PS1 and TrkB.T1 genotypes on midbrain dopamine neurons and stimulated dopamine release in vivo

brain research 1622 (2015) 452–465

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Research Report

Opposing effects of APP/PS1 and TrkB.T1 genotypes on midbrain dopamine neurons and stimulated dopamine release in vivo E. Ka¨rkka¨inena,n,1, L. Yavichb,c,1, P.O. Miettinena, H. Tanilaa,1 a

A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland School of Pharmacy, University of Eastern Finland, Kuopio, Finland c Invilog Research Ltd, Kuopio, Finland b

ar t ic l e in f o

abs tra ct

Article history:

Brain derived neurotrophic factor (BDNF) signaling disturbances in Alzheimer's disease

Accepted 2 July 2015

(AD) have been demonstrated. BDNF levels fall in AD, but the ratio between truncated and

Available online 11 July 2015

full-length BDNF receptors TrkB.T1 and TrkB.TK, respectively, increases in brains of AD

Keywords:

patients and APPswe/PS1dE9 (APP/PS1) AD model mice. Dopaminergic (DAergic) system

Alzheimer's disease

disturbances in AD and detrimental effects of BDNF signaling deficits on DAergic system

Dopamine

functions have also been indicated. Against this, we investigated changes in nigrostriatal

BDNF

dopamine (DA) system in mice carrying APP/PS1 and/or TrkB.T1 transgenes, the latter line

Amyloid protein precursor

modeling the TrkB.T1/TK ratio change in AD. Employing in vivo voltammetry, we found

Presenilin-1

normal short-term DA release in caudate-putamen of mice carrying APP/PS1 or TrkB.T1

Transgenic mice

transgenes but impaired capacity to recruit more DA upon prolonged stimulation. However, mice carrying both transgenes did not differ from wild-type controls. Immunohistochemistry revealed normal density of tyrosine hydroxylase positive axon terminals in caudate-putamen in all genotypes and intact presynaptic machinery for DA release and reuptake, as shown by unchanged levels of SNAP-25, α-synuclein and DA transporter. However, we observed increased DAergic neurons in substantia nigra of TrkB.T1 mice resulting in decreased tyrosine hydroxylase per neuron in TrkB.T1 mice. The finding of unchanged nigral DAergic neurons in APP/PS1 mice largely confirms earlier reports, but the unexpected increase in midbrain DA neurons in TrkB.T1 mice is a novel finding. We suggest that both APP/PS1 and TrkB.T1 genotypes disrupt DAergic signaling, but via separate mechanisms. & 2015 Elsevier B.V. All rights reserved.

n

Corresponding author. E-mail address: elisa.karkkainen@fimnet.fi (E. Kärkkäinen). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.brainres.2015.07.006 0006-8993/& 2015 Elsevier B.V. All rights reserved.

brain research 1622 (2015) 452–465

1.

Introduction

Brain derived neurotrophic factor (BDNF) signaling through its main receptor TrkB is suggested to play a significant role in the pathophysiology of AD (Castrén and Tanila, 2006). The levels of BDNF and its main receptor TrkB are decreased in the hippocampus of AD brains (Connor et al., 1997; Fahnestock et al., 2002; Hock et al., 2000; Holsinger et al., 2000; Michalski and Fahnestock, 2003). In contrast, the truncated, dominantnegative isoform of TrkB (TrkB.T1) that lacks the cytoplasmic tyrosine kinase catalytic domain (Barbacid, 1995; Middlemas et al., 1991) and seems to act as an inhibitory modulator of BDNF signaling (Eide et al., 1996), has been shown to be increased (Connor et al., 1996; Ferrer et al., 1999). Importantly, BDNF levels are reduced already at the preclinical stages of the disease (Peng et al., 2005). Furthermore, the association of impaired BDNF-signaling to memory deterioration does not seem to limit to AD, since a study in aged nondemented patients indicated that low levels of serum BDNF are connected to both smaller hippocampal volumes and associated memory impairments (Erickson et al., 2010). We have recently demonstrated that similarly to findings in AD brains, the ratio between the truncated TrkB.T1 and full-length TrkB.TK receptors is increased in the common APPswe/PS1dE9 (APP/PS1) mouse model of AD (Kemppainen et al., 2012). Functionally this changed ratio affects the spatial memory impairment observed in APP/PS1 mice around the age of 12 months, and we have found that TrkB.T1 overexpression (Kemppainen et al., 2012) and BDNF deficiency (Rantamäki et al., 2013) in APP/PS1 mice leads to aggravated memory impairment, while overexpression of the TrkB.TK receptor had an opposite effect (Kemppainen et al., 2012). In AD brains the DAergic system displays varying extent of typical AD neuropathological changes such as neurofibrillary tangles and Aβ plaques (Burns et al., 2005; Parvizi et al., 2001; Reyes et al., 1996), and neuron loss in the substantia nigra (SN) (Burns et al., 2005; Horvath et al., 2014; Lyness et al., 2003; Reyes et al., 1996) and the putamen (Horvath et al., 2014). Over a third of AD patients present extrapyramidal symptoms such as bradykinesia and rigidity (Lopez et al., 1997; Soininen et al., 1992), and levels of brain DA (Storga et al., 1996) and DA receptors (Kemppainen et al., 2000; Kumar and Patel, 2007; Pizzolato et al., 1996) are decreased in AD. In fact, PET studies have found that decreased DA receptor availability in the hippocampus correlates with memory performance in AD patients (Kemppainen et al., 2003), which is in accordance with the finding that administration of DA agonists to AD patients has positive effects on cortical neurotransmission and synaptic plasticity mechanisms (Koch et al., 2014; Martorana et al., 2013). Experimental data examining possible DAergic system changes in APP transgenic mice are still scarce. Two studies have reported unchanged levels of DA (Manaye et al., 2007; Szapacs et al., 2004) and its metabolites DOPAC and HVA (Szapacs et al., 2004) as assessed by HPLC, while another study found an age-dependent decrease in striatal DOPAC but not DA and HVA levels in APP/PS1 mice (Perez et al., 2005). No decrease in SN DAergic neurons has been found in APP/PS1 mice (Liu et al., 2008; O’Neil et al., 2007; Perez et al., 2005).

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Dopaminergic neuron loss and atrophy in the ventral tegmental area (VTA) of aged APP/PS1 mice was found in one study (Liu et al., 2008), while another study reported unchanged SN-VTA neuron numbers (O’Neil et al., 2007). Interactions between BDNF signaling and the DAergic system have been widely studied. BDNF has been shown to support the survival and maturation of DAergic neurons (Baker et al., 2005; Erickson et al., 2001; Hyman et al., 1991). Although some studies on BDNF-deficient mice have reported no change in DAergic neuron counts in the SN (Baker et al., 2005; Luellen et al., 2007) or fiber density in the striatum, VTA or nucleus accumbens (Luellen et al., 2007), others have found that BDNF or TrkB deficiency results in loss of DAergic neurons, fibers and receptors (Baker et al., 2005; Baquet et al., 2005; Do et al., 2007; Von Bohlen und Halbach et al., 2005). Further, one study reported no change in DA levels in the hippocampus, frontal cortex and hypothalamus of BDNF deficient mice (Chourbaji et al., 2004), whereas in vitro voltammetry studies have found that BDNF deficiency leads to reduced DA release and uptake rates in the mouse striatum (Apawu et al., 2013; Bosse et al., 2012). These findings, together with reports of BDNF-induced increases in DAergic neurons, fibers, receptors and stimulated DA release (Do et al., 2007; Goggi et al., 2002; Spenger et al., 1995), imply an essential role of BDNF on the functions of the DAergic system. Additionally, our previous study found an interesting interaction between APP/PS1 and TrkB.T1 genotypes on spontaneous exploratory activity, such that APP/PS1 genotype alone led to hyperactivity and TrkB.T1 genotype alone was characterized by a depression-like lack of interest to explore a new environment or new objects, whereas mice expressing both genotypes did not differ from wild-type mice (Kemppainen et al., 2012). Hyperactivity/hyperexploration in all transgenic mice overexpressing the APPswe mutation is a well-known feature (Dumont et al., 2004; Rustay et al., 2010; Van Dam et al., 2003), which appears to be present independent of Aβ plaque load (Born et al., 2014) but has no known neural cause so far. Since manipulation of DA biosynthesis and receptors has dramatic effects on spontaneous activity (Del Arco and Mora, 2008; Kobayashi, 2001), the DAergic system is an obvious candidate to look for neurobiological changes underlying the hyperactivity vs. hypoactivity phenotypes of APP/PS1 and TrkB.T1 mice, respectively. The above mentioned findings led us to take a special interest in the separate and combined effects of APP/PS1 and TrkB.T1 genotypes on DA signaling in the mouse brain. The TrkB.T1 mouse models the BDNF signaling changes occurring in AD, and to our knowledge, the status of nigrostriatal DA system in TrkB.T1 mice has not been reported in the literature so far. We employed both functional and morphological approaches to study the neuropathological changes in the nigro-striatal DA system in these mice. We used in vivo voltammetry to assess the dynamics of DA release upon prolonged stimulation, whereas the morphological studies focused on the number of midbrain DAergic neurons and their striatal terminals and also the density of key proteins for DA release and reuptake in DAergic nerve terminals by means of immunohistochemistry. Our prediction was to find clear disruption in DAergic signaling of both APP/PS1 and TrkB.T1 mice, with even more prominent impairments in mice having both transgenes.

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Fig. 1 – Histological sections from an AþT1 mouse (a) FLAG-staining at the level of the dopaminergic midbrain, (b) FLAGstaining at the level of the head of caudate-putamen, (c) Enlarged image from the rectangle in (a) showing FLAG-positive neurons and fibers in substantia nigra, (d) TH-staining of the adjacent section to that in (a), showing TH-positive neurons and fibers in substantia nigra.

2.

Results

2.1. TrkB.T1 is expressed in cortical areas and midbrain dopaminergic nuclei, but not in the striatum The distribution of TrkB.T1 protein expression has been described earlier (Saarelainen et al., 2000a). To verify that the expression pattern still holds after several generations of TrkB.T1 mice, we used the FLAG staining to tag the TrkB.T1 receptors at two levels: the head of caudate-putamen (CPu) and the dopaminergic midbrain. TrkB.T1 was expressed only in mice with the TrkB.T1 transgene, and could be found in cortical areas and the midbrain dopaminergic nuclei, but not in the striatum (Fig. 1).

2.2. Impact of APP/PS1 and TrkB.T1 genotypes on dopamine release We have reported earlier that prolonged stimulation of medial forebrain bundle results in recruitment of dopamine in CPu to the readily releasable pool by either mobilization of storage vesicles

or enhanced reuptake (Yavich et al., 2004). Here we applied this model to determine the combined effect of APP/PS1 and TrkB.T1 transgenes on dopamine release and reuptake dynamics. The results are summarized in Fig. 4. We found that both wild-type (AwTw) (F1,5 ¼ 11.7, p ¼0.019, ANOVA-RM; Fig. 4A) and doubly transgenic (AþT1) (F1,4 ¼ 6.2, p ¼0.067, ANOVA-RM; Fig. 4D) mice showed a relative increase in dopamine overflow after prolonged stimulation, as was expected for the wild-type mice. However, mice with either APP/PS1 or TrkB. T1 transgene showed a deficit in dopamine mobilization within presynaptic terminals, i.e. the relative increase in dopamine overflow was not present (all p40.27, ANOVARM; Fig. 4B and C). Importantly, we found a significant interaction between the stimulation train and both A and T genotypes (F1,17 ¼ 9.8, p¼ 0.006, ANOVA-RM), whereas no interaction between the stimulation train and A or T genotype was found (all p40.50, ANOVA-RM). We also measured the frequency response for peak DA overflow, and found that AwTw and AþT1 mice had slightly lower peak DA overflow in response to high-frequency stimulation than AwT1 and AþTw mice, but this difference

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Fig. 2 – Striatal sections from AþT1 mice, showing regions of interest for the optic density measurements for caudateputamen (CPu) (a) Tyrosine hydroxylase (TH) staining (b) SNAP-25 staining (c) DAT staining (d) α-syn-staining.

was not statistically significant (all p for interactions between stimulation frequency and A and/or T genotypes40.10, ANOVA-RM) (Fig. 4E).

2.3. TrkB.T1 genotype associates with increased dopaminergic neuron density in the substantia nigra To find a structural correlate for the genotype differences in the DA release dynamics, we first analyzed optic density of THstained DA terminals in the CPu and counted TH-positive cells in SN in all subgroups of APP/PS1  TrkB.T1 mice. There was no main genotype effect on TH-positive DA terminals in CPu (p40.35, ANOVA; Fig. 5A) and no interaction between the genotypes (p¼ 0.92). However, we found a significantly increased

number of THþ cells in SN in T1 mice (F1,51 ¼10.4, p¼0.002; Fig. 5D) but no main effect of the APP/PS1 genotype (p¼0.85) and no interaction between the genotypes (p¼ 0.81). Further, the ratio between CPu DA terminals to SN neuron count was decreased in T1 mice (F1,51 ¼ 4.7, p¼0.035; Fig. 5F) while there was no main effect of the APP/PS1 genotype (p¼ 0.37).

2.4. APP/PS1 and TrkB.T1 genotypes have no effect on SNAP-25, α-synuclein or dopamine transporter densities To assess distinct and combined effects of APP/PS1 and TrkB.T1 genotypes on the function of the synaptic vesicular exocytotic machinery on the presynaptic plasmalemmae of striatal neurons, we analyzed optic density for SNAP-25 and α-synuclein that

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associates with the SNARE-complex. To confirm that SNAP-25 also measures DA terminals, we plotted optic density of SNAP-25 against TH staining. The correlation proved to be strong and positive (Pearson rho¼0.73, po0.0001). However, there was no genotype main effect on SNAP-25 density in CPu (p40.42, ANOVA; Fig. 6A) and no interaction between genotypes (p¼ 0.86). Neither did we find a genotype main effect on α-syn optic density (p40.075; Fig. 6C) or an interaction between the genotypes (p¼0.63). Finally, we also assessed possible

Fig. 3 – TH-stained sections at the level of substantia nigra (SN) from an AwTw mouse, showing regions of interest for the cell number quantification in SN and THþ cells inside the 250 lm  250 lm square.

contribution of dopamine transporter (DAT) mediated reuptake to differences in dopamine release dynamics in APP/PS1  TrkB. T1 mice to determine the effect of these genotypes on DA reuptake. There was no genotype main effect on DAT density (p40.32; Fig. 6B) or an interaction between the genotypes (p¼ 0.77).

3.

Discussion

This study set out to investigate the changes in the nigrostriatal DA system in mice carrying APP/PS1 and/or TrkB.T1 transgenes. We also wanted to elucidate the neurobiological underpinnings of the hyperactivity vs. hypoactivity phenotypes that we have earlier revealed in APP/PS1 and TrkB.T1 mice, respectively (Kemppainen et al., 2012). The focus on nigro-striatal dopaminergic (DAergic) system was natural for many reasons. First, the DAergic system in AD is susceptible to neuronal loss (Burns et al., 2005; Horvath et al., 2014; Lyness et al., 2003; Reyes et al., 1996) and displays typical pathological hallmarks of AD, neurofibrillary tangles and Aβ plaques (Burns et al., 2005; Parvizi et al., 2001; Reyes et al., 1996). Second, BDNF has been shown to support the survival and maturation of DAergic neurons (Baker et al., 2005; Erickson et al., 2001; Hyman et al., 1991) while BDNF or TrkB deficiency seems to result in loss of DAergic neurons, fibers and receptors (Baker et al., 2005; Baquet et al., 2005; Do et al., 2007; Von Bohlen und Halbach et al., 2005), and reduced striatal DA release and uptake (Apawu et al., 2013; Bosse et al., 2012; Dluzen et al., 2002). Third, modifications of DA biosynthesis or receptors significantly influence spontaneous activity in mice and rats (Del Arco and Mora, 2008; Kobayashi, 2001). To our knowledge, the status of the nigro-striatal DA system in TrkB.T1 mice has not been reported in the literature so far.

Fig. 4 – Caudate-putamen stimulated dopamine overflow (a) in AwTw, (b) in AwT1, (c) in AþTw, (d) in AþT1 mice (e) as function of increasing stimulation frequency. For images (a)–(d) data are presented for the 1st and 3rd stimulus train as percentage of the evoked release after the 1st burst in the train; the six bursts shown on the x-axis comprise one stimulus train. For image (e) data are presented as molar concentration (lM/l). * ¼significant main effect of stimulus train in AwTw mice (p¼0.019, ANOVA-RM), # ¼near-significant main effect of stimulus train in AþT1 mice (p ¼0.067, ANOVA-RM).

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Fig. 5 – Dopaminergic neuron quantification in TH-stained sections (a) CPu optic density, mean7SEM, (b) SN cell count, mean7SEM, n ¼ significant main effect of T1 genotype (p¼ 0.002, ANOVA), (c) CPu OD divided by SN cell count, mean7SEM, n ¼ significant main effect of T1 genotype (p¼ 0.035, ANOVA). TH¼ tyrosine hydroxylase, CPu¼caudate-putamen, OD¼ optic density, SN¼ substantia nigra.

Fig. 6 – Quantification of caudate-putamen optic density in (a) SNAP-25 stained sections, mean7SEM, (b) DAT-stained sections, mean7SEM, (c) α-syn-stained sections, mean7SEM. SNAP-25 ¼ synaptosome-associated protein of 25 kDa, CPu¼ caudate-putamen, OD¼ optic density, DAT¼ dopamine transporter, α-syn¼alpha synuclein.

3.1. The effects of APP/PS1 and TrkB.T1 genotypes on dynamics of dopamine release In the in vivo voltammetry analysis we found that short-term release of DA in CPu did not differ between the genotypes but a prolonged stimulation revealed an interesting difference. Both wild-type (AwTw) and doubly transgenic (AþT1) mice showed a relative increase in DA overflow after prolonged stimulation, while mice with either the APP/PS1 or the TrkB. T1 transgene did not show this increase. Importantly, there was a significant interaction between the stimulation train and both A and T genotypes. This finding indicates that both APP/PS1 and TrkB.T1 single transgenic mice have compromised capacity to maintain sufficient DA release under prolonged stimulation. This finding is compatible with observed hypoactivity in TrkB.T1 mice and the normalization of spontaneous activity in doubly transgenic APP/PS1 x TrkB. T1 mice. However, decreased recruitment of stored/reuptaken DA in APP/PS1 single transgenic mice cannot be accounted for the observed spontaneous hyperactivity. In human postmortem AD brains reduced levels of DA, its precursor L-DOPA and metabolite DOPAC (Storga et al., 1996) and reduced DA receptors (Kemppainen et al., 2000; Kumar

and Patel, 2007; Pizzolato et al., 1996) have been reported. In AD model mice, however, mixed results have been reported. TgCRND8 mice (with APPswe and APPind mutations) show elevated DA levels in the striatum and frontal cortex but reduced DA levels in the hippocampus (Ambrée et al., 2009). On the other hand, all reported studies in the same APP/PS1 mouse model as ours consistently have found unchanged levels of striatal DA (Manaye et al., 2007; Perez et al., 2005; Szapacs et al., 2004). However, a close association between DAergic pathology and amyloid deposition in this mouse model was reported by revealing dystrophic DAergic neurites adjacent to Aβ plaques (Perez et al., 2005). Recent studies in neural retina have also shown an in vivo association between the BDNF/TrkB receptor and the abnormal processing of APP leading to both functional and morphological changes (Gupta et al., 2014; Ning et al., 2008). The effects of impaired BDNF signaling on brain DA levels have been studied mainly in vitro. Earlier voltammetry studies in CPu slices of BDNFþ/  mice revealed reduced DA release and uptake rates when compared to wild-type controls (Apawu et al., 2013; Bosse et al., 2012). In addition, superfusion with BDNF led to partial recovery of DA release in BDNFþ/  mice, but this recovery was blocked by treatment

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with a TrkB inhibitor, indicating that it is TrkB activation that enables DA release (Apawu et al., 2013). In fact, although increased CPu DA levels have been reported in adult BDNFþ/  mice compared to wild-type littermates (Dluzen et al., 1999; Joyce et al., 2004), the stimulated DA release in BDNFþ/  mice was decreased (Dluzen et al., 2002). Furthermore, one group found unchanged striatal, cortical, hippocampal and hypothalamic DA and HVA levels in BDNFþ/  mice using HPLC (Chourbaji et al., 2004). To our knowledge, this is the first study on the combined effects of AD transgenes and impaired BDNF-signaling on dynamics of striatal DA release.

3.2. TrkB.T1 genotype affects dopaminergic neuron number but not striatal fiber density We found an increased number of THþ cells in SN in mice with the TrkB.T1 transgene but no significant effect of the APP/PS1 genotype. SN dopaminergic neuron loss in AD patients is well established, especially in those showing extrapyramidal symptoms (Burns et al., 2005; Horvath et al., 2014; Lyness et al., 2003; Reyes et al., 1996), far fewer studies have been conducted in AD model mice. In line with our results, striatal TH staining intensity was found unchanged in an earlier study (Perez et al., 2005). While one study has reported SN neuron losses in 3xTgAD mice (Sun et al., 2012), SN neuron numbers in APP/PS1 mice in the present study and three earlier studies (Liu et al., 2008; O’Neil et al., 2007; Perez et al., 2005) were found to be unchanged. BDNF has been shown to support the survival and maturation of DAergic neurons (Baker et al., 2005; Erickson et al., 2001; Hyman et al., 1991). Although some studies performed on BDNF-deficient mice have reported no change in DAergic neuron counts in the SN (Baker et al., 2005; Luellen et al., 2007) or fiber density in striatum of adult mice (Luellen et al., 2007), others have found losses of DAergic SN dendrites (Baker et al., 2005) or SN neurons (Baquet et al., 2005). Also TrkB deficiency has been shown to result in losses of SN neurons and striatal THþ fibers (Von Bohlen und Halbach et al., 2005). These findings, together with reports of BDNFinduced increases in THþ neurons and fibers (Spenger et al., 1995), receptors (Do et al., 2007) and stimulated DA release (Goggi et al., 2002), imply that BDNF deficiency causes brain DAergic network impairments. Why we, then, found an increased number of THþ SN neurons? The most parsimonious explanation is that this results from tyrosine kinase independent signaling through the TrkB.T1 receptor which has been reported to regulate both neurite growth (CarimTodd et al., 2009) and apoptosis (Dorsey et al., 2006). In any event, this is the very first observation of TrkB.T1 dependent increase in a particular neuronal subtype in the adult brain. How does the finding of altered numbers of midbrain DAergic neurons in our transgenic mice fit with the voltammetry findings? First, there is a notable difference between spontaneous DA release, which reflects the number of active neurons, and evoked release by electrical MFB stimulation. Independent of the neuronal number in the DAergic nucleus, an optimally placed electrode stimulates a similar-sized subset of DA fibers in the bundle. Second, we may envision that if TrkB.T1 mice have same amount of TH in CPu as APP/ PS1 mice despite a higher DA neuron number in SN, TH

downregulation at the terminals must have occurred in TrkB. T1 mice and/or corresponding TH upregulaton in APP/PS1 mice. When a fixed number of DA fibers are artificially stimulated, these compensatory changes likely lead to the observed failure of DA recruitment upon prolonged stimulation. Since the compensatory mechanisms are opposite in APP/PS1 and TrkB.T1 mice, it is conceivable that they are absent in the double transgenic mice, thus leading to normal phenotype in this particular assay.

3.3. Unaltered SNAP-25, DAT and α-synuclein levels in APP/PS1 and TrkB.T1 mice The immunohistochemical analysis of CPu SNAP-25 staining found no main genotype differences. Given the central role of SNAP-25 in the SNARE-complex function, this finding speaks for unaltered synaptic vesicular exocytotic machinery in striatal neurons in our mouse models, although we cannot exclude a selective deficit in some other key proteins in the SNARE complex. Further, the observed high positive correlation between CPu SNAP-25 and TH optic densities suggests that no major genotypes differences were present in SNAP-25 levels in the DAergic terminals, either. Earlier studies have shown decreased SNAP-25 protein (Greber et al., 1999; Minger et al., 2001; Shimohama et al., 1997) levels in postmortem AD patient brain samples. However, also unchanged (Sze et al., 2000) SNAP-25 protein levels or regionally specific changes (Clinton et al., 1994) in AD brain samples have been reported. Decreased SNAP-25 levels have been reported in TrkB  /  mice (Carmona et al., 2003; Martínez et al., 1998) whereas SNAP-25 protein levels have not been found to increase after exposure of mouse (Robinet and Pellerin, 2011) or rat (Matsumoto et al., 2006) neurons to BDNF. By and large, striatal SNAP-25 expression does not seem to explain the observed phenotype differences between APP/PS1 and TrkB.T1 mice. We found no main genotype effects on DAT optic density in CPu. Efficacy of DA uptake through DAT is one of the major determinants of increased DA recruitment during prolonged stimulation (Chadchankar et al., 2011). Thus, finding no genotype difference in striatal DAT levels rules out the possibility that decreased DA recruitment in single transgenic mice would have resulted from genotype effects on DAT expression. Consistent with our findings, postmortem studies of DAT levels in the AD brain have revealed unchanged protein and mRNA levels (Chen et al., 2011), while DA uptake has been found to be either unchanged (Piggott et al., 1999) or reduced (Allard et al., 1990; Kemppainen et al., 2001; Rinne et al., 1998) in AD. BDNFþ/  mice show unchanged DAT protein levels, like in our TrkB.T1 mice, but reduced dopamine transport via DAT when compared to wild-type littermates (Boger et al., 2011). We found no significant genotype effect on striatal optic density of α-syn, a negative regulator of DA release (Yavich et al., 2004). Earlier, both unchanged (Irizarry et al., 1996) and increased (Jellinger, 2003; Mikolaenko et al., 2005) α-syn protein levels have been reported in AD patients. Further, AD patients with extrapyramidal symptoms such as bradykinesia, rigidity, tremor and parkinsonian gait display more severe SN neuron loss and α-syn pathology (Burns et al.,

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2005). α-Synþ neurites have also been demonstrated in AD model mouse brains (Yang et al., 2000). However, interactions of changed BDNF-signaling and α-syn levels have been less thoroughly studied. One study found increased α-syn accumulation in SN neurons in heterozygous TrkBþ/  mice (Von Bohlen und Halbach et al., 2005), but exposure to BDNF in vitro did not affect α-syn mRNA expression (Satoh and Kuroda, 2001). In conclusion, the present study found normal short-term CPu DA release in mice carrying APP/PS1 and/or TrkB.T1 transgenes. This is compatible with finding of normal density of TH-positive axon terminals in CPu in all genotypes and intact presynaptic machinery for DA release and reuptake, as evidenced by unchanged levels of DAT, SNAP-25 and α-syn. However, upon prolonged stimulation mice carrying either the APP/PS1 or the TrkB.T1 gene displayed compromised capacity to recruit more DA for release as wild-type animals do. This may be linked to a novel observation of increased number of SN DA neurons in TrkB.T1 mice, which is compensated for by decreased TH terminals per neuron. In contrast, APP/PS1 mice, consistent with previous studies, have a preserved number of SN DA neurons and unchanged TH levels per neuron at the CPu axon terminals. Thus the ultimate reason for compromised capacity to recruit DA upon prolonged stimulation in APP/PS1 mice remains open. We suggest that both APP/PS1 and TrkB.T1 transgenes cause DAergic system impairment, but via different mechanisms.

4.

Experimental procedures

4.1.

Animals

The APPswe/PS1dE9 (APP/PS1) founder mice were obtained from D. Borchelt and J. Jankowsky (Dept. Pathology, Johns Hopkins Univ., Baltimore, MD, USA) and a colony was established at the University of Eastern Finland (Kuopio, Finland). These mice were generated by co-injection of chimeric mouse/human APPswe (mouse APP695 harboring a human Aβ domain and mutations K595N and M596L linked to Swedish familial AD pedigrees) and human PS1-dE9 (deletion of exon 9) vectors controlled by independent mouse prion protein promoter elements (Jankowsky et al., 2004). This mouse line was originally maintained in a hybrid C3HeJ  C57BL6/J F1 background, but the mice used in the present study were derived from backcrossing to C57BL6/J for 12 generations. The development of mice overexpressing the truncated TrkB receptor (TrkB.T1) specifically in neurons ( Z 2-fold overexpression throughout cortex and hippocampus) has been described previously by Saarelainen et al. (Saarelainen et al., 2000a, 2000b). Expression of the transgenic receptor in the TrkB.T1 mouse is highest in the cerebral cortex and hippocampus (Saarelainen et al., 2000a), thus overlapping with brain areas with the highest amyloid load in the APP/PS1 mouse (Jankowsky et al., 2004). The TrkB.T1 transgenic line has transgene expression also in the thalamus (Saarelainen et al., 2000a). This mouse line was originally maintained in a hybrid BALB/c  DBA/2 background, but mice used in the

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present study were derived from backcrossing to C57BL6/J for 10 generations. Crossing of these two mouse lines yielded four genotypes, indicated as: AwTw¼ wt  wt AwT1¼ wt  TrkB.T1 AþTw¼APP/PS1  wt AþT1¼APP/PS1  TrkB.T1

The housing conditions (National Animal Center, Kuopio, Finland) were controlled (temperature þ22 1C, light from 07:00 to 19:00; humidity 50–60%), and fresh food and water were freely available. The experiments comprised two separate groups of male mice. Voltammetry was performed on a group of 23 4- to 6month-old mice, while histological analysis was performed on an additional group of 55 13-month-old mice.

4.2.

Ethics statement

The experiments were conducted according to the Council of Europe (Directive 86/609) and Finnish guidelines, and approved by the State Provincial Office of Eastern Finland. The protocol was approved by the Animal Experiment Board of Finland (Permit number: 07-04388). Voltammetry was performed under chloral hydrate anesthesia, and animal suffering was minimized by all possible means.

4.3.

In vivo voltammetry

4.3.1.

Preparing the animals

The 4- to 6-month-old mice (25–30 g) were anesthetized with chloral hydrate (450 mg/kg, i.p.) and fixed in a stereotaxic frame. Anesthesia was maintained by bolus injections of the drug at 100 mg/kg every 45–60 min. Rectal temperature was kept at 37 1C. The carbon-fiber working electrode was inserted through a skull opening to the caudate nucleus (0.5 mm anterior, 2.0 mm lateral and 3.2 mm ventral from bregma), and a bipolar stimulating electrode was implanted in the medial forebrain bundle (MFB) (A 2.0 mm, L 1.2 mm, V 5.1– 5.3 mm). The exact placement of the stimulating electrode in the dorsoventral coordinate was adjusted for maximal DA release. An Ag/AgCl reference electrode in a saline bridge was placed on the skull. A stainless steel screw as the auxiliary electrode was fixed into occipital bone. After the experiments, the working electrodes were calibrated as described below, except in cases in which the locations of the working electrodes were verified histologically after electrolytic lesions (6 V for 15 s).

4.3.2.

Electrochemical technique

Stimulated DA overflow was measured at 0.4 V vs. an Ag/AgCl reference electrode by constant potential amperometry with carbon fiber working electrodes 32 mm in diameter and 300 mm in length in glass insulation (Invilog Research, Kuopio, Finland) and In Vivo Voltammetry Setup from the same company. After the experiments, the working electrodes were

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rinsed with deionized water and calibrated for DA in PBS containing ascorbic acid (200 mM/l) at pH 7.4.

4.3.3.

Electrical stimulation and experimental protocol

The stimulation train consisted of six repeated 2-s bursts (50 Hz; biphasic constant-current pulses; 1 ms each) that were separated by 5-s intervals. In total, three trains were delivered, separated by 90-s intervals. Stimulation was applied using In Vivo Voltammetry Setup from Invilog Research. To ensure response stability, this stimulation pattern was repeated two times at 60 min intervals, and data from the last set are presented. The evoked DA overflow following stimulation of the MFB at increasing frequencies was analyzed using 2-s stimulus bursts (1 ms bipolar pulse) delivered at 10, 20, 30, 40 and 50 Hz. The interval between consecutive trains of stimulation was increased from 3 to 5 min with increasing frequency.

4.3.4.

Data presentation

Peak DA release is expressed in molar concentrations (mM/l) on the basis of postcalibration data or as percentage of the evoked release. In the latter case, dynamics of DA decline within each stimulus train is expressed as the percentage of the evoked release after the first burst in that particular train. Dynamics of DA decline within the second and third train in this case were statistically indistinguishable. For this reason, data on the second train are not shown in the Resultssection.

4.4.

Immunohistology

4.4.1.

Preparing the animals

The mice used for immunohistology were deeply anesthetized with pentobarbiturate-chloral hydrate cocktail (60 mg/ kg each) and perfused transcardially with 50 ml heparinized ice-cold 0.9% saline (10 ml/min) followed by 9 min perfusion with 4% paraformaldehyde (PFA). Brains were transferred to 4% PFA solution for 4 h and then to 30% sucrose solution overnight, after which brains were stored in cryoprotectant at 20 1C. Brains were cut on a sliding/freezing microtome into 35 mm coronal sections and stored in cryoprotectant at 20 1C. Sections were selected using a mouse brain atlas (Franklin, 1997) as a reference. Desired section levels lay at specified distances from bregma, and sections were chosen as close to the chosen levels as possible, at the same time not compromising section quality.

4.4.2.

Tyrosine hydroxylase staining

Six sections at the level of substantia nigra (SN) from approximately  3.52 mm to 3.16 mm from bregma and two striatal sections from the range of bregma þ0.50 mm to þ0.62 mm (same location as for voltammetry measurements of DA release) were stained for tyrosine hydroxylase (TH). TH is an enzyme that catalyzes the conversion of L-tyrosine to LDOPA, a precursor for DA (Nagatsu et al., 1964). Sections were rinsed with Tris-Buffered Saline with Triton (TBS-T) and pretreated with 10% normal goat serum (NGS, Biowest, Nuaillé, France,cat.no S2000) in TBS-T for 30 min. Sections were stained with polyclonal rabbit anti-TH antibody (antiTH, 1:7000, Millipore, Temecula, CA, USA, cat.no AB152) in 1%

NGS and incubated for 72 h at 4 1C on a shaker table. Following this, sections were rinsed and incubated for 2 h with the secondary antibody, biotinylated goat anti-rabbit (1:500, Vector Laboratories, Burlingame, CA, USA, cat.no BA1000). After this, sections were rinsed and transferred to Streptavidin-horseradish peroxidase conjugate (1:1000, GE Healthcare, Buckinghamshire, UK, cat.no RPN1231V) solution for 2 h. Visualization of DAergic and noradrenergic neurons was achieved by incubation with DAB–Ni solution. Stained sections were rinsed with PBS and mounted on gelatincoated slides, cleared with xylene (VWR International, Helsinki, Finland) and mounted in DePeX (BDH Chemicals via VWR International, Helsinki, Finland).

4.4.3.

SNAP-25-staining

To assess the integrity of synaptic vesicle exocytosis machinery in our mouse model, two striatal sections from the range of bregma þ0.50 mm to þ0.62 mm were stained for synaptosome-associated protein of 25 kDa (SNAP-25). SNAP25 is a component of the soluble NSF (N-ethylmaleimide sensitive fusion protein) attachment protein (SNAP) receptor (SNARE) complex, which regulates the release of neurotransmitters into the synaptic cleft by regulating membrane fusion between synaptic vesicles and plasma membranes (Söllner et al., 1993). Sections were rinsed with TBS-T and pretreated with 10% NGS in TBS-T for 30 min. Sections were stained with polyclonal rabbit anti-SNAP-25 antibody (SNAP-25, 1:4000, Synaptic systems, Goettingen, Germany, cat.no 111002) in 1% NGS and incubated overnight at room temperature (RT) on a shaker table. Following this, sections were rinsed and incubated for 2 h with the secondary antibody, biotinylated goat anti-rabbit (1:500, Vector Laboratories, Burlingame, CA, USA, cat.no BA-1000). Further treatment and visualization of presynaptic SNAP-25 was as for TH staining.

4.4.4.

Dopamine transporter staining

To indirectly assess DA reuptake activity, three striatal sections from the range of bregma þ0.02 mm to þ0.38 mm were stained for dopamine transporter (DAT). DAT is a transmembrane protein that provides the main mechanism through which DA is cleared from synapses back into the presynaptic DAergic nerve terminals (Torres et al., 2003). Sections were rinsed with TBS-T and pretreated with 0.3% H2O2 in TBS-T for 30 min. After rinsing, sections were pretreated with 1.5% NGS in TBS-T, rinsed and stained with monoclonal rat anti-DAT antibody (anti-DAT, 1:10000, Millipore, Temecula, CA, USA, cat.no MAB369), incubating for 48 h at 4 1C on a shaker table. Following this, sections were rinsed and incubated for 2 h with the secondary antibody, biotinylated sheep anti-rat (1:500, AbD Serotec, Oxford, UK, cat.no AAR10B). Further treatment and visualization of DAT-containing presynaptic DAergic nerve terminals was as for TH staining.

4.4.5.

α-synuclein-staining

To visualize alpha-synuclein (α-syn) load, one striatal section from approximately bregma þ0.14 mm (range þ0.02 to þ0.38 mm) was stained for α-syn. This protein is well known for its association with Parkinson's disease and some other neurodegenerative diseases, and has been implicated in synaptic vesicle trafficking and release, since it is associated

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to the SNARE-complex assembly (Burré et al., 2010; Chandra et al., 2005), and kinetics of synaptic vesicle endocytosis (Vargas et al., 2014). Sections were rinsed with PotassiumPhosphate Buffered Saline with Tween (KPBST) and pretreated with 1% H2O2 in KPBST for 30 min. After rinsing, sections were pretreated with 1% Bovine Serum Albumin (BSA, Sigma, St. Louis, MO, USA, cat.no A2153-50G) in KPBST for 30 min. Sections were stained with polyclonal rabbit antiα-syn antibody (α-synuclein/NACP, 1:1000, Enzo Life Sciences, Lausen, Switzerland, cat.no BML-SA3400) in 1% BSA in KPBST and incubated overnight at 4 1C on a shaker table. Following this, sections were rinsed and incubated for 2 h with the secondary antibody, biotinylated goat anti-rabbit (1:500, Vector, Burlingame, CA, USA, cat.no BA-1000). Further treatment and visualization of α-syn-containing presynaptic nerve terminals was as for TH staining.

4.4.6.

TrkB.T1-staining

The transgene in the TrkB.T1 was originally tagged N-terminally with an eight amino acid FLAG peptide as previously described (Haapasalo et al., 1999). To control for tissue expression of TrkB. T1 in the meso-striatal DA system, one section from one animal per genotype was chosen at the level of the head of caudateputamen (approximately bregma 0.22 mm) and DAergic midbrain (approximately bregma  2.70 mm). Sections were rinsed with TBS-T and incubated with anti-FLAG BioM2 antibody (antiFLAG, 1:1000, Kodak, New Haven, CT, USA) overnight at RT on a shaker table. Following this, sections were rinsed and incubated for 2 h with the secondary antibody, biotinylated goat anti-rabbit (1:500, Vector, Burlingame, CA, USA, cat.no BA-1000). Further treatment and visualization of the FLAG peptide was as for TH staining (Fig. 1A to C).

4.5.

Data analysis

For quantification of DAergic axon terminals in caudateputamen (CPu), two TH-stained striatal sections from all mice (n¼ 55; AwTw n¼ 15; AwT1 n¼ 15; AþTw n ¼13, AþT1 n¼ 12) were analyzed for optic density. The first section was chosen at the coronal plane where the anterior commissure is still clearly seen as two separate round formations and the second one 105 mm more rostrally. Sections were photographed using the Zeiss Imager M2 microscope (Zeiss, Oberkochen, Germany) and the attached AxioCam ERc 5s camera with a 2.5  objective. Images were photomerged and transformed to grayscale in Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA), which was also used for image analysis. The optic densities values (0 for black to 255 for white) were determined by placing a 76  76 pixel rectangular marquee on three regions of interest near the CPu lateral edge, preferring the most evenly stained locations and not including the fiber bundles. To control for slightly varying light condition we measured three control optic density values from lateral ventricles. Final optic density values were obtained by subtracting the mean tissue value from the mean control values Thus, the larger the optic density value, the deeper the staining color (Fig. 2A). For quantification of DAergic neurons, six TH-stained sections from all mice (n ¼55) were analyzed for SN cell count. The caudalmost section was chosen at the coronal

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plane where the dentate gyrus is still seen as an open formation, and following sections at 105 mm intervals. For cell count analysis we used the Olympus BX40 microscope (Tokyo, Japan) with a 40  objective and a 250  250 mm grid placed in the ocular. Cells were counted one by one on two occasions from the lateral part of SN. The grid was placed just outside the most lateral THþ cell and then just above the most rostral THþ cell. All cells fully or partly inside the grid were counted (Fig. 3). To quantify the exocytotic fusion complexes on neuronal plasmalemmae, the two striatal SNAP-25-stained sections from all mice were analyzed. The sections were photographed, photomerged and transformed to grayscale and analyzed for optic densities as for the TH-stained striatal sections. Our SNAP-25 antibody strongly stained striatal fiber bundles and other white matter, which seems common for other SNAP-25 antibodies (Kawakami et al., 2012). Therefore care was taken to measure optic density in gray matter, avoiding fiber bundles (Fig. 2B). To quantify the DAT-containing presynaptic DAergic nerve terminals, the three striatal DAT-stained sections from 20 mice (AwTw n¼5, AwT1 n¼ 6, AþTw n¼ 4, AþT1 n ¼5) were analyzed as for the TH-stained striatal sections. Image analysis was identical to that for the TH-stained sections, but using a larger 153  153 pixel marquee (Fig. 2C). To quantify the α-syn load, the striatal α-syn-stained sections from the same 20 mice as for DAT-staining using an identical protocol (Fig. 2D).

4.6.

Statistical analysis

For voltammetry data, the statistical analysis was performed using multivariate ANOVA for repeated measurements (ANOVA-RM) (SPSS Statistics for Windows, Version 17.0, SPSS Inc., Chicago, IL, USA), with repeated electrical stimulation as the within-subject factor (6 bursts  3 trains), and APP/PS1 and TrkB.T1 genotypes as the between-subjects factors. Data are presented as mean7SEM. For immunohistological data, the statistical analysis was performed using two-way ANOVA (IBM SPSS Statistics for Windows, Version 21.0, IBM Corp., Armonk, NY, USA), with APP/PS1 (Aþ vs. Aw) and TrkB.T1 (T1 vs. Tw) genotypes as fixed factors. The two-tailed bivariate correlation test represented by Pearson correlation coefficient was performed on optical density data, and any correlation between 0.3 and 0.5 was considered low, any correlation between 0.5 and 0.7 moderate, and any correlation between 0.7 and 0.9 high.

Acknowledgments We thank Dr. D. Borchelt (Univ. Florida, FL, USA) and Dr. J. Jankowsky (Baylor College of Medicine, Houston, TX, USA) for the APPswe/PS1dE9 breeder mice and Dr. L. Alhonen (Univ. Eastern Finland, Kuopio, Finland) and Dr. E. Castren (Univ. Helsinki, Finland) for the TrkB.T1 breeder mice. The study received funding from North-Savonia Regional Fund (grant number 65132044), Finnish Medical Foundation (grant number 294), Finnish Medical Society Duodecim (grant number 16) and Emil Aaltonen Foundation.

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