Molecular and Cellular Neuroscience 46 (2011) 625–632

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Molecular and Cellular Neuroscience

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GABAergic neurons expressing p75 in rat substantia innominata and nucleus basalis

E. Formaggio ⁎, A.C. Dalfini 1, F. Fazzini 1, G. Fumagalli 1, C. Chiamulera 1

Dept. of Medicine and Public Health, Sect. Pharmacology, University of Verona, P.le Scuro 10, 37134 Verona, Italy

Abbreviations: BDNF, brain-derived neurotrophic faccholine acetyltransferase; GABA, γ-aminobutyric acid;boxylase 67; HDB, horizontal limb of diagonal band; MSbasalis magnocellularis; NGF, nerve growth factor; PAminase; SI, substantia innominata; Trk, tyrosine-kinase;band.⁎ Corresponding author. Present address: Aptuit M

Fleming 4, 37135 Verona, Italy. Fax: +39 045 8219111,E-mail address: elena.formaggio@gmail.com (E. Form

1 Fax: +39 045 8027452.

1044-7431/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.mcn.2011.01.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 September 2010Revised 1 January 2011Accepted 3 January 2011Available online 14 January 2011

Keywords:Cholinergic neuronsGAD67p75 neurotrophin receptorNGFBDNF

In vitro findings suggested a role for the p75 neurotrophin receptor in the maturation of GABAergic neuronsresiding in the basal forebrain (BF), a brain area known to have p75 expression only on cholinergic neurons.We document here the presence of GABAergic neurons which express p75 in the BF in vivo. Colocalization ofp75with the cholinergic marker choline-acetyltransferase (ChAT) and/or the GABAergic marker glutamic aciddecarboxylase-67 (GAD67) was investigated in the BF at birth, at two weeks, and in adulthood. A subset ofGAD67+ neurons was p75+ (p75+/GAD67+) but ChAT− in the substantia innominata and nucleus basalismagnocellularis at birth, whereas all p75+/GAD67+ neurons were also ChAT+ from twoweeks onward. Thesephenotypic features suggest that a subpopulation of GABAergic neurons could be sensitive to neurotrophinsduring brain maturation. To unravel this issue, we then pursued a functional analysis by assessing p75expression profile, and its modulation by nerve growth factor (NGF) or brain-derived neurotrophic factor(BDNF) in primary BF cell cultures. NGF increased p75 expression exclusively in cholinergic neurons, whereasBDNF induced p75 expression only in a subset of GABAergic neurons (p75+/GAD67+/ChAT−) through a p75-and tyrosine-kinase-dependent mechanism. The latter findings point to a selective role of BDNF in theinduction of p75 expression in BF GABAergic neurons. Altogether these results confirm the role ofneurotrophins in the developing and mature circuitry of GABAergic neurons in the BF regions.

tor; BF, basal forebrain; ChAT,GAD67, glutamic acid decar-

, medial septum; NBM, nucleusG, phosphate-activated gluta-VDB, vertical limb of diagonal

edicines Research Centre, Via+39 045 8027452.aggio).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

Introduction

GABAergic and cholinergic neurons of the basal forebrain (BF)influence the development of the cerebral cortex and its function, thusregulating overall brain development (Semba, 2000). The neuro-transmitter phenotype of these two populations of BF neurons isregulated during development by the neurotrophins nerve growthfactor (NGF) and brain-derived neurotrophic factor (BDNF) throughdifferentmechanisms (Lin et al., 2007). In particular, expression of theneurotrophin receptor p75 is required for the recruitment/maturationto a GABAergic, but not to a cholinergic, phenotype. In BF cell culturesprepared from p75-deficient mice (p75−/−), NGF and BDNF increasethe number of cholinergic, but not of GABAergic, neurons (Lin et al.,2007). The expression of a p75 rescue construct or the application ofconditioned medium from p75+/+ cultures restores the neurotro-

phin-induced expansion of GABAergic neurons in p75−/− cultures(Lin et al., 2007).

Despite such indications of an involvement of p75 expression inthe regulation of GABAergic neurons in the BF, p75 has been reportedto be expressed only in cholinergic neurons residing in this brain area(Hartikka and Hefti, 1988; Heckers et al., 1994). Therefore, currentlyavailable data suggest a non-cell-autonomous mechanism for theregulation of the GABAergic phenotype, in which p75 expression incholinergic neurons could modulate the release of soluble factorsacting on GABAergic neuron maturation. However, in culturesvalidated as a model of cholinergic BF cells, it has been recentlyreported that p75 is expressed also in neuron subsets which canrelease ACh and GABA, or GABA only (Huh et al., 2008). The presenceof non-cholinergic p75+ GABAergic neurons within the BF may revealan alternative, cell-autonomous mechanism for the regulation of theGABAergic phenotype, but the existence of such neuron populationhas not yet been verified in vivo.

On this basis, the aim of the present study was to verify whethernon-cholinergic p75+ GABAergic neurons reside in the rat BF. To thispurpose, we investigated here in rats at birth, at two postnatal weeksand in adulthood the co-localization in BF neurons of p75, choline-acetyltransferase (ChAT), and glutamic acid decarboxylase-67(GAD67), the GABA synthetic enzyme isoform which was used hereas GABAergic neuron marker. Both p75+/ChAT+/GAD67+ and p75+/ChAT−/GAD67+ neuron populations were found in BF regions atdifferent stages of brain maturation.

626 E. Formaggio et al. / Molecular and Cellular Neuroscience 46 (2011) 625–632

We then aimed to assess whether BF GABAergic neurons could bedirectly sensitive to neurotrophin-induced maturation. Thus, using anin vitro approach, we pursued a functional study, investigatingwhether NGF or BDNF exposure modulates p75 expression incholinergic and/or GABAergic neurons from the rat BF.

Results

p75+ GABAergic neurons in the rat BF

Brain sections obtained from rats at P0 and P15, as well as adultrats were processed for p75/ChAT/GAD67 triple immunofluorescence.

In the adult, outside the BF, i.e. in the caudate putamen and in thecerebral cortex, cholinergic (ChAT+) neurons were also p75−, aspreviously reported (Levey et al., 1984; Mesulam, 2004), (not shown).The typical complete co-localization pattern of p75 and ChAT wasdetected both in the MS/VDB/HDB and in the NBM/SI (Fig. 1A, B).Interestingly, in the most ventral portions of the NBM/SI, a smallpopulation of intensely labelled triple-positive neurons (p75+/ChAT+/GAD67+)was detected (Fig. 2A). Suchneuronswere not detected in theMS/VDB/HDB.

BF sections from P15 rat pups showed a similar expression patternfor p75/ChAT/GAD67 immunofluorescence (Fig. 2A), but the p75+/ChAT+/GAD67+ cell population was detected in more dorsal portionsof the NBM/SI.

At P0, the ChAT immunosignal was weaker than at P15 or in theadult, as also described in previous reports on ChAT immunoreactivity

MS

VDB

HDB

0.72 mmBregmaA

B

Fig. 1. p75 colocalizes with ChAT in adult rat MS/VDB/HDB. A, graphic representation of adul(left). Black vertical lines in this picture correspond to points of Bregma (0.72, −0.60, −2.16right). B, magnification of the coronal section at Bregma 0.72 mm (left). The black square inshown on the right. Representative confocal images of p75/ChAT/GAD67 triple-immunoflumerged in the upper left picture (merge). Black and white pictures represent the indicated siChAT. No colocalization is found for GAD67 with p75 or ChAT stainings. Scale bar is 20 μm.

during perinatal development (Molnar et al., 1998; Li et al., 1995; Kohand Higgins, 1991). In theMS/VDB/HDB, aswell as in the NBM/SI, about50% of the p75+ neurons were also ChAT+. In the NBM/SI, ChAT+

neurons were mostly distributed in clusters (not shown). Interestingly,in theNBM/SI, a p75+/GAD67+neuron subsetwas detected, but showedno ChAT immunoreactivity (p75+/ChAT−/GAD67+) (Fig. 2A, B). GAD67immunosignal intensity in these neuronswas higher in the dorsal of theNBM/SI than in its ventral portions (Fig. 2B). These data show theexistence of p75+/GAD67+ neurons in the rat NBM/SI since early stagesof development. Such neurons express a non-cholinergic phenotype atbirth anda cholinergic phenotype fromP15 onwards, indicating that thisphenotypic feature matures over time.

BDNF induces p75 expression in a GABAergic neuron subset in vitro

NGF and BDNF have been described to increase GABAergicneurons' number in BF cell cultures (Lin et al., 2007), thus pointingto a role of these neurotrophins also in the phenotypic maturation ofthe BF GABAergic cell component. We investigated here whether NGFand BDNF modulated p75 expression in the BF cholinergic and/orGABAergic neurons, using awell standardized in vitro system of BF cellcultures (Hartikka and Hefti, 1988; Auld et al., 2001). Cells grown for12 days in the absence of exogenously added neurotrophins weretreated for 72 h with vehicle, or BDNF, or NGF (100 ng/ml each). Theeffect on the expression of p75was analyzed by counting the number ofimmunofluorescence-positive neurons. In particular, we used double-

merge GAD67

p75 ChAT

-0.60 mm -2.16 mm

t rat BF sections analyzed in this study. A longitudinal section of the rat brain is depictedmm) where coronal sections of the brain were cut (see correspondent sections on the

dicates the portion of the section containing the MS/VDB/HDB and analyzed in picturesorescence (right) in adult rat. GAD67 (green channel), p75 (red) and ChAT (blue) arengle-channel acquisitions. In the MS/VDB/HDB p75 shows complete colocalization withGraphic representations adapted from Paxinos and Watson, 2004.

merge GAD67

p75 ChAT

merge GAD67

p75 ChAT

merge GAD67

p75

ADULT P15

P0

A

BGAD67merge

p75

ChAT

NBMRt

ic

Fig. 2. p75 is expressed in a small subpopulationofGABAergic neurons in theNBM/SI. Representative confocal imagesof p75/ChAT/GAD67 triple-immunofluorescence in adult, P15 andP0rat BF sections of theNBM/SI.A,magnificationof the schematic representationof adult rat brain (left) takenasa general reference forBF slice cuttingand identification (seealso Fig. 1A). Theblack squares indicate the brain areas where confocal pictures were acquired (reticular thalamic nucleus, Rt; internal capsule, ic). Adult images shown are from ventral portions (lowerblack square), P15 and P0 images from corresponding dorsal portions (upper black square). Coloured pictures (merge) represent merged GAD67 (green channel), p75 (red) and ChAT(blue) images. Black andwhite pictures represent the indicated single-channel acquisitions. In adult aswell as in P15 rats, a small subpopulation of neurons in theNBM/SI is p75+/ChAT+/GAD67+. This identifies a new set of p75-positive cholinergic and GABAergic neurons in the BF in vivo. In P0, subpopulations of p75+/GAD67+/ChAT- neuronswere identified. ChAT signalwas hardly detectable in these sections. p75+/GAD67+neurons, indicated by arrowheads, are characterized by intense labelling for bothmarkers. Arrows indicate p75+/GAD67−neuronsdetected in the same section. B, confocal images of the ventral portions of the NBM/SI neurons (corresponding to brain sections at Bregma−0.6 mm in Fig. 1A) were acquired followingp75/ChAT/GAD67 immunofluorescence. P0 rats (merge), showed weak GAD67 staining (green channel) of p75+ neurons (red) in this region. ChAT signal was not detected in theseneurons (not shown). Magnified single-channel pictures (corresponding to the white square) are shown on the right. Scale bar is 20 μm.

627E. Formaggio et al. / Molecular and Cellular Neuroscience 46 (2011) 625–632

immunolabelling with ChAT to identify cholinergic neurons, and withGAD67 to label GABAergic neurons.

In vehicle-treated cultures, we observed complete co-localizationof p75 and ChAT immunoreactivity, so that p75+ neurons were alsoChAT+ and vice versa. BDNF and NGF significantly increased thenumber of p75+ neurons, and BDNF treatment showed the highesteffect (Fig. 3A) (Table 1). Interestingly, while NGF effects wererestricted to ChAT+ neurons typically characterized by a large soma,the BDNF-induced increase of p75+ neurons involved a ChAT− small-soma neuron population undetectable under control (vehicle)conditions. The newly detected population of p75+/ChAT− neuronswas characterized by GAD67 immunostaining (Fig. 3B), and did notexpress the glutamatergic marker PAG (not shown). This effect of

BDNF appeared specific for BF cell cultures, since no p75+ GABAergicneurons were detected in hippocampal cell cultures similarly treatedwith BDNF (data not shown).

Modulation of ChAT immunolabelling by the neurotrophinsshowed that NGF increased the number of ChAT+ neurons (Fig. 3C),while BDNF had no significant effect.

Neither NGF nor BDNF induced modifications of the total number ofneurons in culture, as verified by counts after immunolabelling withanti-NeuN antibody (NS vs. control, unpaired t-test, n=12 for controlandNGF, n=11 for BDNF). Furthermore, in both treated and control cellcultures, upon 24, 48 and 72 h of treatment, undifferentiated nestin+

cells were found to acquire progressively a glial (nestin+/GFAP+), butnot a neuronal phenotype (no nestin+/MAP2+ detected) (not shown).

control BDNF NGF0

100

200

300

400

500

600

cholinergiccholinergic+GABAergic

** **

*****

p75+

neur

ons

(% c

ontr

ol)

A B

control BDNF NGF0

100

200

300

400

500

**

ChA

T+

neur

ons

(% c

ontr

ol)

C

Fig. 3. p75 and ChAT immunoreactivity in BF cholinergic and GABAergic neurons following in vitro treatments. A, BDNF and NGF differently affected p75 immunoreactivity incholinergic and GABAergic neurons in BF cultures. BF cultures at d12 were treated for 72 h as indicated in the abscissa. Data are expressed asmean percentage of control±SEM. Eightindependent experiments were performed. (total cholinergic+GABAergic population, grey bars, BDNF: pb0.001***, NGF: pb0.01** vs. control, n=21; cholinergic population, openbars, BDNF: NS, NGF: pb0.01** vs. control, n=21). B, representative confocal image of p75/GAD67 immunofluorescence staining after BDNF treatment of mature BF cultures. p75signal is red and GAD67 signal is green, yellow indicates colocalization of the markers. p75 is present both on large-soma (presumably cholinergic) neurons (red only, upper leftcorner of the picture) and on small GAD67+ GABAergic neurons (yellow). Note that only a subset of the GAD67+ neurons expresses p75 after BDNF treatment. Scale bar is 20 μm.C, BDNF and NGF differently affected ChAT immunoreactivity in BF cultures. BF cultures at d12 were treated for 72 h as indicated in the abscissa. Cells were immunostained for ChATand observed by confocal microscopy. ChAT+ cells were counted as indicated in the Experimental methods section. Data are expressed as mean percentage of control±SEM. Fiveindependent experiments were performed (NGF=pb0.01**; BDNF: NS, vs. control, n=8).

*

nsit

yol

)

A

B

control BDNF NGF0

50

100

150

100

150

******

p75

inte

nsit

y(%

con

trol

, AU

)

Immunofluorescence

FACS

628 E. Formaggio et al. / Molecular and Cellular Neuroscience 46 (2011) 625–632

These data indicate that in our conditions exposure to neurotrophinsdid not stimulate either neuronal proliferation or neuronal differenti-ation. This also suggests that modulation in the number of p75+ andChAT+ neurons could result from upregulation of p75 or ChATexpression levels in neurons formerly detected as p75− or ChAT−.This hypothesis was confirmed by quantitative data on the fluorescencesignal intensity of p75 and ChAT. Double-immunofluorescence (p75/ChAT) analysis showed that, following treatmentwith NGF but notwithBDNF, p75 immunoreactivity was increased at the single cell level incholinergic neurons (Fig. 4A). Similarly, FACS analysis of ChATexpression levels showed that NGF, but not BDNF, significantlyincreased the mean fluorescence intensity of ChAT+ neurons, suggest-ing an increase in mean ChAT expression levels per cholinergic neuron(Fig. 4B).

Altogether these data demonstrate that neurotrophins, in the time-frame considered, are ineffective in promoting neuronal proliferation orcommitment of undifferentiated cells to a neuronal phenotype. On thecontrary, our data suggest that NGF and BDNF may promote theexpression of a cholinergic/p75+ phenotype in ChAT−/p75− neurons,and the induction of a p75+phenotype in a subpopulation of GABAergicneurons, respectively.

Activation of both Trks and p75 is necessary for BDNF-induced p75expression in GABAergic neurons

To determine whether the effects of BDNF were mediated by TrkBor p75 activation, we incubated cells with BDNF in the presence of theselective Trks inhibitor K252a or the anti-p75 antibody Ab9651 (an

Table 1Cholinergic and GABAergic subsets of p75+ neurons. Mean number (mean±SEM) ofp75+ neurons per mm2 (n=21 for each treatment) detected in BF cultures following72 h of incubation with vehicle (control), NGF or BDNF. p75+ neurons weredistinguished in cholinergic (ChAT+, large soma) or GABAergic (GAD67+, smallsoma) neurons. Note that p75+ GABAergic neurons are absent in control conditions.

p75-positive neurons/mm2

Cholinergic GABAergic Total

Control 1.23±0.20 0.00 1.23±0.20BDNF 1.14±0.22 5.00±1.10⁎⁎⁎ 6.14±1.06⁎⁎⁎

NGF 2.61±0.36⁎⁎ 0.04±0.04 2.66±0.38⁎⁎

⁎⁎ pb0.01.⁎⁎⁎ pb0.001.

antibody directed against p75 binding site for neurotrophins),respectively. Both agents, used at doses known to inhibit their targets(Knüsel and Hefti, 1992; Auld et al., 2001; Huber and Chao, 1995),completely blocked the BDNF-induced p75 expression in GABAergicneurons (Fig. 5). Each treatment (alone or in combination with BDNF)had no effects on the total number of neurons as determined byNeuN immunolabelling (n=5–6, NS vs. control or BDNF), indicatingthat neuronal survival was not compromised. The data indicate that

ChA

T in

te(%

con

tr

control BDNF NGF0

50

Fig. 4. NGF selectively increases p75 and ChAT expression in cholinergic neurons.A, mean fluorescence intensity measurements (arbitrary units) of p75 in cholinergicneurons following the indicated treatments. Five independent experiments wereperformed (BDNF: NS, NGF:, pb0.001*** vs. control, n=5–13). B, the mean fluorescenceintensity of ChAT+ population wasmeasured by FACS analysis following control, NGF andBDNF treatments. Data are expressed as mean percentage of control±SEM. Threeindependent experiments were performed. (NGF: pb0.05* vs. control; BDNF: NS vs.control, n=10–19).

BDNF

BDNF+K252a

BDNF + A

b0

50

100

150

****

p75+ /

GA

D67

+ ne

uron

s(%

BD

NF

)

Fig. 5. BDNF-induced p75 expression in BF GABAergic neurons is regulated by both p75-and Trk-dependent mechanisms. K252a and Ab9651 inhibit BDNF induction of p75expression in GABAergic neurons. K252a (100 nM) or Ab9651 (Ab, 7.5 μg/ml) were added1 h before BDNF treatment. Data are expressed asmean percentage of BDNF±SEM. Threeindependent experiments were performed. (BDNF+K252a: pb0.01** vs. BDNF, BDNF+Ab9651: complete inhibition of p75-positive GABAergic neurons, n=6–9).

629E. Formaggio et al. / Molecular and Cellular Neuroscience 46 (2011) 625–632

the effects of BDNF on p75 expression in the newly found GABAergicpopulation are mediated by the activation of the TrkB and p75receptors.

Neither K252a, nor Ab9651 affected p75 expression in cholinergicneurons in BDNF-treated cultures (n=6–9, NS vs. BDNF).

Both NGF and BDNF increase the cell body size in cholinergic neurons

BDNF showed no significant effects on the modulation of theexpression levels of p75 and ChAT in cholinergic neurons, whereas itexerted a trophic action on the soma size of these neurons, aspreviously reported (Martinez-Serrano et al., 1996). By measuring thesize of p75+/ChAT+ cell bodies (clearly discernible from the p75+/GAD67+ neurons on the basis of their morphology), we found that100 ng/ml BDNF indeed induced a robust hypertrophic response(BDNF+32.1±8.7% increase in soma size vs. vehicle-treated controls,n=49 and 53 for control and BDNF, respectively), similar to thatinduced by NGF (NGF+53.9±5.7% vs. control, n=49 and 65 forcontrol and NGF respectively) (Fig. 6). These results indicate that inour conditions, BDNF retained its function on the stimulation oftrophic responses on cholinergic neurons.

Discussion

In the present study, the p75 receptorwas found to be expressed inChAT+/GAD67+ and ChAT−/GAD67+ neuron populations of the BF atdifferent stages of brainmaturation. In the functional study performed

control BDNF NGF0

250

500

**********

Cel

l bod

y ar

ea (

µm2 )

Fig. 6. BDNF and NGF increase cell body area of cholinergic neurons. After NGF or BDNFtreatment, BF cultures were subjected to p75 and ChAT double-immunofluorescencestaining and subsequent confocal microscope observation. Cell body area of p75+

cholinergic neurons was then measured in at least three independent experiments(n=49 neurons measured for control, n=65 for NGF, n=53 for BDNF) (pb0.01**,pb0.001*** vs. control).

in vitro, we documented that NGF expanded the number of p75+/ChAT+ neurons by upregulating the expression of these proteins,whereas BDNF, ineffective on the cholinergic cell population,selectively induced p75 expression in ChAT−/GAD67+ neurons.These findings point out that BDNF and NGF differentially modulatethe maturation of cholinergic and GABAergic BF neuron populations.

GABAergic neurons of the BF express p75 since birth

Although GABAergic BF neurons have not been previouslyreported to express p75, yet a role for this protein in GABAergicneurons has been recently suggested by p75-rescue experimentsperformed on postnatal BF cultures obtained from p75−/− animals(Lin et al., 2007). In particular, a soluble factor secreted by p75-expressing (bona fide cholinergic) neurons was found to stimulateGABAergic neurons maturation. This suggested that p75 could act in anon-cell-autonomous manner for the modulation of GABAergicneuron development (Lin et al., 2007).

In the present study, we show that both during postnataldevelopment (P0, P15) and in the adult brain, a subset of p75-positive neurons in the BF is GABAergic. Previous reports haveidentified a small proportion of p75+/ChAT− neurons only in BF cellcultures from P10–13 rats (Huh et al., 2008), and such neurons havebeen electrophysiologically characterized as GABA-releasing cells. Thepresent finding in vivo suggests a possible direct involvement of p75in the development of GABAergic neurons. In particular, our findingsindicate that a cell-autonomous mechanism could promote a p75-dependent maturation of GABAergic neurons which reside in theNBM/SI.

At birth, p75+/GAD67+ neurons did not exhibit ChAT immuno-reactivity (p75+/ChAT−/GAD67+), whereas at P15 and in the adult BFonly triple-immunoreactive (p75+/ChAT+/GAD67+) neurons wereidentified. This suggests that p75+/ChAT−/GAD67+ neurons acquire acholinergic phenotype during the first two postnatal weeks. Actually,we observed generalized low immunoreactivity for ChAT at P0, inaccordance with previous reports dating at P8 the stabilization ofChAT immunoreactivity to the adult levels (Molnar et al., 1998; Liet al., 1995), and describing an increase in the number of ChAT-positive neurons during the normal postnatal maturation of the BF(Ward and Hagg, 1999). An alternative view of p75/GAD67 co-expression is that from P15 onwards cholinergic neurons can have ornot have GAD67. In that case, the p75+/ChAT+/GAD67+ populationwould not be a new population of GABAergic neurons expressing p75,but rather a subset of the cholinergic population.

Of notice, in this manuscript we used GAD67 immunolabelling forthe identification of GABAergic neurons. It is well known that GABAsynthesis involves two isoforms of GAD named GAD65 and GAD67,that are independently encoded by different genes (Erlander et al.,1991), and that present a different subcellular distribution. In severalbrain regions, including the MS, GAD65 is preferentially found in axonterminals, while GAD67 is primarily found in the soma of GABAergicneurons (Esclapez et al., 1994; Castaneda et al., 2005). Differences inthe distribution of the two isoforms have been suggested to reflect adifferent metabolic function and presumably a different role incognitive processes (Soghomonian and Martin, 1998). Our choicefor GAD67 as a marker for GABAergic neurons is due to its expressionin neuron somas that suits to colocalization studies with ChAT andp75 markers, both characterized by a prevalent somatic staining.

We found a different localization of p75+/GAD67+ neurons atdifferent time of brain development. A more dorsal or ventrallocalization was detected respectively for P0–P15 and adult rats.Differences in the distribution of a given neuronal population duringdevelopment may be explained by cell migration and/or the constitu-tion of a specific environmental niche whose stimuli–the neurotrophicsupport, the expression profile of the nearby populations, etc.–may befinely regulated both spatially and over time (Giehl, 2007).

630 E. Formaggio et al. / Molecular and Cellular Neuroscience 46 (2011) 625–632

Functional significance of the newly identified p75+/GAD67+/ChAT+ neuronal population in adult rat BF still remains undefined.Further studies are needed to understand the electrophysiologicalcircuitries they can modulate, both during development and in adultlife. An attracting, though speculative, possibility is that expression ofp75 in these neurons and stimulation with NGF or BDNFmodulate therelease of distinct neurotransmitter pools, as found for sympatheticneurons. Specifically, in sympathetic neurons NGF stimulates norepi-nephrine, whereas BDNF acetylcholine release, resulting in a p75-controlled functional switch between excitatory and inhibitoryneurotransmission in single neurons (Yang et al., 2002). Functionalstudies should be performed in order to analyze if a similarmechanism is on stage for p75+/GAD67+/ChAT+ neuronal popula-tion, potentially capable of releasing both acetylcholine and GABAneurotransmitters.

NGF and BDNF exert a differential effect on cholinergic and GABAergicneurons of the BF

The effects exerted by NGF and BDNF on BF cholinergic neuronshave been extensively characterized in vitro (Hartikka and Hefti,1988; Alderson et al., 1990; Svendsen et al., 1994), as well as in vivo,both during development (Li et al., 1995), and in adult intact(Martinez-Serrano et al., 1996; Koliatsos et al., 1994; Klein et al.,1999) or injured (Morse et al., 1993; Koliatsos et al., 1994) brain. Onthe other hand, only limited data are available for the effects of NGFand BDNF on BF GABAergic neurons, probably because this neuronalpopulation was considered to be insensitive to the effects ofneurotrophins. In the light of findings showing that GABAergic cellmaturation is neurotrophin-driven and p75-dependent (Lin et al.,2007), information about the role of neurotrophins on the maturationof GABAergic neurons in the BF are needed. NGF ability to upregulateChAT and p75 immunoreactivity in cholinergic neurons both in vitroand in vivo is well established (Koliatsos et al., 1994; Svendsen et al.,1994), and in the present study the effects of NGF served as areference to monitor cholinergic neuron changes. When analyzinghow NGF and BDNF modulated p75 and ChAT neuron numbers in ourBF cell cultures, we found that NGF increased ChAT and p75immunoreactivity exclusively in cholinergic neurons, whereas BDNFselectively modulated p75 immunoreactivity in a subpopulation ofGABAergic neurons without affecting p75 and/or ChAT expressions inthe cholinergic neurons.

That BDNF did not increase ChAT+ neurons number is in accordancewith previous data produced in vitro under similar experimentalconditions of cell density and delayed exposure to exogenousneurotrophin (Alderson et al., 1990). Alderson and colleagues foundthat a delayed addition of BDNF (no BDNF for 7 days followed by BDNFtreatment for 5 days) induced no increase in the number of AChE-positive cells.

The observed BDNF effect on p75 expression in GABAergic neuronsin vitro has never been reported previously, and appears of particularinterest in view of the identification of p75+/GAD67+ neuronsdetected in vivo in the NBM/SI. In particular, in vitro BDNF inducedp75 expression in a subpopulation of GABAergic neurons which wereChAT−, thus mimicking the situation found at birth. BDNF-inducedp75+/GAD67+ neurons were characterized by a small soma and shortprocesses and thus presented a morphology clearly distinguishablefrom p75+/ChAT+ population showing big somas and very longprocesses. Altogether, these data suggest a putative role for BDNF inthe regulation of the p75-positive phenotype in a GABAergic neuronsubset.

Investigation of themechanisms throughwhich BDNF induced p75expression in GABAergic neurons in vitro, was conducted by selectiveinhibition of p75 and TrkB activation with Ab9651 and K252arespectively. When K252a and Ab9651 were used alone, neuronalviability was not compromised, and p75 expression was not detected

in GABAergic neurons, but only in big-soma cholinergic neurons,which were not affected in number. Therefore application of K252a orAb9651 resulted in a situation identical to control conditions. Whenused in combination with BDNF, Ab9651 or K252a completely blockedthe BDNF effect on p75 induction in GABAergic neurons. Consideringthat blockade of p75 by Ab9651 does not compromise BDNFinteraction with TrkB–and similarly blockade of Trks activation byK252a does not compromise BDNF interaction with p75–our resultssuggest that both p75 and TrkB neurotrophin receptors are necessaryto induce p75 expression in GABAergic neurons by BDNF. It alsoappears that p75 and TrkB are not individually sufficient to stimulateBDNF effect on GABAergic neurons. We can speculate that co-expression of both p75 and TrkB may be required for the BDNF effecton GABAergic neurons. Considering that cholinergic neurons expressboth receptor types both in vivo and in vitro, and that p75 (this study)and TrkB (Fryer et al., 1996; Molnar et al., 1998) expression has alsobeen detected in non-cholinergic cells, a complex network ofinteractions between various cellular components of the BF may beon stage for the modulation of GABAergic neuron phenotype. Onepossible view is that cholinergic neurons stimulated by BDNF mayinduce the release of soluble factors stimulating p75 expression inGABAergic neurons. For example, it has been recently shown thatbone morphogenetic factor 9 (BMP9), a soluble factor regulating thedifferentiation of the forebrain, induces p75 expression in non-cholinergic neurons of cultured BF cells (Schnitzler et al., 2008).Additional experiments would be necessary to investigate if BMP9release may be stimulated by BDNF and/or neurotrophin receptorsactivation on cholinergic neurons. Another possibility, or additionalmechanism to that described above, is that p75 receptor expressed byGABAergic neurons may be actively involved in the regulation of itsown expression–together with neurotrophin receptors expressed bycholinergic neurons–and acts as a feedback to modulate its own levels.Another interesting though speculative perspective is that TrkB is co-expressed with p75 in the p75+/ChAT−/GAD67+ population identifiedin this study. In this case, BDNF couldmodulate p75 expression througha cell-autonomous mechanism, involving exclusively neurotrophinreceptors residing in GABAergic neurons. Further investigations arenecessary to establish if TrkB is expressed in p75+/ChAT−/GAD67+

population and to study which mechanisms and neuron types areinvolved in the modulation of p75 expression in this population.

BDNF was not totally ineffective on cholinergic neurons, since,similar toNGF, it increased cholinergic neuron's soma size. In agreementwith our BDNF data, in vivo experiments have shown that BDNF-secreting cell transplantation in BF NBM produced a hypertrophicresponse increasing the cell body size of p75+/ChAT+ neurons, withoutaffecting their number and density (Martinez-Serrano et al., 1996).

Altogether these data show that NGF and BDNF differentially affectvarious phenotypic parameters of cholinergic and GABAergic neurons,and suggest that these neurotrophins may play still undisclosed rolesin the development and physiological functioning of BF circuitry.

Experimental methods

Animals

Male Sprague Dawley rats (Charles River, Wilmington, MA) wereused. The experiments were performed with the approval of theinstitutional animal care committee and the ItalianMinistry of Health,following the NIH Guide for the Use and Care of Laboratory Animalsand in accordance with the European Communities Council Directive(86/609/EEC).

Immunohistochemical phenotyping of neurons in the basal forebrain

Rats at birth (postnatal day, P, 0; n=3), at P15 (n=3) and adultones (n=4) were used in this part of the study. The animals were

631E. Formaggio et al. / Molecular and Cellular Neuroscience 46 (2011) 625–632

deeply anesthetized with chloral hydrate and transcardially perfusedwith 4% paraformaldehyde (PFA) in phosphate buffer, pH 7.4. Thebrains were dissected, postfixed for 2 h at 4 °C in the same fixativesolution, washed and soaked overnight in 30% sucrose in phosphatebuffer. From each brain, coronal sections were cut on a freezingmicrotome at a 40 μm thickness. Sections containing the cholinergicareas of themedial septum (MS), vertical limb of diagonal band (VDB)and horizontal limb of diagonal band of Broca (HDB) were collectedfrom AP +1.00 to +0.20 mm from Bregma (Paxinos and Watson,2004). Sections containing the nucleus basalis magnocellularis (NBM)and the substantia innominata (SI) were collected from AP −1.60 to2.80 mm from Bregma. Sections were collected in series of every thirdor fourth section and processed free-floating for triple immunofluo-rescence staining with primary antibodies directed against p75(rabbit polyclonal 9992 antibodies, 1:500; Promega, Madison, WI,USA), ChAT (goat polyclonal, 1:100; Chemicon/Millipore, Billerica,MA, USA), and GAD67 (mouse monoclonal, 1:1000; Chemicon),diluted in phosphate-buffered saline (PBS) containing 0.5% bovineserum albumin (BSA) and 0.5% Triton X-100 (overnight incubation at4 °C). After washing, the sections were incubated for 2 h at roomtemperature in the following fluorochrome-conjugated secondaryantibodies (all from donkey and diluted 1:1000): anti-rabbit Cy3(Amersham, Piscataway, NJ, USA), anti-goat AlexaFluor-633 (Chemicon),anti-mouse AlexaFluor-488 (Chemicon).

Cell cultures

Rat primary cultures of BF neurons were prepared from Sprague–Dawley pups at embryonic day (E) 20. BF cholinergic regions weredissected and processed for in vitro culturing as previously described(Formaggio et al., 2010). Briefly, chemical dissociation was performedby incubation with 0.1% trypsin (Gibco/Invitrogen, Carlsbad, CA, USA)plus 0.1 mg/ml DNAse treatment for 18 min at 37 °C. Upon trypsinblockingwith 10% fetal bovine serum (BioWittacker/Lonza Group Ltd.,Basel, Switzerland) and extensive washings, cells were mechanicallydissociated with fine-pipette tips in Hank's Balanced Salt Solution(Gibco). Neurons were plated on poly-D-lysine-coated coverslips at adensity of 1500 cells/mm2 in Neuron Chow (2% B27 Supplement,0.5 mM L-glutamine in Neurobasal medium) supplemented with12.5 μM glutamate, 100 U/ml penicillin and 100 μg/ml streptomycin(all from Gibco). Cells were grown at 37 °C, 5% CO2 and half of themedium was changed after 4 days in culture with Neuron Chowwithout glutamate. BF cultures were grown for 12 days in the absenceof exogenously added neurotrophins and then exposed to thetreatments (see Cell culture treatments) or processed for phenotyp-ical and morphological characterization (see Immunofluorescence ofBF cell cultures).

We previously reported that in these culturing conditions BF culturesat day 12 show the expression profile, morphological features, andprotein colocalization pattern typical of BF cells (Formaggio et al., 2010).Specifically, BF cultures express mRNAs for all neurotrophin receptors,(p75, Trk-A, -B and -C) and for cholinergic (ChAT) andGABAergic (GAD67)transcripts. Immunostaining analysis shows that ChAT+ neurons exhibitlarge somata and well-developed neurites. All ChAT+ neurons are alsop75+ and vice versa, thus indicating complete co-expression of the twomarkers in basal conditions (Formaggio et al., 2010).

We further determined here by immunofluorescence and confocalmicroscopy analysis that the total neuronal population was composedof glutamatergic (phosphate-activated glutaminase-positive, PAG+),GABAergic (GAD67+) and cholinergic (ChAT+) neurons (Supplemen-tary material 1).

Cell culture treatmentsCultures were treated for 72 h with 100 ng/ml NGF or 100 ng/ml

BDNF (both from Sigma, St. Louis, MO, USA) and then analyzed byimmunofluorescence. In some experiments, cultures at day 12 were

treated with the blocking anti-p75 antibody Ab9651 (antibodydirected against the neurotrophin binding site of p75 receptor; kindlyprovided by M. Chao, Skirball Institute, New York University, NewYork) or Trks inhibitor K252a (Sigma). Ab9651 (7.5 μg/ml) and K252a(100 nM) were administered 1 h before treatment with BDNF. K252a(100 nM) was re-added to the culture after 36 h.

Immunofluorescence of BF cell culturesBF cultures grown on coverslips were fixed and permeabilized by

3 min incubation with cold methanol, followed by 30 min incubationin 5% FBS in HBSS (quenching).

The following protocol was used for GAD67 immunofluorescence:20 min in a solution of 4% PFA and 4% sucrose in phosphate buffer,30 min blocking in quenching, 10 min permeabilization in 5% FBS and0.1% Triton X-100 in HBSS. Primary and secondary antibodies werediluted in quenching and applied for 1 h and 45 min, respectively. Thefollowing primary antibodies were used: rabbit polyclonal anti-p75antibodies 9992 (1:500; Promega), mouse monoclonal anti-p75 192IgG (1:500; Alomone Labs, Jerusalem, Israel), goat polyclonal anti-ChAT (1:100, Chemicon), mouse monoclonal anti-neuronal nuclei(anti-NeuN, 1:200; Chemicon), mouse monoclonal anti-microtubule-associated protein 2 (anti-MAP2, 1:300; Sigma), rabbit polyclonalanti-MAP2 (1:100; Chemicon), rabbit polyclonal anti-glial-fibrillay-acidic protein (anti-GFAP, 1:1000, Sigma), mouse monoclonal anti-PAG (a marker for glutamatergic neurons, 1:500; kindly provided byTakeshi Kaneko, Kyoto University, Kyoto, Japan), mouse monoclonalanti-GAD67 (1:1000; Chemicon), and mouse monoclonal anti-nestin(a marker for undifferentiated cells, 1:1000; BD Biosciences, San Jose,CA, USA). The following secondary antibodies were then used (alldiluted 1:1000): goat anti-rabbit Cy3 (Amersham), chicken anti-rabbit488 (Amersham), goat anti-mouse 488 (Amersham), donkey anti-goat546 (Invitrogen, Paisley, UK), and donkey anti-mouse 488 (Invitrogen).

Flow cytometry (FACS)Cultured cells were detached using 1.25% trypsin, washed with

Neurobasal Medium+10% FBS, and resuspended at 105 cells/100 μLin PBS. Cells were permeabilized by Fix and Perm (Caltag/Invitrogen)and incubated with anti-ChAT antibodies (Chemicon) at roomtemperature for 15 min. Cells were washed in 0.3% BSA in PBS andthen incubated with fluorochrome-conjugated secondary antibodyfor 10 min. At least 10,000 events were acquired by flow cytometry(FACScalibur, BD Biosciences). Morphological gate on forward vs. sidescatter (FSC vs. SSC) dot plot was drawn and the ChAT meanfluorescence intensity was evaluated. Analysis was performed usingFlowJo software (Tree Star, Ashland, OR, USA).

Data analysis

Confocal microscopySections and/or coverslips were mounted on glass slides with 1,4-

diazabicyclo [2.2.2] octane (Sigma) in PBS containing 50% vol/volglycerol. Brain sections were observed with a LSM710 confocalmicroscope, whereas cell cultures were observed with a LSM510confocal microscope (both from Carl Zeiss, Jena, Germany). Bothmicroscopes were equipped with argon and helium/neon excitationlasers, allowing excitation at multiple wavelengths. Confocal imageswere acquired with 1 airy unit pinhole aperture for all channels used(488, 546, and 633). Sequential acquisition mode and conditionsavoiding channel overlapping were used. For each immunostainingcondition, the same parameter settings (detector gain, amplificationgain, detector offset, laser intensity, pinhole aperture, objective, etc.)were kept constant for all images acquired.

Image quantificationIn vitro immunofluorescence experiments were replicated at least

5 times using independent sets of cell cultures. For each replication, at

632 E. Formaggio et al. / Molecular and Cellular Neuroscience 46 (2011) 625–632

least 2 glass slides (accommodating 1 cell coverslip each) percondition were analyzed. For cell counting, 5 regions of interest(ROIs) per slide, each of 460.7 μm2, were randomly acquired with a20× objective from BF cultures double-stained for p75/NeuN and p75/ChAT. Cell counting analysis was performed with NIH Scion program(www.scioncorp.com). Positive cells for each marker (p75, ChAT, andNeuN) were defined as cells reaching fixed intensity threshold values.p75/ChAT double staining images were used also formeasurements ofthe soma size of p75+ cholinergic neurons. The area of soma wasquantified manually using standard tools of LSM510 software.Measurements of p75 fluorescence signal intensity were obtainedfrom the analysis of 5 ROIs (230.3 μm2 each) per slide, randomlyacquired with a 40× objective following p75/NeuN and p75/ChATstaining. Mean fluorescence signal intensity was measured on totalstained neuronal area with NIH Scion program.

StatisticsStatistical significance was analyzed by Student's t tests using the

GraphPad Prism, v.4,02 (GraphPad Software, San Diego, CA, USA). Forimmunofluorescence counting and FACS analysis, n values corre-sponded to the total number of slides, and samples analyzed for eachcondition, respectively. For neuron soma size and signal intensitymeasurements, n values corresponded to the number of p75-positiveneurons analyzed.

For data presented in Table 1, statistical analysis was separatelyperformed for each population (or column) in order to compare theeffect of treatment vs. its control. For GABAergic population onesample t-test showed significant difference between the mean valuefor the BDNF treatment and zero (assumed as control condition). Forcholinergic and total population, statistical analysis was made withunpaired t-test vs. control as described above.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.mcn.2011.01.002.

Acknowledgments

The authors thank Prof. Marina Bentivoglio, University of Veronafor her critical reading of the manuscript, useful editing support andhelpful discussion. Technical assistance of Dott. Francesco Bifari withflow cytometry is also acknowledged.

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