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Originally published In Press as doi:10.1074/jbc.M308287200 on August 1, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42313-42320, October 24, 2003
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Cerebellar Deficits and Hyperactivity in Mice Lacking Smad4*

Yong-Xing Zhou{ddagger}§, Mingrui Zhao§, Dan Li{ddagger}§, Kazuhiro Shimazu¶, Kazuko Sakata¶, Chu-Xia Deng{ddagger}||, and Bai Lu¶**

From the {ddagger}Mammalian Genetics Section, NIDDK and the Section on Neural Development and Plasticity, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 29, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Smad4 is a central mediator of TGF-{beta} signals, which are known to play essential roles in many biological processes. Using a Cre-loxP approach to overcome early embryonic lethality, we have studied functions of TGF-{beta}/Smad4 signals in the central nervous system (CNS). No obvious deficits were detected in mice carrying the targeted disruption of Smad4 in the CNS. The overall morphology of the hippocampus appeared normal. There was no change in the proliferation of neuronal precursor cells, nor in several forms of synaptic plasticity. In contrast, deletion of Smad4 resulted in a marked decrease in the number of cerebellar Purkinje cells and parvalbumin-positive interneurons. Accompanied by the abnormality in the cerebellum, mutant mice also exhibited significantly increased vertical activity. Thus, our study reveals an unexpected role for Smad4 in cerebellar development and in the control of motor function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transforming growth factor-{beta} (TGF-{beta})1 superfamily consists of several subfamilies of more than 40 ligands (1). The TGF-{beta} subfamily has 3 members: TGF-{beta}1, {beta}2, {beta}3. In the brain, TGF-{beta}1 is expressed mainly in the meninges (2, 3). During early development, TGF-{beta}2 and {beta}3 are widely expressed in the CNS (4). However, their expression is generally not associated with areas enriched with dividing progenitor cells, suggesting that TGF-{beta}s are not involved in controlling mitosis in the CNS (except in cerebellar granule neurons of external granular layer) (3). In the adult brain, TGF-{beta}2 immunoreactivities are found in astrocytes as well as in large neurons such as pyramidal neurons in the hippocampus, cortex, spinal motor neurons, and cerebellar Purkinje neurons (5). TGF-{beta}3 is widely distributed throughout the postnatal brains although at very low levels (2, 3). In the cerebellum, TGF-{beta}2 is the predominant isoform produced by Purkinje cells and by both proliferating and postmitotic granule cells (6). TGF-{beta}2 regulates the proliferation of cultured cerebellar neurons (6, 7). TGF-{beta}2 has also been shown to promote neurite growth and prevent neuronal death induced by ischemia or neurotoxic agents in cultured hippocampal neurons (8-13). The bone morphogenetic proteins (BMPs) belong to the second and the largest subfamily that contains many members. BMPs and their receptors are expressed in brain (14). Treatment of neural plate explants with BMPs induces the expression of cerebellar granule cell markers, and BMP-treated neural cells form mature granule cells after transplantation into the postnatal cerebellum (15). BMP-7 enhances dendritic growth in cultured hippocampal neurons (16). The third subfamily, the activins, is mostly involved in neural induction at the early stage of development (17), but has been shown to support the survival of hippocampal neurons in culture (18). Despite extensive studies using cultured cells, the functional role of TGF-{beta}s in vivo has not been well established. One of the major difficulties is that mice lacking TGF-{beta}s die early in development (19, 20).

All TGF-{beta} signal via the same heteromeric, serine-therine kinase receptor complex containing type I (T{beta} RI) and type II (T{beta} RII) receptors (1). Ligand binding induces association of T{beta} RI and T{beta} RII, leading to unidirectional phosphorylation and activation of T{beta} RI by T{beta} RII. The signal transduction mechanisms of TGF-{beta}s require Smad proteins, which constitute a family of 8 members (21, 22). Smads are divided into three major classes. The first class is called receptor-regulated, or R-Smads, which includes Smads 1, 2, 3, 5, and 8. These Smads are directly phosphorylated by T{beta} RI, and can be further divided into two categories: Smad2 and Smad3 respond to TGF-{beta} and activins (23-27), while Smad1, Smad5, and Smad8 function in BMP signaling pathway (28-32). The second class is called inhibitory Smads, which includes Smad6 and Smad7 and counteract the effects of R-Smads and antagonize TGF-{beta} signaling (33-35). The third class is called Co-Smad, and contains only one member, Smad4, also known as DPC4. Smad4 forms various heterodimers with the R-Smads, which in turn are translocated to the nucleus to induce or repress the expression of TGF-{beta} target genes. Smad4/DPC4 is therefore the central mediator of the signal transduction of all TGF-{beta}s (28, 29). Mutant mice carrying a targeted disruption of Smad4 died at embryonic day 7, indicating an indispensable role of Smad4 signals in early stages of embryonic development (36, 37). However, a role of Smad4 in postnatal development and adult remains illusive due to the early lethality associated with the Smad4 deficiency.

Very little is known about the expression and distribution of Smad proteins in the brain. Even less is known about the role of Smads in the development and function of the brain. A recent study showed that in cerebellar cultures, BMP4 application elicits nuclear translocation of Smad1 and promotes the differentiation of both neurons and astrocytes, and these effects were attenuated by treatment of the cultures with antisense oligonucleotides to Smad1 or Smad4 (38). Similarly, majority of the studies on functions of TGF-{beta}s in the nervous system have been done using culture models (2, 3). The goal of the present study is to investigate the role of TGF-{beta}s/Smads in the development and/or function of the CNS in vivo. Since Smad4 is the Co-Smad essential for all TGF-{beta}s/Smads signaling, we chose to examine the functional consequences of the inhibition of Smad4 in the brain. To overcome embryonic lethality of the Smad4 knockout, we used the Cre-loxP approach to specifically disrupt Smad4 in the brain. Surprisingly, we found that mice with deletion of Smad4 in the brain were relatively normal. Detailed analyses revealed a significant decrease in the number of Purkinje cells in the cerebellum, and abnormal function in vertical locomotive activity. In contrast, the loss of Smad4 does not interfere with hippocampal development and functions despite of its high expression in this region. These results indicate a critical role of Smad4 in cerebellum development and motor control.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mouse Strains, Genotyping--Mice with two loxP-flanked Smad4 alleles (Smad4Co) were crossed with transgenic mice in which Nestin regulatory element controls Cre recombinase expression (Nes-Cre). We found that the paternally derived Nes-Cre [Smad4Co/Co (female) X Smad4Co/+;Nes-Cre (male)] caused embryonic lethality of Smad4Co/Co; Nes-Cre offspring. To generate adult mice lacking Smad4 in the CNS, Smad4Co/+;Nes-Cre or Smad4Co/Co;Nes-Cre females were used to cross with Smad4Co/Co males. In these crosses, the Smad4Co/Co;Nes-Cre offspring survived to adulthood at a rate closed to predicted. To examine the spatial distribution of Cre-mediated recombination, we crossed female Nes-Cre transgenic mice with male Rosa-26 reporter line that express {beta}-galactosidase upon cre-mediated removal of a stop codon (39). Mice carrying a Smad4 conditional allele were genotyped by PCR with the following primers: Smad4-9: 5'-GGG CAG CGT AGC ATA TAA GA-3', Smad4-10: 5'-GAC CCA AAC GTC ACC TTC AC-3'. For genotyping Nestin-Cre transgene, we used the following primers: CreF: 5'-TGG GCG GCA TGG TGC AAG TT-3', CreR: 5'-CCG TGCTAA CCA GCG TTT TC-3'. All animal procedures were conducted in complete compliance with National Institutes of Health Guidelines.

Southern, Northern, and Western Blots—Southern: Genomic DNAs from hippocampus or cerebellum were digested by EcoRV, and hybridized with 32P-labeled probe indicated in Fig. 2A. Northern: RNAs were isolated from adult brains using RNA STAT-60 according to manufacturer's instruction (Tel-Test, Inc.). About 30 µg of total RNA from each sample was separated on an agarose gel and transferred to a Gene-Screen filter. The probe, a 5'-end cDNA fragment (800 bp), was labeled with [{alpha}-32P]dCTP by nick translation to a specific activity of around 109 cpm/µg. The filters were hybridized with the probe at high stringency (65 °C), washed at 54 °C in 0.1x SSC, and exposed to Kodak XAR-5 films, which were scanned. The intensities of the bands were quantified using NIH Image software. Signals from mutant mice were calculated as the percentage of the average signals in the wild type mice in the same blot. Western: Proteins from homogenized cerebellar tissues were separated by SDS-PAGE, and transferred to nitrocellulose filter. Parvalbumin was detected by a mouse monoclonal antibody against parvalbumin (Sigma, 1:1000), followed by a peroxidase-labeled sheep anti-mouse IgG (Amersham Biosciences; 1:5000), using a standard ECL procedure.



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  		FIG. 2.
Disruption of Smad4 in adult brain. A, a diagram showing the cross between Smad4 conditional mice with Nestin-Cre mice and Smad4 conditional allele before and after Cre-mediated recombination. B, Southern blot showing Cre-mediated recombination. DNA samples were prepared from brain tissue of wild-type mice (first lane), and Smad4Co/Co mice (second lane), and Smad4Co/Co;Nes-Cre (Mutant) mice (third and fourth lanes). DNAs were digested with EcoRV and probed with a 2.2-kb HpaI-EcoRV fragment. C, Northern blot analysis of RNAs isolated from adult brain tissue of Smad4Co/Co (first lane) and Smad4Co/Co;Nes-Cre mutant mice (second lane). GAPDH was used for loading control.

 

X-Gal Histochemistry, Histology, and Immunohistochemistry—For X-gal staining, animals were anesthetized and transcardially perfused with 10 ml of phosphate-buffered saline (PBS), 5 ml of 2% paraformaldehyde in PBS, and then. In all cases, animals were anesthetized and transcardially perfused with 10~20 ml of PBS, 40 ml of 2~4% paraformaldehyde in PBS, and then 5~20 ml of PBS. For X-gal staining, the brains were processed through a series of sucrose-PBS solutions (15~30%), sectioned into 10-20-µm thick (coronal or sagittal) sections on a vibratome. The sections were mounted on gelatin-coated slides, air dried, and fixed again in 2% paraformaldehyde, 0.125% glutaraldehyde in PBS for 3 min. The sections were rinsed in PBS containing 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, and incubated in X-gal-staining buffer (containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-D-galactopyranoside) at room temperature overnight. For hematoxylin and eosin (H&E) staining of hippocampus and cerebellum, the brains were postfixed with 4% paraformaldehyde for 6-16 h and embedded in paraffin. Thin sections (10-20 µm) were cut and processed according to standard protocol. For immunocytochemistry, the brains were postfixed overnight then were cryoprotected in 30% sucrose, and sectioned at 40 µm in a cryostat. The following primary antibodies were used: monoclonal antibodies against parvalbumin D-28k (Sigma, 1:1000) and glial fibrillary acidic protein (GFAP, DAKO). Positive cells were detected by the Zymed Laboratories Inc. HistoMouse-SP kit (Zymed Laboratories Inc., or by FITC-conjugated secondary antibody. To measure apoptotic cells on tissue sections, we used a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay detection system from Intergen according to the manufacturer's directions.

BrdU Labeling—Mice received an intraperitoneal injection of BrdU (Sigma, 300 mg/kg body weight) and were killed 3 h later by transcardial perfusion of 4% paraformaldehyde. Frozen sections (40 µm) were cut and processed for BrdU immunohistochemistry with diaminobenzidine (DAB) following the ZYMED BrdU staining protocol (Zymed Laboratories Inc.). In brief, endogenous peroxidases were inactivated in methanol/H2O2 (196 ml/4 ml) for 20 min at room temperature. Sections were treated with trypsin for 25 min and denatured by HCl (4 N) for 30 min then incubated in blocking solution for 20 min at room temperature. After incubating overnight at 4 °C with biotinylated monoclonal mouse anti-BrdU, the slices were incubated with streptavidin-peroxidase for 10 min and DAB mixture for 10 min. All slices were counterstained with Hematoxylin. The slides were dehydrated in a graded series of alcohol, cleared with xylene and cover-slipped under Histomount.

Cell Counting—Cells were counted in a one-in-six series of sections (40 µm per section, 240 µm apart) from Smad4Co/CoNes-Cre and Smad4Co/Co mice by the optical fractionator method (40). The stereology setup consists an Olympus IX70 microscope fitted with a Ludl AMG 5000 XYZ motorized stage (Ludl Electronic Products Ltd) and a Micro-Max 1300 cooled CCD camera (Roper Scientific, Inc.). Images were acquired by IPLab software (Scannalytics). Positive cells (BrdU or parvalbumin labeled) were counted under an oil immersion objective (60x, numerical aperture = 1.25) by a Gundersen's unbiased counting frame (41) within the optical dissector height, which was set at the middle 2/3 of total tissue thickness for each section. The sampling was performed according to unbiased stereological principles (42). The total positive cell number (N) was estimated as in Equation 1,

(Eq. 1)
where sum Q- is the total number of cells actually counted in the disectors; tsf is the thickness sampling fraction; asf is area sampling fraction; and ssf is the section sampling fraction.

Slice Preparation and Electrophysiology—Transverse hippocampal slices (400 µm) were prepared from control and mutant mice (4-5-week-old). The slices were maintained in an interface chamber for both recovery (2 h) and recording, and exposed to an artificial atmosphere of 95% O2, 5%CO2 (43). The slices were perfused in artificial cerebrospinal fluid (ACSF, 34 °C) containing (in mM): NaCl 124, KCl 3.0, CaCl2 2.5, MgCl2 1.5, NaHCO3 26, KH2PO4 1.25, glucose 10, ascorbic acid 2, pH 7.4, at a rate of 15 ml/h. Field excitatory postsynaptic potentials (EPSPs) were evoked in molecular layer by stimulating medial or lateral perforant pathways (MPP, LPP, respectively) in dentate gyrus with Teflon-insulated monopolar stainless steel electrodes and recorded with ACSF-filled glass pipettes (<5 M{Omega}) using an Axoclamp-2B amplifier (Axon Instruments). Test stimuli consisted of monophasic 200 µsec pulses of constant current delivered by stimulus isolation units. Basal synaptic transmission was monitored at every 30 s with a stimulus intensity that produced a quarter-maximal response. EPSPs were digitized (10 kHz), filtered at 3 kHz using acquisition system pClamp6, stored on magnetic media and analyzed off-line using Clampfit 8.0 (Axon Instruments) and Microsoft Excel visual basic programming (Microsoft Corp.). Tetanic stimulation (100 Hz, 500 ms duration, repeated every 30 s for 4 times) at a stimulus intensity that elicited the maximal EPSP was used to induce LTP. The positions of the stimulating and recording electrodes were adjusted to obtain selective stimulation of MPP or LPP, based on the following two criteria (44). First, the polarity of EPSPs reverses across the outer and middle third of the molecular cell layer by stimulation of the LPP or MPP, reflecting an inward current sink or an outward current source. The sharp polarity reverse indicates that a fine local stimulation of LPP or MPP fibers. Second, the LPP-dentate synapses exhibit paired-pulse facilitation (PPF) of the EPSP whereas the MPP-dentate synapses exhibit paired pulse depression (PPD) at certain intervals (45). For response to high frequency stimulation (HFS), changes in EPSPs over time were obtained by normalizing successive EPSP slopes to the first EPSP slope in the train. EPSP slopes were plotted against the number of the stimulus in each HFS.

Behavioral Tests—(1) Open-field activity. Animals were individually placed in a Digiscan open-field box (RXYZCM, AccuScan Instruments, Inc.) equipped with two rows of infrared beams that automatically detect and quantify their activities. The horizontal (locomotor activity) and vertical (rearing) activities were measured by the number of times an animal broke the first (lower) and the second (upper) row infrared beams, respectively (2). Balance bar task. Mice were held upside-down by their tails and then placed against a wooden rod allowing mice to grasp the rod only with their hind limbs. The tails were then released and the time it took the mice to pull themselves into a balanced position upon the rod was recorded (3). Rotarod test. Mice were placed on a rotating drum and the time they were able to maintain their balance on the rod was measured at low (3 rpm) and high (23 rpm) speeds. The latency and rotation speed at which the animal falls off were recorded.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cre-mediated Inactivation of Smad4 in Adult Central Nervous System—To specifically inactivate Smad4 in the CNS, we crossed mice carrying a Smad4 conditional allele (Smad4Co) (46) with mice carrying a Nes-Cre transgene (47). The Nes-Cre mice have been used in a number of previous studies, all of which used male Nes-Cre mice to cross with female mice carrying conditional alleles. Several groups (48-50) reported high efficiency (70-100%) of Cre-mediated deletion in CNS during late embryonic stages. In our initial crosses, we found that when the Nes-Cre was from male [Smad4Co/Co (female) X Smad4Co/+;Nes-Cre (male)], the Smad4Co/Co;Nes-Cre offspring died at early post-implantation stages exhibiting defects very similar to the those of Smad4-/- mice (37), Table I). These results not only confirm the importance of Smad4 in early embryos, but also suggest that paternally derived Nes-Cre is active at very early stages. In contrast, when the Nes-Cre was from female [Smad4Co/+;Nes-Cre (female) X Smad4Co/Co (male)], the Smad4Co/Co;Nes-Cre mice survived to adulthood. Since our goal was to study the role of Smad4 in the CNS in the adult, the latter cross was used in the remaining studies.


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  		TABLE I
Mating scheme and frequency of mutant mice

Data from more than 200 littermates are compiled here. M: male; F: female.

 

To determine whether and when the maternally derived Nes-Cre is expressed during CNS development, we crossed female Nes-Cre transgenic mice with male Rosa-26 reporter line that express {beta}-galactosidase upon cre-mediated removal of a stop codon (39). The Cre gene was expressed as early as E11.5 (Fig. 1A), and gradually increased during development (not shown). In 4-week-old mice, the Cre-expressing cells were found throughout the brain (Fig. 1B). Strong expression was detected in the granule cell layer of the dentate gyrus and CA1-CA3 regions of the hippocampus (Fig. 1C), and in the Purkinje cell layer of the cerebellum (Fig. 1D). The expression of Nes-Cre in the hippocampus and cerebellum was maintained and perhaps slightly increased as animals grew older (e.g. Fig. 1E, 7-week-old hippocampus, Fig. 1F, 10-week-old cerebellum). These results suggest that the maternally derived Nes-Cre is expressed in somatic cells in the CNS, albeit slightly later in embryonic development than that derived paternally. This delayed expression of Nes-Cre may explain why the [Smad4Co/+;Nes-Cre (female) X Smad4Co/Co (male)] crosses were not embryonically lethal (Table I). In the mean time, we have achieved effective deletion of the Smad4 gene postnatally in the brain.



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  		FIG. 1.
Expression of maternally derived Nes-Cre gene in mouse brain. The expression of Nes-Cre was determined in offspring of [Nes-Cre/+ (female) X Rosa-26/+ (male)]. The expression of Cre recombinase was detected by X-gal staining. All sections were counter-stained by fast red. A, a low power (1x objective) view of a sagittal section of E11.5 embryo. Note the sporadic blue cells in various areas in the CNS. B, a low power (1x objective) view of a coronal section of the forebrain of a 4-week-old mouse. C, a higher magnification (4x objective) of the same section showing hippocampus. D, a coronal section at high magnification (4x objective) showing cerebellum. E, a sagittal section (4x objective) of hippocampus of a 7-week-old mouse. F, a sagittal section (4x objective) of cerebellum of a 10-week-old mouse.

 

Fig. 2A shows the structures of wild-type allele, conditional allele, and Nes-Cre-mediated deletion of the exon 8 of the Smad4 gene. Southern and Northern blot analyses of Smad4 were performed to determine the extent of Cre-mediated recombination. Southern blot analysis on DNA isolated from hippocampus and cerebellum indicated that the deletion of Smad4 was incomplete (Fig. 2B). Further, Northern blot revealed that the levels of Smad4 transcripts in hippocampus and cerebellum of the conditional mutants (Smad4Co/Co;Nes-Cre) were reduced by ~80% (Fig. 2C). Since the Cre-mediated deletion of Smad4 occurred effectively in postnatal Purkinje cells in the cerebellum and granule cells in hippocampus, we focused on the analysis of the development of these areas in the Smad4Co/Co;Nes-Cre mice.

Lack of Effects of Smad4-deficiency on Hippocampus Development and Function—The shape and the size of brains from Smad4Co/Co;Nes-Cre mice appeared normal (not shown). Examination of histological sections throughout the brain did not reveal any gross morphological abnormality (not shown). Since the expression of Smad4 and Nes-Cre recombinase activity was relatively high in the hippocampus, we analyzed this region in some details. The cytoarchitectural organization of the granule cell layer of the dentate gyrus and pyramidal cell layer of CA1-CA3 regions in Smad4Co/Co;Nes-Cre mice was almost identical to that of control (Smad4Co/Co) mice (Fig. 3, A and B). Thus, the inactivation of Smad4 does not seem to elicit overt abnormality in hippocampal anatomy or development.



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  		FIG. 3.
Normal hippocampal morphology and proliferation of neuronal precursor cells in Smad4 mutants. A and B, low power (4x) images of hippocampi from the control and mutant mice. H&E staining reveals similar morphology of the control (left) and mutant (right) hippocampus. C and D, medium power (20x) images of BrdU-labeled cells in the control (left) and mutant (right) dentate gyrus. The sections were processed for BrdU immunohistochemistry followed by Nissl counter stain. The proliferating cells were labeled by BrdU (arrows). E, summary of BrdU cell counts in the dentate using stereological techniques. Total number of BrdU-labeled cells per brain (2 dentates) is presented. Open bar, control; filled bar, Smad4 mutant. n = 5 pairs of animals (2-month-old). No significant difference was found between the two groups (Student's t test, p = 0.2942).

 

TGF-{beta}s are generally considered as anti-mitotic factors in many systems. It has been reported that TGF-{beta}2 inhibits the proliferation of neuroendocrine chromaffin cells (51, 52) and precursors of cerebellar granule cells (6) in culture. The dentate gyrus of the hippocampus is one the few regions in the adult brain where prominent neurogenesis could be observed. We therefore tested whether inhibition of TGF-{beta} signaling by Smad4 deletion affects adult neurogenesis by measuring the incorporation of bromodeoxyuridine (BrdU) into the dividing neuronal precursor cells. Three hours after BrdU was injected intraperitoneally the brains of the injected animals were fixed and processed for BrdU histochemistry. We found that the proliferating neuronal precursors were mainly concentrated in the granule cell layer of the dentate gyrus in the adult hippocampus (Fig. 3, C and D). We counted the dentate BrdU-labeled cells using stereological techniques. There was no difference in the number of BrdU positive cells in the dentate gyrus between mutant (Smad4Co/Co;Nes-Cre) and control (Smad4Co/Co) mice (Fig. 3E, Student's t test, p = 0.2942). These results suggest that TGF-{beta}/Smad4 signals are not involved in controlling neuronal mitosis in the adult hippocampus.

To further study functional role of TGF-{beta}/Smad4 in the hippocampus, we performed a series of electrophysiological experiments. Field excitatory postsynaptic potentials (EPSPs) were recorded by stimulating medial or lateral perforate pathways (MPP or LPP) of the hippocampal slices. MPP and LPP synapses can be distinguished not only by their afferent inputs, but also by their responses to paired pulse stimuli. When two consecutive stimuli are applied within a range of inter-pulse interval (ISI), the MPP synapses usually exhibit paired pulse depression (PPD), i.e. the second response is smaller than the first one. In contrast, the LPP synapses undergo paired pulse facilitation (PPF). These are short-term forms of synaptic plasticity that reflect changes in the probability of transmitter release (Pr) (53). We measured paired pulse responses using the ratios of the second and the first EPSP slopes at ISIs of 10, 20, 50, 80, and 100 ms. As shown in Fig. 4A, PPF profiles of the LPP synapses in slices from control and mutant mice were almost identical, exhibiting a typical change at different ISIs: close to 100% at 10 ms, peaked at around 50 ms, and low again thereafter (control: n = 11 slices, n = 7 animals; mutant: n = 9 slices, n = 5 animals). No difference was observed between PPD at the MPP synapses from the control and the mutant mice at all ISI examined (Fig. 4A, lower panel, control: n = 18 slices, n = 7 animals; mutant: n = 8 slices, n = 5 animals). Another short-term plasticity was synaptic fatigue at the LPP synapses induced by a brief, high frequency stimulation (HFS, 100Hz, 50 pulses). This is a good measure for the readily releasable pool of synaptic vesicles (54, 55). The EPSP slopes during the entire HFS were recorded from CA1 synapses of control and mutant mice, normalized to the first EPSP slope, and were plotted against stimulus numbers. Fig. 4B shows the averaged responses to HFS. The EPSP slopes exhibited a continuous decline over time, indicative of a gradual depletion of readily releasable pool vesicles. The LPP synapses from control and mutant mice exhibited the same responses to HFS (n = 10 slices/2 animals for both Smad4Co/Co and Smad4Co/Co;Nes-Cre). Next we determined whether Smad4 mutation affects long-term synaptic plasticity in the dentate. At the LPP synapses, a brief tetanic stimulation (100 Hz, 1 s) induced an enhancement of synaptic efficacy that lasted more than 1 h in control slices. Deletion of Smad4 had no effect on long-term potentiation (LTP) (Fig. 4C, upper panel; control: n = 5 slices, n = 4 animals; mutant: n = 8 slices, n = 4 animals). At MPP synapses, robust LTP was induced only when the GABAergic transmission was inhibited by bicucculine (10 µM). Again, there was no difference in the magnitude of LTP at these synapses between the control and mutant slices (Fig. 4C, lower panel; control: n = 9 slices, n = 4 animals; mutant: n = 10 slices, n = 6 animals). Taken together, deletion of Smad4 does not seem to affect synaptic transmission and plasticity at the dentate synapses.



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  		FIG. 4.
Normal synaptic plasticity in hippocampal dentate gyrus in Smd4 mutant. Field EPSPs were recorded at dentate gyrus by stimulating MPP or LPP. Data (open circles, control; filled circle, Smad4 mutant, 4-5-week-old) from multiple animals were pooled and expressed as mean ± S.E. A, paired pulse responses. The ratios of the second and first EPSP slopes were calculated, and mean values are plotted against different interpulse intervals (10-100 ms). PPF was observed at LPP synapses while PPD at MPP synapses. B, synaptic response to high frequency stimulation. A train of tetanic stimulation (100 Hz, 50 pulses) was applied at LPP. The slopes of all EPSPs induced by the train were measured, normalized to the first EPSP, and plotted against the number of the pulses. C, LTP at LPP and MPP synapses. Tetanic stimulation was applied to LPP or MPP at time 0, and EPSP slopes recorded 20 min before and up to 60 min after the tetanus was measured and plotted against time. For MPP recording, GABAa receptor antagonist bicucculine (10 µM) was included in the ACSF.

 

Effects of Smad4 Mutation on Cerebellar Neurons—The cerebellum is the second region where Smad4 is highly expressed. The Nes-Cre recombinase activity is also relatively high (Fig. 1, C and F). We therefore also analyzed this region in some details. The gross morphology of the cerebellum of the mutant mice, however, was not changed. There were no obvious deficits in size or foliation of the cerebellar cortex at one to two months of age (Fig. 5, A and B). Purkinje cells typically are located in a single row at the border of the granular and the molecular layer. The cytoarchitectural organization, including the molecular cell layer (MCL), the granule cell layers (GCL), and the Purkinje cell layer (PCL), appeared normal in sections derived from mutant mice (Fig. 6A).



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  		FIG. 5.
Normal morphology and radial glial cells in Smad4 mutant. A and B, low power (4x) images of cerebellum from the control and mutant mice. H&E staining reveals no difference in morphology of the control (A) and mutant (B) cerebellum. The sections from the two panels were cut slightly at different angles. C and D, medium power (20x) images of radial glia, stained by anti-GFAP antibody, in cerebella from a pair of control (C) and mutant mice (D) (2-month-old).

 


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  		FIG. 6.
Reduction in parvalbumin-positive in the Smad4 mutant cerebellum. A, a thin section (20 µm) showing the cytoarchitectural organization of the mutant cerebellum at postnatal day 10. PCL, Purkinje cell layer. MCL, molecular cell layer. The section was stained with anti-parvalbumin antibody, and counterstained with Hematoxylin. B, summary of cell counting results. Open bar, control; filled bar, Smad4 mutant. n = 5 pairs of adult animals (2-month-old). *, p < 0.05, Student's t test. C, Western blot showing a decrease in the levels of parvalbumin in cerebellum in Smad4 mutant. Top, a representative Western blot showing specific band of parvalbumin. Bottom: summary of all results. Protein samples extracted from the cerebella of wild type and mutant mice were run on an SDS gel. The blot was scanned and the intensities of the bands were quantified using NIH Image software. Open bar, control; filled bar, Smad4 mutant. n = 4 pairs of adult animals (2-month-old). *, p < 0.05, Student's t test.

 

Previous studies have suggested a potential role of TGF-{beta} in the cerebellar radial glial cells (3), based mostly on the effects of TGF-{beta} on cultured astrocytes (56). To determine whether TGF-{beta} signaling is important for radial glia in vivo, we examined these cells in the Smad4 mutant cerebellum. Radial glia were detected by immunocytochemistry using an antibody against glial fibrillary acidic protein (GFAP). As shown in Fig. 5, C and D, the distribution and morphology of the radial glia appeared to be quite normal. TGF-{beta} has also been implicated in regulating the survival of a number of neuronal populations (3, 56). We used terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay to measure apoptosis in cerebellum. No difference was found in the number of apoptotic cells in the cerebelli between control and mutant mice (data not shown).

There was a debate whether TGF-{beta}2 stimulates or inhibits the proliferation of cerebellum neurons in cultures (3, 6, 7). It is possible that inhibition of TGF-{beta}2 signaling by Smad4 deletion could interfere neuronal mitosis during development, leading to a change in cell numbers. We examined whether deletion of Smad4 affects the number of Purkinje cells. To reliably quantify the changes in Purkinje cells, we performed immunocytochemistry using parvalbumin antibody, which detects both Purkinje cells and some GABAergic interneurons in the molecular layer (Fig. 6A). Using the optical fractionator method (40), the parvalbumin-containing Purkinje cells and interneurons in the entire cerebellum were counted in serial sections from 5 pairs of control and mutant mice. There was a significant decrease in the number of Purkinje cells in the cerebellar sections from the Smad4Co/Co;Nes-Cre mice (Fig. 6B). On average, the number of parvalbumin-containing Purkinje cells per cerebellum in the mutant mice was only 43% of that in the control littermates (Fig. 6B, Student's t test, p < 0.05). Further, the number of parvalbumin-positive interneurons in the molecular layer was also decreased by 34.4% (Fig. 6B, p < 0.05). Consistent with this, Western blot analysis indicated that the levels of parvalbumin proteins were significantly reduced (Fig. 6C). These results suggest a role of TGF-{beta}/Smad4 signals in regulating neuronal cell number in the cerebellum.

Increased Vertical Activity Observed in Smad4 Mutant Mice—Cerebellum is known to be involved in motor controls. To determine whether the decrease in the number of parvalbumin-positive neurons in the cerebellum could lead to any motor deficits, we performed a number of behavior tests for motor function on the control and Smad4 mutant mice. One such test was the balance bar task (57). Mice were placed upside-down to grasp a horizontal bar 30 cm above ground only with their hind limbs. The tails were then released and the amount of time it took for the mice to pull themselves into a balanced position upon the rod was recorded. No obvious differences were observed between the control and mutant mice (Fig. 7A, n = 8 mice for each genotype, p > 0.78). We next performed rotarod assay, which tests animal's ability to remain on a rotating rod at low (3 rpm) and high (23 rpm) speed. The mean time spent on the rotarod did not differ significantly between the control and mutant mice (n = 8 pairs of mice, data not shown). Finally, we measured open field activity. Mice were placed in a plastic box with two rows of infrared light beams mounted on the sides to detect and distinguish horizontal and vertical movements. Horizontal activity (first row beam breaks) and rearing (second row beam breaks) were measured in a 5-min period. Smad4Co/Co;Nes-Cre (n = 8) and Smad4Co/Co (n = 8) showed very similar horizontal movements, as measured by the number of times the horizontal beams were broken (Fig. 7B). In contrast, the mutant mice exhibited substantially higher vertical activity as compared with the control mice (Fig. 7B, Student's paired t test, p < 0.05).



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  		FIG. 7.
Elevated vertical activity of Smad4 mutant mice. A, balance bar test. Open bar, control; filled bar, Smad4 mutant. B, open field activity. Horizontal and vertical beam breaks were measured in 1-min intervals over a 5-min test session. Open circle, control; filled circle, Smad4 mutant. Note that the mutant mice exhibit a substantially higher vertical activity as compared with the control mice.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study addresses the functional role of Smad4 in the brain in vivo. We used a Cre-loxP system to delete Smad4, the central mediator all TGF-{beta}s/Smads signaling, in the CNS. While the brains of Smad4 mutant mice were relative normal, we did reveal two unexpected and potentially related phenotypes: (1) a significant decrease in the number of cerebellar Purkinje neurons; and (2) abnormality in vertical locomotive behavior. These results indicate a critical role of Smad4 in cerebellum development/function and provide a potential link between cerebellar Purkinje cells and vertical locomotion behavior. Moreover, this is, to our knowledge, the first study on the function of Smads in the brain in vivo.

A somewhat unexpected finding was the lack of morphological and physiological deficits in the hippocampus of the Smad4 mutant mice. Several studies showed that TGF-{beta}s promote neurite sprouting and elongation in culture (9, 16, 18, 58). Another repeatedly reported phenomenon is that TGF-{beta}s protect cultured hippocampal neurons from damages by glutamate excitotoxicity (59, 60), ischemia (8, 12), or {beta}-amyloid (10, 11, 61). There was even a report showing a suppressive effect of activin on LTP in the dentate gyrus (62). We did not detect any morphological impairment in the hippocampus of the Smad4 mutant mice. Electrophysiological recordings demonstrated completely normal synaptic plasticity including LTP in the dentate. BrdU experiments indicated that adult neurogenesis was also normal. There are several possible explanations for the apparent discrepancy between the results obtained from in vitro and in vivo studies. One is that the TGF-{beta}s/Smads signals were not completely inactivated in our Smad4 mutant mice, as indicated by Northern blot showing residual Smad4 RNA in the hippocampus (Fig. 2C). Low levels of Smad4 may be sufficient for certain TGF-{beta}s/Smads functions. Alternatively, hippocampal neurons may exhibit their responsiveness to TGF-{beta} when they are damaged. The current study did not examine the response of the mutant hippocampal neurons to any damaging insults. The third possibility is that the results obtained from culture studies may not completely reflect the in vivo situation. The neurons in cultures are derived from embryonic brains and are living in artificial environments. Finally, we cannot completely exclude the possibility that TGF-{beta}s can still signal in the absence of Smad4.

The lack of phenotypes in the Smad4 mutant cerebellum is also unexpected. TGF-{beta}s have been shown to elicit profound effects on the actin cytoskeleton and stress fibers, enhance the expression of GFAP, and inhibit the proliferation in cultured cerebellar astrocytes (63-65). The Smad4Co/Co;Nes-Cre mice, however, did not exhibit any obvious morphological deficits in the GFAP positive astrocytes in the cerebellar sections. In contrast to a previous report showing that TGF-{beta}s accelerate the apoptosis of cerebellar granule neurons in culture (66), our TUNEL staining did not found any difference in the number of apoptotic cells between wild-type and Smad4 mutant mice. TGF-{beta}s may stimulate or inhibit the proliferation of granule cells depending on culture conditions (6, 7). Moreover, several previous studies showed that BMPs may induce postmitotic neurons to differentiate into cerebellar granule cells in culture (15, 38) and some of these effects are thought to be mediated by Smads (38). While it was difficult to count the number of granule cells, careful examination of the cerebellar sections indicated that the internal granule cell layers from the control and Smad4 mutant mice were almost identical. Once again, our study suggests the importance of confirming culture work by in vivo approaches.

Despite the lack of effects on cerebellar astrocytes or granule cells, the deletion of Smad4 does result in a significant reduction in the number of parvalbumin-positive neurons including the Purkinje cells. Our knowledge about the role of TGF-{beta} s/Smads in Purkinje cell development and/or function is very limited. There was only one report showing that treatment of cultured Purkinje neurons with TGF-{beta} 1 prevented kainite-induced cell death (60). The present study showed that there were significantly fewer Purkinje cells in Smad4 mutant mice, providing the first evidence for a critical role of TGF-{beta} signaling in controlling the Purkinje cell number in vivo. Another very interesting phenotype of the Smad4Co/Co;Nes-Cre mice was increase in vertical (or rearing) activity in the open field test. The difference between genotypes was remarkably selective, while the horizontal activity was not affected at all. What is the relationship between Purkinje cells and spontaneous (voluntary) locomotive activity? The GABAergic Purkinje neurons, the sole output of the cerebellar cortex, send powerful inhibitory controls to the deep cerebellar nuclei. This system is known to be critical in controlling the ongoing execution as well as coordinating the planning of limb movement. Waite et al. (67) infused intraventricularly OX7, the immunotoxin that selectively destroys Purkinje cells. Although the vertical versus horizontal activities were not distinguished, they observed that the OX7-treated rats exhibited hyperactivity when tested in open field. Thus, it is conceivable that impairments of Purkinje cells would reduce the inhibitory output of the cerebellar cortex, leading to an elevated locomotive activity. The mechanisms and implications of the increase in rearing activity seen in the Smad4 mutant animal remain unknown. Rearing on the hind legs in the open field is believed to reflect exploratory behavior (68, 69). An increase in rearing is often associated with enhanced fear, stress or anxiety (70). It may be useful to test whether the Smad4Co/Co;Nes-Cre mice, with specific deficits in the number of Purkinje cells and rearing behavior, can be used as a model for these mental illnesses.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ The first three authors contributed equally to this study. Back

|| To whom correspondence may be addressed. Tel.: 301-402-7225; Fax: 301-480-1135; E-mail: chuxiad{at}bdg10.niddk.nih.gov.

** To whom correspondence may be addressed. Tel.: 301-435-2970; Fax: 301-496-1777; E-mail: bailu{at}mail.nih.gov.

1 The abbreviations used are: TGF-{beta}, transforming growth factor-{beta}; BMPs, bone morphogenetic proteins; BrdU, bromodeoxyuridine; GFAP, glial fibrillary acidic protein; MPP or LPP, medial or lateral perforate pathways; PPF or PPD, paired pulse facilitation or depression; LTP, long-term potentiation; MCL or PCL, molecular or Purkinje cell layer; DAB, diaminobenzidine; CNS, central nervous system; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; X-gal; 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We thank Brian Bates and Andreas Trumpp for the Nes-Cre transgenic mice, and Daniel Abebe for helps with the behavior experiments and Cuiling Li for animal maintenance.



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