements upon full rotation of the images.

Fig. 5Fig. 5
Vesicular acetylcholine transporter (VAChT)+, vesicular GABA transporter (VGAT)+ or vesicular glutamate transporter (VGluT2)+ BF terminals in relation to pyramidal cells and interneurons in the PFC. Projected views of merged z-series (large panels, composed ...

In BDA/VAChT/NPNF material, BDA+/VAChT+ axonal varicosities were often detected in the vicinity of NPNF+ cell bodies or dendrites, but never in direct contact with the NPNF+ profiles (Fig. 5A). Similarly, in BDA/VAChT/PV series, BDA+/VAChT+ varicosities could be seen in close proximity though not in contact with PV+ cell bodies or proximal dendrites (Fig. 5B). In BDA/-VAChT/CB series, BDA+/VAChT+ terminals were seen close and sometimes apposed to CB+ cells bodies (Fig. 5C).

In BDA/VGAT/NPNF material, BDA+/VGAT+ axonal varicosities were often detected in the vicinity of NPNF+ cell bodies or dendrites, and also in contact with the cell body or dendrites (Fig. 5D). In BDA/VGAT/PV series, BDA+/VGAT+ varicosities could also be seen in close association with PV+ cells, and occasionally encircling the soma to form multiple contacts in a perisomatic innervation (Fig. 5E). In BDA/VGAT/CB series, it was also found that BDA+/VGAT+ varicosities could form appositions with CB+ cell bodies (Fig. 5F).

In BDA/VGluT2/NPNF series, BDA+/VGluT2+ varicosities were detected only in the vicinity of NPNF+ cells (Fig. 5G). Similarly, in BDA/VGluT2/PV series, BDA+/VGluT2+ varicosities were only seen near and never contacting PV+ cells (Fig. 5H). In BDA/VGluT2/CB series, it was found that BDA+/VGluT2+ varicosities could occasionally form appositions with CB+ cell bodies (Fig. 5I).


The present study shows that the BF projection to the cortex is formed by glutamatergic in addition to GABAergic and cholinergic contingents. Through these contingents and their differential innervation of cortical neurons, the BF would have a multifarious influence in the PFC, and contribute differentially to modulation of its activity and the cognitive and behavioural functions it regulates.

Technical considerations

BF fibres were labelled by anterograde transport of BDA-10 000 MW. The labelled varicosities in the PFC were considered to represent the full axonal arborization of the labelled BF cells as the survival period of 5–6 days should be largely adequate for axonal transport to the PFC from the BF, approximately 4 mm distance in the rat. Indeed, estimates for anterograde transport rates for proteins range from 2 mm/day (for slow) up to 400 mm/day (for fast) (Roy et al., 2007), and BDA was originally found to be transported to distant targets in the brain within 3 days and to remain unchanged therein for 14 days (Rajakumar et al., 1993). Because rare cells were labelled by BDA in areas surrounding the BF and none in the PFC in the present study, we also considered that the terminals in the PFC would not likely originate from collaterals of retrogradely labelled cells, but would originate predominantly if not exclusively from the BF neurons in the MCPO and SI that were labelled in the injection site (Henny & Jones, 2006b).

Pre- and postsynaptic markers were employed here to assess whether BF terminals contain the VTPs for Glu, GABA or ACh, whether they can form excitatory or inhibitory synapses, and whether they contact interneurons or pyramidal cells in the PFC. To determine colocalization of the VTPs with BDA in varicosities, overlap of Cy3 with Cy2 was evaluated by epifluorescent microscopy, which with the chromogens employed should provide adequate lateral resolution (< 300 nm distance between two points for their differentiation) given the approximate size in diameter of the varicosities (~0.5–1.0 μm; Wouterlood, 2006; Wouterlood et al., 2007). For confirmation of this colocalization and evaluation of contacts between elements, confocal laser-scanning microscopy was performed, as for the three chromogens employed for this application, Cy2, Cy3 and Cy5, it allows greater resolution (~150–250 nm) and permits three-dimensional rendering and rotation of images along with projection of orthogonal single planes, for assessment of overlap, touching and/or separation between the stained elements (Wouterlood et al., 2003, 2007; Wouterlood, 2006). On the other hand, it is well recognized that actual synapses (0.2–0.3 μm) are below the resolution of the confocal microscope and can thus only be visualized by electron microscopy. Here, the use of PSPs as markers for synapses, however, greatly enhances the probability that the appositions viewed by confocal laser-scanning microscopy between the pre- and the labelled postsynaptic elements reflect synapses (Wouterlood et al., 2003; Henny & Jones, 2006a,b).

With epifluorescent microscopy, estimates of the proportions of glutamatergic, GABAergic and cholinergic terminals were performed using random, unbiased sampling by stereology that permits quantitative estimates of elements irrespective of shape, size and distribution (West, 2002). With confocal microscopy, the assessment of contacts between varicosities and PSP-stained elements or cortical neurons was performed in a semiquantitative manner, as stereology could not be applied in the confocal microscope.

Glutamatergic BF cortically projecting neurons

Employing immunostaining for the VTPs for Glu, VGluT1, 2 and 3, which mediate vesicular uptake and associated release of Glu (Fremeau et al., 2001, 2002; Fujiyama et al., 2001), we established here that cortically projecting BF neurons contain VGluT2 protein in their axonal varicosities, and thus have the capacity to take up and release Glu from their terminals in the cortex. Our results are supported by previous reports showing by in situ hybridization the presence of mRNA for VGluT2 in the cell bodies of BF neurons, including some retrogradely labelled from the cortex (Lin et al., 2003; Hur & Zaborszky, 2005). These findings also conform to the principle that in contrast to VGluT1, which is expressed predominantly by cortical neurons, VGluT2 is expressed predominantly by subcortical neurons (Fremeau et al., 2001; Kaneko et al., 2002).

VGluT3 was not found in the terminals of cortically projecting BF neurons here, nor had it been found previously by us in the terminals of hypothalamically projecting BF neurons (Henny & Jones, 2006b). Yet, neurons in the BF have been shown to contain VGluT3 in their cell bodies (Harkany et al., 2003; Gritti et al., 2006), and might accordingly release Glu from cell bodies or dendrites as shown for cortical neurons (Harkany et al., 2004).

VGluT2 was not found to be colocalized with VAChT or VGAT in the BF axonal varicosities. These findings thus do not support the conjecture previously based upon colocalization of ChAT and GAD with PAG in the cell bodies, that BF neurons might co-release ACh and Glu or GABA and Glu from their axon terminals (Manns et al., 2001). On the other hand, cholinergic and some GABAergic, along with glutamatergic, BF neurons might release Glu from somatodendritic compartments, given the presence of VGluT3 in the soma and proximal dendrites of ChAT+, GAD+ and PAG+ BF cells (Harkany et al., 2003, 2004; Gritti et al., 2006).

VGAT was found to be colocalized with VAChT in varicosities, however, only in a very small proportion (< 4%) of the BDA-labelled BF varicosities in the cortex. Within the rat cortex, GAD is known to be colocalized with ChAT in cholinergic interneurons (Bayraktar et al., 1997), which could give rise to many of the VGAT+/VAChT+ terminals seen here. Within rat and cat BF, however, GAD was found to be colocalized with ChAT only in a minute proportion (1–2%) of cholinergic neurons (Brashear et al., 1986; Fisher & Levine, 1989), which would presumably give rise to the BDA-labelled VGAT+/−VAChT+ BF terminals seen here. Indeed, within the cat cortex in which there are no cholinergic interneurons, only a small percentage of ChAT+ terminals was found to be immunostained for GABA (< 8%; Beaulieu & Somogyi, 1991). Thus, GABA might be utilized in a minute proportion of cholinergic BF terminals.

Overall, the present results show that the BF projection comprises three virtually non-overlapping, phenotypically distinct groups of glutamatergic, GABAergic and cholinergic varicose fibre systems in the PFC. The fibres of these three contingents are difficult to distinguish morphologically, however, the glutamatergic and GABAergic tend to have larger and often more round varicosities than the cholinergic fibres. Accordingly, the original distinction of two types of BF fibres (type I and II), studied using anterograde tracing with Phaseolus vulgaris leucoagglutinin, and identifying the GABAergic as the larger and the cholinergic as the smaller (Freund & Gulyas, 1991; Freund & Meskenaite, 1992), might have included the glutamatergic varicosities with the, albeit more numerous, GABAergic terminals in the cortex.

Proportions and laminar distribution of glutamatergic, GABAergic and cholinergic BF axon terminals in PFC

We have found that glutamatergic, GABAergic and cholinergic BF terminals represent ~15%, ~52% and ~19% of BF terminals in IL and similar proportions in VLO/LO of the medial and lateral PFC. The proportions of VGAT+ and VAChT+ terminals are similar to the proportions of GAD+ (43%) and ChAT+ (23%) neurons retrogradely labelled in the MCPO after injections in the medial PFC (Gritti et al., 1997). Yet, the proportion of VGluT2+ terminals (~15%) is less than the proportion of ChAT−/GAD− neurons (~34%) in the MCPO of that study (Gritti et al., 1997), and also of the average proportion of VAChT−/VGAT− terminals (~29%) in the present study, suggesting that another contingent of cortically projecting neurons must utilize a neurotransmitter other than ACh, GABA or Glu. This contingent could use another type of excitatory neurotransmitter, such as aspartate, which is not recognized as a substrate by the VGluTs (Bellocchio et al., 2000; Fremeau et al., 2001, 2002). It must also be considered that the proportions of the three VTP+ varicosities might not total 100% because the amount of VTP in some varicosities is below the threshold for positive immunohistochemical staining.

Within the IL, VGluT2+, VGAT+ and VAChT+ BF varicosities were distributed across all layers, but in highest density within the deep, V–VI layers of the cortex. These results indicate that, as described for BF fibres (Saper, 1984; Freund & Gulyas, 1991) and the cholinergic fibres (Houser et al., 1985; Mechawar et al., 2000), the glutamatergic and GABAergic fibres also reach all cortical layers while terminating most densely in the deep layers. In this manner, they would be able to modulate input and processing in all layers and prominently influence output to subcortical targets, including the autonomic and limbic areas to which IL projects (Gabbott et al., 2005). They could also shape the corticofugal feedback of the PFC to the BF (Gaykema et al., 1991).

PSPs in relation to glutamatergic, GABAergic and cholinergic BF terminals

We have shown in this study that the majority of BF glutamatergic and GABAergic axon terminals in the cortex are, respectively, associated with the PSPs, PSD-95 and Geph, providing evidence that these terminals, respectively, form excitatory and inhibitory synapses (Sassoe-Pognetto & Fritschy, 2000; Sassoe-Pognetto et al., 2000, 2003; Sheng, 2001).

A majority of the VGluT2+ BF varicosities were associated with PSD-95+ profiles (~53%) opposite their terminals in the PFC. These results indicate that BF terminals likely form glutamatergic, excitatory synapses, as PSD-95 is associated with N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and is concentrated at asymmetrical junctions (Sheng, 2001; Sassoe-Pognetto et al., 2003). That an even higher incidence of associations with PSD-95 elements for VGluT2+ terminals was not evident may not be surprising given that PSD-95 was reported to stain ~70% of asymmetric synapses in the cerebral cortex (Valtschanoff et al., 1999). In both cases, the incomplete staining could be due to relatively low sensitivity of immunostaining for PSD-95 with the techniques routinely employed (Sassoe-Pognetto et al., 2003). For the BF VGluT2+ varicosities, the incidence could also reflect the possibility that not all the VGluT2+ varicosities form synapses. A small proportion of VGluT2+ terminals was also seen associated with Geph+ profiles (~17%) here in the cortex. Given the lack of colocalization between VGluT2 and VGAT in the varicosities, it is difficult at present to hypothesize what the function of this association might be. It should be noted, nonetheless, that CNS glutamatergic neurons have the capacity to change their phenotype to that of GABAergic under certain conditions (Gomez-Lira et al., 2005), for which the presence of Geph in association with some glutamatergic axons might be indicative.

The large majority of BF VGAT+ varicosities were associated with Geph+ profiles (~72%) facing their terminals in the PFC. These results clearly indicate that the BF terminals form GABAergic inhibitory synapses, as Geph is a protein that forms part of the molecular scaffolding of GABAA receptors and is present at symmetric, commonly inhibitory synapses (Sassoe-Pognetto & Fritschy, 2000; Sassoe-Pognetto et al., 2000). The present results are substantiated by previous electron microscopic evidence that BF GABA-immunoreactive terminals in the cortex form symmetric junctions with postsynaptic elements (Freund & Gulyas, 1991). A very small proportion of BF VGAT+ varicosities were associated with PSD-95 (~13%) in the present study, which as for the association of VGluT2+ varicosities with Geph+ elements (above) might reflect a potential for plasticity of these terminals (Gomez-Lira et al., 2005).

The majority of VAChT+ terminals were not associated with PSPs (~70%) in the PFC. These results could be interpreted to mean that the majority of cholinergic terminals do not form synapses in the cortex, as has been suggested by some, according to electron microscopic studies of ChAT-immunoreactive terminals (Umbriaco et al., 1994). Such non-synaptic transmission would be consistent with the diffuse postsynaptic distribution of the G-protein-coupled, muscarinic ACh receptors (Volpicelli & Levey, 2004). Alternatively, the present results indicate that cholinergic terminals and receptors are associated with other, specific and as yet unidentified PSPs. Because nicotinic ACh receptors form ligand-gated ion channels, they might be expected to be associated with PSPs, such as PSD-95 (Sheng & Pak, 2000). A small proportion of VAChT+ varicosities were seen here in association with PSD-95+ profiles (~14%). PSD-93, a closely related protein member of the PSD-95 family, has been found in association with neuronal nicotinic receptors and cholinergic synapses in the periphery (Conroy et al., 2003; Parker et al., 2004). Notably, some ChAT-immunoreactive terminals in the cortex have been reported to form asymmetric junctions (Houser et al., 1985; Turrini et al., 2001), which are generally associated with PSD-95 (above; Sheng, 2001). On the other hand, we observed a higher incidence of VAChT+ terminals associated with Geph+ profiles (~30%), and one which far exceeded the colocalization of VAChT and VGAT in BF terminals (above). As examined in avian peripheral ganglia (Allaire et al., 2000), Geph is not known to be associated with or play a role in the assembly of any known ACh receptor type. Most significantly, however, ChAT-immunostained terminals in the cortex have been found to form junctions primarily of the symmetric type (Houser et al., 1985; Umbriaco et al., 1994; Turrini et al., 2001), which are commonly associated with Geph (Sassoe-Pognetto & Fritschy, 2000). Clearly, more research is needed to understand the associations of VAChT+ terminals with PSD-95 and Geph+ profiles.

We conclude that the majority of glutamatergic and GABAergic BF terminals contact and likely, respectively, form excitatory and inhibitory synapses in the PFC. A minority of cholinergic BF terminals might also form such synapses.

Relation of glutamatergic, GABAergic and cholinergic BF terminals to pyramidal cells and interneurons

Using cell marker proteins for pyramidal cells and interneurons, we found that only GABAergic BF varicosities contacted pyramidal cells. Glutamatergic along with cholinergic and GABAergic BF terminals contacted CB+ interneurons, and GABAergic BF terminals also contacted PV+ interneurons in the PFC. Forming largely non-overlapping groups of GABAergic interneurons with calretinin cells, the CB interneurons, which comprise double bouquet, Martinotti and other cell types, are thought to provide inhibitory input to different regions of the dendrites of pyramidal cells, and PV interneurons, which comprise basket and chandelier cells, to provide inhibitory input to the soma, proximal dendrites or axon initial segment of pyramidal cells (Kawaguchi & Kubota, 1993; Kubota et al., 1994; DeFelipe, 1997; Gabbott et al., 1997; Wang et al., 2004).

VGluT2+ BF terminals could only be detected in apposition to CB+ interneurons. These appositions could correspond to a portion of the contacts by large (type 1) BF terminals previously all considered to be GABAergic (above) and described as being primarily upon CB or somatostatin neurons, which comprise a large subset of CB interneurons in the rat (Freund & Gulyas, 1991).

VGAT+ BF terminals were seen in apposition to both CB+ and PV+ interneurons, and to NPNF+ pyramidal cells. These results differ somewhat from previous studies in the rat in which the large, considered GABAergic, BF varicosities were characterized as being predominantly upon CB or somatostatin interneurons, rarely on PV+ interneurons and not on pyramidal cells (Freund & Gulyas, 1991). In the cat, a more substantial innervation of PV interneurons in addition to somatostatin interneurons by BF terminals was found (Freund & Meskenaite, 1992). The present results would thus add to these previous findings, providing evidence that BF GABAergic fibres innervate both CB and PV interneurons and also, importantly, pyramidal cells.

VAChT+ BF varicosities could be detected in apposition only to CB+ interneurons, and were otherwise seen close to but not contacting PV+ interneurons or pyramidal cells. These results agree in general with studies showing a preferential innervation by cholinergic terminals of GABAergic interneurons in the cortex (Houser et al., 1985; Beaulieu & Somogyi, 1991). The low incidence of appositions onto other cell types may also be indicative of the low incidence of synaptic specializations that cholinergic axons in the cortex have been reported to form (see above), and the assumed slow, modulatory action of extrasynaptically released ACh (Umbriaco et al., 1994). As shown in vitro, ACh produces a slow depolarization of pyramidal cells through a muscarinic receptor, which, however, is preceded by a fast inhibitory postsynaptic potential, mediated by local interneurons (McCormick & Prince, 1986). Interneurons with characteristics of CB cells (Kawaguchi & Kubota, 1993) have been shown to be rapidly excited by ACh through nicotinic receptors (Xiang et al., 1998). Cholinergic BF terminals could thus provide spatially and temporally punctate, in addition to diffuse, influences in the cortex.

Functional considerations

The important roles of the BF in stimulating cortical activation and promoting attention, learning and memory were once attributed entirely to the cholinergic BF neurons. It is well known from pharmacological studies that ACh promotes fast cortical activity typical of cortical activation and blocks slow wave activity typical of slow wave sleep (SWS; Metherate et al., 1992; Jones, 2004; 2005). The cholinergic BF neurons discharge selectively in association with gamma and theta cortical activity during active or attentive waking and during rapid eye movement sleep, whereas they cease firing with irregular slow wave activity during SWS (Manns et al., 2000b; Lee et al., 2005). By their selective discharge and postsynaptic actions, the cholinergic cells can thus promote cortical activation, which underlies processes of attention and arousal (Sarter et al., 2003) and learning and memory (Weinberger, 2003). Here, it appears that the major influence of the cholinergic input to the cortex would be largely through the innervation of interneurons, as was previously concluded by other investigators (Beaulieu & Somogyi, 1991), and possibly also by diffusion of ACh onto nearby pyramidal and other cells as proposed by some (Umbriaco et al., 1994). Perhaps through contacts upon interneurons and nicotinic receptors, the cholinergic cells can shape or pace activity in the cortex to stimulate rhythmic theta during cortical activation (Lee et al., 2005), or through diffusion and muscarinic receptors, stimulate the slow depolarization of multiple cortical cells to promote fast activity and prevent slow wave activity (Metherate et al., 1992). Lesions of cholinergic BF neurons have been associated with decreased amplitude of theta and gamma activity (Lee et al., 1994; McGaughy et al., 1996; Gerashchenko et al., 2001; Berntson et al., 2002), yet not to the extent that non-specific lesions or inactivation of the total BF cell population had previously been shown to do (Stewart et al., 1984; Stewart & Vanderwolf, 1987; Dunnett et al., 1991; Wenk, 1997; Cape & Jones, 2000; Jones, 2004). The other GABAergic and glutamatergic BF cell groups thus also appear to make an important contribution to these processes.

The role of GABAergic BF neurons in modulating cortical activity has been more difficult to discern. This difficulty may be due to the fact that in contrast to the cholinergic cells, the GABAergic BF cells are physiologically heterogeneous and comprise different subgroups that discharge maximally either with cortical activation or conversely with cortical deactivation (Manns et al., 2000a, 2003; Jones, 2005). Moreover, the GABAergic cells manifest distinct patterns of discharge including regular tonic rapid firing or phasic firing, some cells in association with rhythmic theta activity during cortical activation, other cells in association with irregular slow wave activity during cortical deactivation. Regular tonic or phasic activity by GABAergic neurons could be associated with pacing or gating of activity in target neurons through GABAA-mediated fast inhibitory postsynaptic potentials, as is the case with cortical interneurons (Freund & Buzsaki, 1996; Klausberger et al., 2003; Tamas et al., 2004; Somogyi & Klausberger, 2005), instead of simple inhibition or disinhibition, as was once thought most likely to be the case with BF GABAergic input to cortical neurons (Freund & Gulyas, 1991). Indeed, as shown by reduced amplitude of hippocampal theta following selective lesions of GABAergic neurons in the septum, the GABAergic septo-hippocampal projection cells clearly participate together with the cholinergic in stimulating theta activity (Yoder & Pang, 2005). Accordingly, through the innervation of different cortical cells documented in the present study, different BF GABAergic cell subgroups could have the capacity to stimulate or pace different activities, including theta or delta, in cortical neurons and networks, and in association with different behavioural states.

Little is known concerning the role of glutamatergic BF neurons in shaping cortical activity, though a potential role of non-cholinergic and non-GABAergic neurons in modulating cortical, like hippocampal, rhythmic slow activity or theta has been postulated (Manns et al., 2003; Yoder & Pang, 2005). Putative glutamatergic BF neurons, identified as ChAT−/GAD− and PAG+ cells comprise diverse cells, most of which discharge in association with cortical activation, including rhythmic slow activity (Manns et al., 2000a, 2003). From the results of the present study, such cells could act upon CB interneurons, which they could possibly drive through NMDA or AMPA receptors and thus excite in a phasic or tonic manner for promoting cortical activation.

The BF projections were documented here to the PFC, which is a particularly important region of the cortex for shaping cortical activity across behavioural states (Schwierin et al., 1999; Massimini et al., 2004). It is also the region of the cortex that through the influence of the BF input is critical for promoting attention during learning (Sarter et al., 2001) and for coordinating different autonomic responses (Dalley et al., 2004; Resstel & Correa, 2006), which may be determinant for the memory and application of emotionally salient associations (Chu et al., 1997; Bechara & Van Der Linden, 2005). In these processes, it is likely that the cholinergic, GABAergic and glutamatergic BF cell groups or subgroups play diverse and complementary roles.


We are grateful to Robert H. Edwards and Robert T. Fremeau (Departments of Neurology and Physiology, University of California San Francisco School of Medicine, San Francisco, CA 94143, USA) for kindly supplying the antibodies for VGluT1 and 2. We thank Lynda Mainville for her excellent technical assistance. The research was supported by research grants (to B.E.J.) from Canadian Institutes of Health Research (CIHR 13458) and U.S. National Institutes of Health (NIH R01 MH 060119).


α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
biotin dextran amine
basal forebrain
choline acetyltransferase
γ-aminobutyric acid
glutamic acid decarboxylase
infralimbic cortex
lateral orbital cortex
magnocellular preoptic nucleus
normal donkey serum
non-phosphorylated neurofilament
phosphate-activated glutaminase
prefrontal cortex
postsynaptic density-95 kDa
postsynaptic protein
substantia innominata
slow wave sleep
Triton X-100
vesicular acetylcholine transporter
vesicular GABA transporter
vesicular glutamate transporter
ventrolateral orbital cortex
vesicular transporter protein


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