University of Groningen

New neurons in the adult brainvan der Borght, Karin

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Input from the medial septum regulates adult hippocampal neurogenesis

Karin Van der Borght1, Jan Mulder1, Jan N. Keijser1, Bart J. L. Eggen2, Paul G. M. Luiten1, Eddy A. Van der Zee1

1) Department of Molecular Neurobiology, Graduate school of Behavioural and Cognitive Neurosciences, and 2) Department of Developmental Genetics, Groningen

Biomolecular Sciences and Biotechnology Institute; University of Groningen, P. O. Box 14, 9750 AA, Haren, Th e Netherlands

Brain research Bulletin (2005), 67(1-2): 117-125

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Abstract

Neural progenitors in the subgranular zone of the hippocampal formation form a continuously proliferating cell population, generating new granule neurons throughout adult life. Between ten days and one month after their formation, many of the newly generated cells die. Th e present study investigated whether a partial lesion of one of the main nuclei projecting to the hippocam-pus, the medial septum (MS), aff ects survival and diff erentiation of cells during this critical pe-riod. Rats were injected with BrdU and fi ve days later excitotoxic lesion of the MS was applied by infusion of either 30 nmol or 60 nmol of N-methyl-D-aspartate (NMDA). One week after the lesion, quantifi cation of immunopositive cells revealed that the number of GABAergic cells was signifi cantly reduced in both lesioned groups, whereas a decline in cholinergic cell number was observed only after injection of 60 nmol of NMDA. Th e partial septohippocampal denerva-tion signifi cantly reduced hippocampal neurogenesis. Survival of newly generated neurons was decreased by approximately 40%. Th e MS lesion did not aff ect proliferation of hippocampal progenitors. Th e present study indicates the importance of a functional septohippocampal path-way for the regulation of hippocampal neurogenesis and it highlights the potential role of GABA as a mediator in this phenomenon.

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Introduction

Th roughout adult life, neural progenitors in the subgranular zone (SGZ) of the dentate gy-rus (DG) give rise to new neurons in the granule cell layer (GCL) (Altman and Das, 1965; Kaplan and Hinds, 1977; Stanfi eld and Trice, 1988). Adult hippocampal neurogenesis has been described in several species, including rodents, non-human primates and humans (Er-iksson et al., 1998; Gould et al., 1997; Kornack and Rakic, 1999). Double labeling of the thymidine analogue 5’-bromo-2’-deoxyuridine (BrdU) and the proliferation marker Ki-67 at diff erent time points after injection with BrdU showed that cell progenitors remain proliferating for approximately four days (Dayer et al., 2003). After the cells become post-mitotic, they diff erentiate into granule cells, send axons to the CA3 region (Hastings and Gould, 1999) and receive input from other cells (Markakis and Gage, 1999). About 50% of the newly generated neurons die between ten days and one month after their birth (Dayer et al., 2003; Hastings and Gould, 1999). Interestingly, there is evidence that many of the newly formed cells can be rescued by training animals in a hippocampus-dependent learn-ing task or housing them under enriched conditions (Ambrogini et al., 2000; Gould et al., 1999a; Kempermann et al., 1997). However, it is still unknown which factors determine if a newly formed cell will survive and be integrated in the hippocampal circuitry. Th e present study was aimed at investigating whether the input from the medial septum (MS) into the hippocampus is involved in the regulation of newly formed cell survival and diff erentiation. Th e MS consists of cholinergic neurons as well as gamma-aminobutyric acid (GABA)-producing interneurons and projection neurons. Th e cholin-ergic neurons innervate the three major cell types of the hippocampus, i.e. the pyramidal, non-pyramidal and granule cells (Frotscher, 1989; Frotscher and Misgeld, 1989; Gaykema et al., 1990), whereas GABAergic projection neurons mainly innervate hippocampal in-terneurons (Freund and Antal, 1988; Gulyas et al., 1991). Th e septohippocampal pathway has been shown to be pivotal for proper hippocampal functioning. Memory performance can be correlated with cholinergic activity in the hippocampus, measured by high-affi nity choline uptake or choline acetyltransferase (ChAT) activity (Decker et al., 1988; Dunbar et al., 1993). Moreover, mechanical disruption of the septal projections to the hippocampus (Alvarez-Pelaez, 1973; Olton, 1977) or lesions of the MS (Hagan et al., 1988; Johnson et al., 2002) disturb memory retention. Considering the key role of the basal forebrain in cognitive functions, the learn-ing-induced increase of adult neurogenesis (Ambrogini et al., 2000; Gould et al., 1999a) could potentially be caused by an increased activation of the septohippocampal pathway. Moreover, during aging a loss in MS cholinergic and GABAergic cells has been reported, which leads to an impairment in hippocampal functioning (Apartis et al., 2000; Fischer et al., 1989; Fischer et al., 1992; Krzywkowski et al., 1995). Concomitantly with the decrease of cell number in the MS, hippocampal neurogenesis signifi cantly declines during aging (Heine et al., 2004a; Kuhn et al., 1996). Additional data also suggest a role for the MS in the regulation of adult hippocampal neurogenesis. Wheel running, for instance, which

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has been shown to evoke hippocampal theta waves, induced by synchronized signaling of septal cholinergic and GABAergic neurons (Teitelbaum et al., 1975), robustly increases hippocampal neurogenesis in mice and rats (Kim et al., 2003; Van Praag et al., 1999b). Moreover, a recent study by Cooper-Kuhn et al. (2004) has shown that 192IgG-saporin infusion into the lateral ventricle, causing cholinergic lesion of the basal forebrain includ-ing the MS, reduces hippocampal neurogenesis. In the present study, rats were injected with BrdU in order to gain a deeper insight into the role of MS-derived cholinergic and GABAergic projections to the hippocampus on the regulation of hippocampal neurogenesis, Five days after the last BrdU injection, N-methyl-D-aspartate (NMDA) was infused into the MS to induce an excitotoxic lesion. One week after the lesion, the impact of a reduced septohippocampal innervation on hip-pocampal neurogenesis was investigated using immunocytochemistry for BrdU and the proliferation marker Ki-67.

Materials and Methods

Animals and experimental procedureAdult male Wistar rats (300-350 g, n=21), bred in our own facilities, were used. Rats were housed individually, had free access to water and food and were kept under a 12/12h light/dark cycle (lights on at 08.00h) in a temperature-controlled environment (21±2°C). Ani-mals were injected intraperitoneally with 100 mg/kg of BrdU (Sigma, St. Louis, MO, USA) dissolved in saline (20 mg/ml, pH 7.0), daily for three consecutive days. Seven days after the fi rst BrdU injection, animals were deeply anesthetized with 2%-2.5% isofl urane and mounted in a stereotaxic frame (Narishige, Japan). Using a Hamilton microsyringe (Bona-duz, Switzerland), 1 μl of 0.01 M phosphate-buff ered saline (PBS) containing 30 nmol (n=7) or 60 nmol (n=7) of NMDA (Sigma) was slowly injected into the MS (coordinates from Bregma: AP: 0.2 mm, ML: 0.0 mm DV: 6.3 mm and 6.0 mm (Paxinos and Watson, 1986)) at an angle of 5° with the vertical plane, in order to avoid the sagittal sinus. Control animals (n=7) were injected with 0.01 M PBS. One week after surgery, animals were sac-rifi ced under deep anesthesia with 1 ml of sodium-pentobarbital by transcardial perfusion with saline, followed by a fi xative solution consisting of 2.5% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buff er (pH 7.4). Prior to perfusion, after the thorax had been opened, a blood sample was taken from the heart. Blood samples (approximately 0.5 ml) were collected in prechilled Eppendorf tubes containing EDTA as anticoagulant. After centrifugation at 2600 rpm for 15 min, the plasma was collected, and stored at -80°C for later radioimmunoassay analysis of corticosterone (ICN Biomedicals, Costa Mesa, USA). Brains were placed in 0.01 M PBS overnight and subsequently cryoptrotected in 30% sucrose at 4°C. Th ree series of coronal sections (30 μm-thick) spanning the MS (Bregma 1.20 to –0.26) and fi fteen series through the entire extent of the hippocampus (Bregma –2.12 to –6.30) were cut on a cryostat and collected in 0.01 M PBS containing

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0.1% sodium azide. All procedures concerning animal care and treatment were in accor-dance with the regulations of the ethical committee for the use of experimental animals of the University of Groningen (DEC number 2729) and European Community Council Directives.

ImmunocytochemistryAll immunocytochemical procedures were performed on free-fl oating sections. Immu-nocytochemistry for glial fi brillary acidic protein (GFAP), glutamic acid decarboxylase (GAD) 65/67, Ki-67 and ChAT was performed following a similar protocol. In brief, sec-tions were pretreated with 0.3% H2O2 for 30 min. Nonspecifi c binding of immunoreagents was blocked with 3% normal goat serum (Zymed, San Francisco, CA, USA). Subsequently, sections were incubated overnight at 4°C with mouse monoclonal anti-GFAP (1:1000) or rabbit polyclonal anti-GAD65/67 (1:5000; both purchased from Sigma, St Louis, MO, USA), for 48 h with mouse monoclonal anti-Ki-67 (1:200; Novocastra, Newcastle upon Tyne, UK) or for 72 h with goat polyclonal anti-ChAT (1:200; Chemicon, Harrow, UK). After a second blocking step, the biotinylated secondary antibodies (goat-anti-mouse, goat-anti-rabbit and rabbit-anti-goat IgGs, all 1:400; Jackson Immunolabs, West Grove, PA, USA) were added. Th is was followed by incubation with avidin-biotin-complex (1:400; ABC Elite kit, Vector Laboratories, Burlingame, CA, USA). Cells were visualized using diaminobenzidine (DAB) as chromogen (20 mg/100 ml). BrdU immunocytochemistry started with DNA denaturation procedures (van der Borght et al., 2005a). In brief, sections were incubated for 2 h at 65°C in 2 x saline sodium citrate (2xSSC) containing 50% formamide. After rinses with 2xSSC, 2 M HCl (37°C for 30 min) and 0.1 M borate buff er (pH 8.5), sections were exposed to the primary antibody (rat monoclonal anti-BrdU, 1:800, Oxford Biotechnology, Oxfordshire, UK) overnight at 4°C. As a secondary antibody biotinylated donkey-anti-rat IgGs (1:400; Jackson Immuno-labs) were used. Th e staining was developed using DAB and H2O2. For triple labeling for BrdU, GFAP and the neuronal marker NeuN, a similar DNA denaturing procedure as described above was used. Subsequently, sections were in-cubated for 72 h in the primary antibody solution containing rat monoclonal anti-BrdU (1:200, Oxford Biotechnologies), rabbit polyclonal anti-cow GFAP (1:400; DAKO, Glos-trup, Denmark) and mouse monoclonal anti-NeuN (1:400; Chemicon, Temecula, CA, USA). Th e following secondary antibodies were used: biotinylated donkey-anti-rat, Cy5-conjugated donkey-anti-rabbit and rhodamine red-conjugated donkey-anti-mouse F(ab’) fragments (1:200; all from Jackson Immunolabs). BrdU staining was made visible by incu-bation with Fluorescein (DTAF)-conjugated streptavidin (1:200; Jackson Immunolabs). Data analysis Th e material was examined in bright-fi eld illumination and confocal microscopy (Zeiss LSM510 confocal laser and scanning microscope) was used for multiple immuno-fl uorescence. NMDA infusion into the medial septum results in widespread neuronal dam-age, often exceeding the target nucleus (Harkany et al., 2000). Following injury, astro-cytes start to proliferate and become activated, a process which is called reactive gliosis.

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Th erefore, the presence of dense GFAP-immunoreactivity can be considered as a suitable indicator of neuronal damage (Ridet et al., 1997). In the intact animal, hardly any GFAP-positive astrocytes in the MS are present. After infusion of NMDA, a very dense cluster of GFAP-positive cells is visible at the injection site. Th is area we called the core of the lesion. Th e less-dense GFAP-positive rim surrounding the core was considered to be the penum-bra zone (Horvath et al., 2002). Guided by the GFAP staining, animals were selected in which the area with the highest density of GFAP-positive cells was located in the MS and only those animals were included in further analyses (30 nmol NMDA: n=6, 60 nmol of NMDA; n=5). Quantitative analyses were based on cell counts, which were performed blindly as to the treatment of the animals. For the quantifi cation of the number of ChAT and GAD65/67-positive cells in the MS, 4-6 representative sections of each animal, ranging from Bregma 1.20 to –0.26 and equally spaced and at matched anteroposterior levels, were used. All immunopositive cells in the MS were counted with a 20x objective. Cells were counted throughout the entire z-axis of every section. To avoid the inclusion of fragmented cell profi les, only cells with clear cytoplasmic staining and discernible, unstained nuclei were counted. Only immunopositive cells in the MS, and not in the vertical limb of the diagonal band of Broca, were included. Whenever the border between the MS and the vertical limb of the diagonal band was unclear, only those cells were counted that were located dorsally from the anterior commissure. In addition, the surface area of the MS was measured using a computer-based image analysis system (Quantimet W500, Leica, Rijswijk, Th e Netherlands). By calculating the number of counted cells/mm2, we obtained a standardized measure for the average cell density in the MS of each animal. For the quantifi cation of the number of Ki-67- and BrdU-positive cells in the GCL and the SGZ of the DG every 15th section containing the hippocampus was taken, revealing a total of 12 sections per animal throughout its anteroposterior extent. Quantifi -cations were performed using a 40x objective. Only Ki-67 and BrdU immunostained cell nuclei, throughout the entire thickness (30 μm) of the section, on the border of the hilus and the GCL were counted, including cells that were located one cell diameter deviating from this border. BrdU-positive cells laying in the GCL were counted as well, since newly formed cells that are 12 to 14 days of age may have migrated into the GCL. Th e total num-ber of counted cells was multiplied by fi fteen to obtain an estimation of the total number of positive cells per DG (Gould et al., 1999a; Malberg et al., 2000). A multi-track analysis of the BrdU/GFAP/NeuN triple staining was performed us-ing a Zeiss LSM510 confocal laser and scanning microscope. From the three experimental groups (n=3 per group), fi fty BrdU-positive cells were randomly chosen and scanned in its entire z-axis, using 1 mm intervals, in order to exclude false double labeling due to an overlay of signals from diff erent cells. With the help of specialized software (Zeiss LSM Image Browser Version 3.2.0.70), each scanned BrdU-positive cell was qualifi ed to be im-munoreactive for GFAP or NeuN.

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StatisticsStatistical analyses were performed to test potential diff erences in group average between the three experimental groups, for the following parameters: 1) ChAT-positive cell numbers and GAD65/67-positive cell numbers in the medial septum, 2) BrdU and Ki-67 positive cell counts in the dentate gyrus, 3) the distribution of the diff erent phenotypes within the BrdU-positive cell population and 4) the plasma corticosterone concentrations. All these parameters were analyzed using one-way analysis of variance (ANOVA). Whenever this revealed signifi cant diff erences, pairwise comparison was performed using the post-hoc Tukey-HSD test. All data are expressed as means ± standard error of the mean (S.E.M.).

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MS

30 nmol

60 nmol

Figure 1: Reconstruction of the lesions. Th e grey area illustrates the size and location of a rep-resentative lesion. Th e penumbra zones of all lesions were located within the area delineated by the black line. Th e core of the lesion was found between Bregma 0.48 and 0.70, whereas the penumbra ranged from Bregma 1.20 to -0.26. Th e anteroposterior extent did not diff er between the two concentrations of NMDA.

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Results

LesionTh e size and location of the lesions were determined on the basis of the GFAP immunos-taining. A lesion was considered to be correctly placed when the core and/or the penumbra of the lesion overlapped with the MS. Based on this criterion, three animals (one of the 30 nmol group and two of the 60 nmol group) were excluded from further analyses, which resulted in a fi nal group size of 7 sham animals, 6 rats with a lesion of 30 nmol of NMDA and 5 rats with a 60 nmol lesion. A representative lesion, as well as the delimitation of the area included in the other lesions are presented in Fig. 1. In order to determine the extent of the damage to the cholinergic system caused by the lesion, an immunocytochemical staining for the acetylcholine-synthesizing enzyme ChAT was performed (Fig. 2A-C). Th e lesion with 60 nmol of NMDA resulted in a 32% and signifi cant reduction of the number of ChAT-positive neurons in the MS (F(2,17)=7.48, P<0.05; post hoc P<0.05). Th irty nmol of NMDA did not aff ect the cholinergic cell number in the MS. Lesion-induced changes in the GABAergic system were visualized by immu-noreactivity for the 65-kDa and the 67-kDa isoforms of GAD, the enzyme that converts glutamic acid into GABA (Fig. 2D-F). A signifi cant diff erence was found between the three groups (F(2,17)=11.93, P<0.001). Post-hoc testing indicated a 62% and signifi cant reduction in GAD65/67- immunopositive cells in rats that were lesioned with 30 nmol of NMDA (P<0.01). Th e 60 nmol concentration caused a 66% and signifi cant reduction of GAD65/67-positive neurons compared to controls (P<0.01). Th ese results demonstrate a potent negative eff ect of NMDA-infusion on the number of GABAergic cells in the MS and a moderate reduction in cholinergic cell number in the MS only after infusion with 60 nmol of NMDA.

Neurogenesis In order to investigate whether a reduced input from the MS to the hippocampus had an eff ect on the survival of newly formed cells, all animals were injected with BrdU one week before surgery and the number of BrdU-positive cells still present in the GCL one week after the lesion was quantifi ed (Fig. 3A,B, page 140). BrdU immunocytochemistry dem-onstrated that NMDA infusion into the MS considerably and signifi cantly reduced the survival of newly formed cells in the GCL (F(2,17)=12. 82, P=0.001). Th e lesion with 30 nmol of NMDA resulted in a 39% and signifi cant decline of the number of BrdU-positive cells (P<0.01), whereas infusion of 60 nmol of NMDA caused a decrease of 40% (P<0.001) suggesting an important role for the MS in the regulation of newly formed cell survival. To determine whether the MS lesion specifi cally aff ected the survival of newly formed astrocytes or neurons, a triple labeling for BrdU, the glial marker GFAP and the neuronal marker NeuN was performed (Fig. 3C,D, page 140). Analysis revealed that the phenotypes of the BrdU-positive cells did not diff er between the 30 nmol and the 60 nmol

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groups, and the data from the two lesioned groups were therefore pooled. Z-series of ran-domly chosen BrdU-positive cells throughout the entire GCL demonstrated that in sham animals as well as in lesioned rats, the percentage of BrdU-positive cells colocalizing with either NeuN or GFAP was similar. Th is result indicates that the MS lesion reduced the number of all newly formed cells, regardless of their phenotype. Mitotic cells express the nuclear marker Ki-67 throughout all phases of the cell cycle (Kee et al., 2002; Scholzen and Gerdes, 2000). Th erefore, staining of Ki-67 positive cells provides information about the number of cells that were in any phase of the cell cycle (except for G0) at the time of termination. In this part of the study, immunocytochemi-cal staining for Ki-67 was used to determine whether a decrease in the septohippocampal input had an eff ect on the number of proliferating cells in the SGZ. No diff erences were detected between the lesioned groups and the sham-operated animals, though the two lesioned groups diff ered from each other (F(2,17)= 4.34, P<0.05; post hoc: P<0.05). Th e

Th e medial septum regulates neurogenesis

sham 30 nmol 60 nmol0

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Figure 2: Impact of NMDA infusion into the MS on the cholinergic and GABAergic cell num-ber A) Th irty nmol of NMDA did not aff ect the number of ChAT-positive cells in the MS, whereas 60 nmol of NMDA caused a signifi cant reduction (* P<0.05, post-hoc analysis fol-lowing ANOVA). Representative photomicrographs of ChAT-immunostaining are shown in panels B (sham) and C (60 nmol of NMDA). Scale bar=80 mm. D) Both concentrations of NMDA caused a signifi cant decline in the number of GAD65/67-positive cells (** P<0.01, post-hoc analysis following ANOVA). Photomicrographs of GAD65/67-positive cells in the MS are shown in E (sham) and F (30 nmol of NMDA). Scale bar=100 μm.

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lesion with 30 nmol of NMDA resulted in 57% more Ki-67 positive cells in the SGZ com-pared to the lesion with 60 nmol of NMDA (Fig. 3E,F, page 140). Since MS lesions occasionally increase plasma corticosterone levels (Alema et al., 1995), which can negatively aff ect hippocampal neurogenesis (Gould et al., 1997; Heine et al., 2004b; Pham et al., 2003), blood samples were taken prior to perfusion to determine plasma corticosterone concentrations. No changes were detected (sham: 19.6 ± 4.6 μg/dl, 30 nmol of NMDA: 23.0 ± 6.1 μg/dl, 60 nmol of NMDA: 20.9 ± 4.1 μg/dl), indicating that the observed changes in neurogenesis were not likely to be caused by changes in corti-costeroid concentrations.

Discussion

Th e present study investigated whether a partial lesion of the MS, a region of the basal forebrain that densely innervates the hippocampal formation, has an eff ect on neurogenesis in the hippocampus of the adult rat. In order to investigate this, the MS was damaged by infusion of two diff erent concentrations of NMDA. Both 30 nmol and 60 nmol of NMDA signifi cantly reduced the survival of cells that had been generated in the GCL fi ve to seven days prior to the lesion. Th is reduced survival was observed both for new neurons and newly formed astrocytes. Excitotoxic lesion of the MS did not aff ect cell proliferation in the SGZ, one week after the lesion. Our data show that the lesion of the MS with 30 nmol and 60 nmol of NMDA caused a similar reduction in the number of GABAergic cells. Th e number of cholinergic cells was reduced only after infusion of 60 nmol of NMDA into the MS. Th e decrease in hippocampal neurogenesis was comparable between the two lesioned groups, which sug-gests that the GABAergic system, and not the cholinergic input to the hippocampus, is in-volved in the regulation of survival of newly generated hippocampal granule neurons. Th e GABAergic system may aff ect survival of newly formed cells either directly or indirectly, via synaptic or nonsynaptic signaling. Th e eff ects of the lesion on survival of newly generated cells may have been caused by nonsynaptic eff ects of GABA. Nonsynaptic GABAergic signaling takes place during various steps of nervous system development (Owens and Kriegstein, 2002), such as synap-togenesis (Belhage et al., 1998), neuronal diff erentiation (Nguyen et al., 2003) and neurite extension (Wolff et al., 1978). It may therefore be possible that GABA also acts as a neuro-trophic factor for immature or nearly mature hippocampal neurons that are formed during adulthood. Because infusion of NMDA into the MS strongly decreased the GABA-ergic input to the hippocampus, it could be hypothesized that this caused a reduction of neuro-trophic support for the survival of newly generated cells in the DG. Alternatively, the eff ects of the lesion on newly formed cell survival may be the result of changes in synaptic signaling. It has been shown that newly generated dentate granule neurons receive synaptic GABAergic input. Studies with acute hippocampal slices

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demonstrate that newly generated granule neurons exhibit postsynaptic responses that are typical for fast neurotransmitters, such as GABA (Van Praag et al., 2002). In intact con-ditions, GABAergic projection neurons in the MS innervate GABAergic interneurons in the hippocampus. Th e GABAergic innervation of hippocampal interneurons is believed to lead to a disinhibition of hippocampal principal cells (Freund and Antal, 1988; Krnjevic et al., 1988; Toth et al., 1997). Following this line of reasoning, a loss of septal GABAergic projection neurons may therefore result in increased inhibition of the hippocampus. Th is is also suggested by other studies, based on the fi nding that a selective lesion of GABAergic septal neurons prevented the induction of theta wave activity in the hippocampus (Wu et al., 2002; Yoder and Pang, 2005). Th e assumption of disturbed disinhibition supports the hypothesis that certain forms of increased hippocampal activity, caused, for instance, by enriched housing conditions or trace eyeblink conditioning, promote the survival of newly generated neurons (Gonzalez-Lima et al., 1994; Gould et al., 1999a; Kempermann et al., 2002; Puurunen et al., 2001). Th e almost exclusive decrease of GABAergic cells cannot rule out the possibility that other factors besides GABA may have accounted for the observed eff ects on the sur-vival of newly formed cells. Neurons of the basal forebrain also express certain peptides that are anterogradely transported to the hippocampus and have been reported to stimulate hippocampal neurogenesis (Lai et al., 2003; Machold et al., 2003). In addition, the cho-linergic and the GABAergic system are closely interconnected (Brauer et al., 1998; Van der Zee and Luiten, 1994; Wu et al., 2000). Th e net output from the MS to the hippocampus depends on the balance between cholinergic and GABAergic activity in the MS, indicat-ing that the loss of GABAergic cells in the MS after the lesion may also have aff ected the properties of the cholinergic septal neurons. Th e proliferation of hippocampal progenitors was not infl uenced by the lesion. In vitro studies indicate a role for acetylcholine in cell progenitor proliferation. Choliner-gic stimulation enhances proliferation of embryonic cortical neural precursors (Li et al., 2001; Ma et al., 2000; Zhao et al., 2003) and of oligodendrocyte progenitors (Larocca and Almazan, 1997). In addition, Lai et al. (2003) demonstrated that disruption of the septo-hippocampal connection by a lesion of the fi mbria-fornix results in a reduced proliferation in the adult mouse DG. Recent studies show that extensive lesion of cholinergic cells in the basal forebrain, by infusion of 192-IgG saporin, strongly reduce cell proliferation in the subventricular zone and the olfactory bulb (Calza et al., 2003) and also leads to a moder-ate, but signifi cant, reduction in hippocampal cell proliferation (Mohapel et al., 2005). In the present study, the eff ect of the MS lesion on hippocampal cell proliferation was less obvious. Although progenitor proliferation in the two lesioned groups diff ered from each other, the results did not deviate from sham animals. Th is may be due to the fact that even the highest concentration of NMDA only moderately reduced cholinergic cell number in the MS, which may not be suffi cient to aff ect cell proliferation. Th e observed decline in hippocampal neurogenesis after the MS lesion may refl ect the changes that occur during aging. Th e MS of aged rats contains less cholinergic and GA-BAergic cells than in younger animals (Fischer et al., 1989; Krzywkowski et al., 1995) and

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the reduced cell number correlates with impairments in hippocampus-dependent learning (Fischer et al., 1989; Fischer et al., 1992). It has also repeatedly been reported that hippo-campal neurogenesis is decreased during aging (Heine et al., 2004a; Kempermann et al., 2002; Kuhn et al., 1996). Twenty-one-month-old rats, for instance, show a 90% reduc-tion in cell proliferation, when compared with 6-month-old animals (Kuhn et al., 1996). However, the survival of newly formed hippocampal neurons is not aff ected during aging (Bondolfi et al., 2004; Heine et al., 2004a). Since we show here that the GABAergic com-ponent of the septohippocampal pathway is unlikely to induce changes in hippocampal cell proliferation, but specifi cally seems to aff ects cell survival, the reduction in cell prolifera-tion occurring during aging is probably not caused by the reduction in GABAergic cell number in the MS. In summary, here we show that NMDA infusion into the MS resulted in a decline in neurogenesis in the DG. Th is is caused by a reduction in the number of surviving newly formed granule neurons and not in the number of proliferating progenitors. Our data sug-gest that the GABAergic system may be one of the factors that determine the fate of newly generated granule neurons. Th ese results provide new insight into the possible mechanisms underlying the regulation of survival of hippocampal granule neurons generated during adulthood.

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