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c-Fos expression after deep brain stimulation of the pedunculopontine tegmental nucleus in the rat 6-hydroxydopamine Parkinson model
Journal of Chemical Neuroanatomy 42 (2011) 210–217
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c-Fos expression after deep brain stimulation of the pedunculopontine tegmental nucleus in the rat 6-hydroxydopamine Parkinson model Assel Saryyeva, Makoto Nakamura, Joachim K. Krauss, Kerstin Schwabe * Department of Neurosurgery, Medical University, MHH, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 June 2011 Received in revised form 3 August 2011 Accepted 3 August 2011 Available online 10 August 2011
Deep brain stimulation (DBS) is used to alleviate motor dysfunction in Parkinson’s disease (PD). The pedunculopontine nucleus (PPN) may be a potential target for severe freezing and postural instability with 25 Hz stimulation being considered more effective than 130 Hz stimulation. Here we evaluated the expression of c-Fos after 25 Hz and 130 Hz DBS of the pedunculopontine tegmental nucleus (PPTg, i.e., the rodent equivalent to the human PPN) in the rat 6-hydroxydopamine (6-OHDA) PD model. Anaesthetized male Sprague Dawley rats with unilateral 6-OHDA-induced nigrostriatal lesions were stimulated with 25 Hz, 130 Hz, or 0 Hz sham-stimulation for 4 h by electrodes implanted into the ipsilateral PPTg. Thereafter the distribution and number of neurons expressing the immediate early gene c-Fos, a marker for acute neuronal activity, was assessed. DBS of the PPTg induced strong ipsilateral c-Fos expression at the stimulation site, with 25 Hz having a more marked impact than 130 Hz. Additionally, c-Fos was strongly expressed in the central gray. In the dorsal part expression was stronger after 25 Hz stimulation, while in the medial and ventral part there was no difference between 25 Hz and 130 Hz stimulation. Expression in the basal ganglia was negligible. In the rat 6-OHDA PD model stimulation of the PPTg did not affect c-Fos expression in the basal ganglia, but had a strong impact on other functional circuitries. PPN stimulation in humans might therefore also have an impact on other systems than the motor system. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Basal ganglia Motor activity Central gray
1. Introduction Deep brain stimulation (DBS) of the pedunculopontine nucleus (PPN) is used to treat severe freezing and postural instability, which are refractory to medical treatment in late stage Parkinson’s disease (PD; Alam et al., 2011; Ferraye et al., 2010; Moro et al., 2010; Plaha and Gill, 2005; Stefani et al., 2007). These symptoms may not respond well to dopamine medication or DBS of the subthalamic nucleus (STN) or globus pallidus internus (GPi), i.e., basal ganglia nuclei, which are established targets for the treatment of parkinsonian symptoms in PD (Benabid et al., 2009; Loher et al., 2002; Rodriguez-Oroz et al., 2005; Stefani et al., 2007). The PPN is an important area within the pontomesencephalic locomotor region in the mesopontine tegmentum. It receives direct input from the cerebral cortex, and is reciprocally connected
Abbreviations: ANOVA, analysis of variance; AP, anterior–posterior; BSA, bovine serum albumin; CG, central gray; DAB, diaminobenzidin; DBS, deep brain stimulation; EPN, entopeduncular nucleus; GPi, globus pallidus internus; L, lateral; NGS, normal goat serum; PBS, phosphate buffered saline; PD, Parkinson’s disease; PPN, pedunculopontine nucleus; PPTg, pedunculopontine tegmental nucleus; SN, substantia nigra; STN, subthalamic nucleus; TH, tyrosine hydroxylase; V, ventral. * Corresponding author. Tel.: +49 511 532 2862; fax: +49 511 532 5864. E-mail address: [email protected] (K. Schwabe). 0891-0618/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2011.08.003
mainly with basal ganglia regions, but also some limbic areas, and it also projects to the thalamus (Matsumura et al., 2000; Steininger et al., 1992). It is also further directly connected to several motor nuclei in the brainstem and spinal cord (Garcia-Rill et al., 2001; Takakusaki et al., 2004, 2008). Post mortem studies of patients with advanced PD showed neuronal degeneration in the PPN that correlated with the severity of pre-mortal gait dysfunction (Hirsch et al., 1987; Jellinger, 1988; Lee et al., 2000; Pahapill and Lozano, 2000). While DBS of the STN or GPi is thought to affect the pathological neuronal activity and abnormally patterned oscillations in the basal ganglia caused by the loss of dopamine in the nigrostriatal system (Brown, 2006; Delong and Wichmann, 2007; Hashimoto et al., 2003; Trottenberg et al., 2007), the circuit related consequences of PPN DBS are not well known. Moreover, there is evidence that PPN DBS with 25 Hz is more effective than with 130 Hz, the frequency, which is usually applied in DBS of the STN or GPi (Plaha and Gill, 2005; Ferraye et al., 2010). While the acute effects of DBS are presumably due to immediate electrophysiological alterations, molecular processes are thought to reflect long-term effects. These molecular changes usually start with the expression of immediate early genes. Of these, c-Fos is a marker for acute neuronal and synaptic activity and has been used to study the effects of DBS at cellular levels (Herdegen and Leah, 1998; Schulte et al., 2006).
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We were interested, which brain regions would be activated by stimulation of the pedunculopontine tegmental nucleus (PPTg, i.e., the rat equivalent to the human PPN), whether there would be differences between 25 Hz and 130 Hz stimulation, and whether there would be differences in the normal compared to the pathophysiological state in a PD model. To address these questions, we quantified the number of c-Fos immunoreactive neurons after 0 Hz (sham-stimulation), 25 Hz and after 130 Hz PPTg DBS in 6hydroxydopamine (6-OHDA) lesioned and sham-lesioned rats, a model that has been successfully used in PD research for many years (Deumens et al., 2002). 2. Experimental procedure 2.1. Animals Adult male Sprague-Dawley rats (n = 47; purchased from Charles River Laboratories, Germany, weighing 200 g upon arrival) housed at the Central Animal Facility of the Medical University of Hannover were used for this study. The rats were kept under artificial conditions of light (14 h/10 h light cycle, light on at 7 a.m.), temperature (22 2 8C) and humidity (55 5%). Animals were kept in groups of 2 in standard Macrolon Type III cages (Techniplast, Hohenpeissenberg, Germany). They received tap water and rat chow (altromin, Altromin GmbH & Co. KG, Lage, Germany) ad libitum. Although PD is a bilateral disease, all rats were unilaterally injected with 6-OHDA or vehicle into the medial forebrain bundle for lesion or sham-lesion since we aimed to induce nearly complete dopamine lesions in the Substantia nigra pars compacta (SNc), which, when induced bilaterally, leads to high incidence of death and sever discomfort in rodents (reviewed e.g., in Deumens et al., 2002). Four weeks later an electrode was stereotactically implanted in the PPTg for stimulation or shamstimulation under general anesthesia. All experiments were carried out in accordance with the European Council Directive of 24 November 1986 (86/609/ EEC). 2.2. Stereotactic surgery For 6-OHDA lesion of the SN the rats were intraperitoneally anaesthetized with chloralhydrate (360 mg/kg; Sigma–Aldrich Chemie GmbH, Steinheim, Germany) and mounted in a stereotactic frame. Additionally, the area for surgery was infiltrated with local anesthesia (Xylocain 2%). For microinjection, 6-OHDA (Sigma Chemical Co.) was dissolved in 0.9% saline solution with 0.02% ascorbic acid added. A dose of 3.6 mg/1 ml was used for injection with a microsyringe (5 ml, Hamilton Company, Nevada, USA) at a rate of 1 ml/min. For injection, a burr hole was drilled above the injection site and the tip of the syringe lowered into the target. Rats were injected into the right hemisphere at two targets with coordinates in mm relative to bregma according to the brain atlas of Paxinos and Watson (1998). Three microliters were injected in the medial forebrain bundle at two sites with the following stereotactic coordinates (1) anterior–posterior (AP): 4.0; lateral (L): 0.8; ventral (V): 7.5 from dura level; incisor bar at +3.4, and (2) AP: 4.4; L: 1.2; V: 7.8 from dura level; incisor bar at 2.4. Sham-lesioned rats (vehicle-injection only) served as controls. After injection the cannula was left in place for 3 min before slowly retracting it. Finally, the skin incision was sutured. 2.3. Electrode implantation and deep brain stimulation Four weeks after injection of 6-OHDA stereotactic surgery was carried out for implantation of bipolar electrodes in the PPTg. Again, rats were intraperitoneally anaesthetized with chloral hydrate (360 mg/kg, followed by additional injection with 120 mg/kg each hour) and placed into a stereotactic frame. Additionally, the area for surgery was infiltrated with local anesthesia (Xylocain 2%). The electrodes were made of two parallel insulated platin-iridium electrode wires (90:10), coated with Teflon (un-coated diameter 76 mm, coated diameter 140 mm, A-M Systems, Inc., Carlsborg, USA). The Teflon coated part of the wires was covered in a 15 mm long cylinder (0.45 mm 0.25 mm, Sterican1 Braun Melsungen AG, Germany) and uncovered at their extremities at a length of 500 mm. The distance between the tips of the two uncoated wires was 500 mm for bipolar stimulation. The other end of the wires was welded to a socket. This type of electrodes has been suggested to be especially suitable for stimulation of the rodent STN (Gubellini et al., 2009) and has been reported not to produce tissue damage after stimulation for several days (Gubellini et al., 2006). Electrodes were inserted into the right PPTg at the following coordinates in mm relative to bregma: AP: 7.8; L: 1.8: V: 7.3 from the cranium surface; tooth bar at 3.3. DBS was applied continuously for 4 h on anesthetized rats and delivered by a stimulator (Multichannel systems, Reutlingen, Germany). For electrical stimulation rectangular biphasic pulses were used with duration of 160 ms (change of polarization after 80 ms). The frequency was set at 130 Hz, 25 Hz or 0 Hz sham-stimulation, and the intensity at 400 mA over the stimulation period for all of the stimulated animals.
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2.4. Histological processing After sham-stimulation, respectively immediately after 4 h 130 Hz or 25 Hz stimulation, rats were deeply anesthetized with an overdose of chloralhydrate (720 mg/kg) and transcardially perfused with phosphate buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4). The brains were removed from the skulls and cryoprotected in 30% sucrose for at least 48 h. Each brain was cut in six series of 40 mm coronal sections on a freezing sliding microtome. For histological processing, one series was used for Nissl-staining, one for tyrosine hydroxylase (TH) immunohistochemistry, and one for c-Fos immunohistochemistry. The sections containing the PPTg were Nissl stained with Thionine to check for correct placement of the electrodes. The sections containing the striatum and SN were processed free floating for immunohistochemical staining for TH. All chemicals were purchased from Sigma–Aldrich GmbH, Steinheim, Germany, except as stated otherwise. First the slices were incubated for 10 min in solution with 1 ml methanol, 1 ml 30% H2O2 and 8 ml 0.1 M PBS, followed by a 3-step washing procedure with PBS with each step lasting 5 min. Subsequently, the slices were incubated in the primary antibody solution (1:5000) with IgG mouse anti tyrosine hydroxylase in 1% bovine serum albumin (BSA) in a PBS/Triton X-100 solution over night, followed by a 3-step washing procedure with PBS. Next, the slices were incubated in the secondary antibody solution (1:200) with biotinylated IgG rabbit anti mouse (DakoCytomation, Glostrup, Denmark) in 1% BSA/PBS/Triton X-100 for 60 min, followed by the washing procedure. Thereafter, the slices were incubated for 60 min by using the ABC-Standard-Kit (1:1000; 1 ml A (AvidinH) + 1 ml B (Biotinyl-Peroxidase) in 1 ml PBS (ABC-Kit, Vector Laboratories, Inc., Burlingame, USA), which was again followed by the washing procedure. Finally, a solution of 3,30 -diaminobenzidin (DAB, DakoCytomation, Glostrup, Denmark), used as the chromogen, in a PBS-ammoniumnickelsulfate solution and H2O2 (20 ml 30% H2O2 solved in 580 ml PBS) was used to stain the slices. To stop the staining reaction, the slices were extensively washed with PBS, mounted on a glass slide and coverslipped with Vitro Clud (Langenbrinck, Emmendingen, Germany). The sections were then examined under a light microscope to determine the loss of nigral dopaminergic neurons and the loss of TH-staining in the striatum. To detect c-Fos immunoreactivity one series of sections was incubated for 1 h in a solution of 10% normal goat serum (NGS), 30% H2O2 and 20% Triton to remove endogenous peroxidase activity, and then incubated in primary antibody against cFos (1:10,000; Calbiochem, Germany) in PBS with 0.02% Triton and 10% NGS overnight at room temperature. After rinsing four times in PBS (1 3 min, 1 5 min, 2 15 min), sections were incubated in PBS with 1% bovine serum albumin for 1 h at room temperature. Subsequently, the sections were incubated in biotinylated goat anti-rabbit secondary antibody (1:2000; DakoCytomation, Glostrup, Denmark) in PBS with 1% BSA and Triton for 1 h, and then washed four times in PBS. Visualization of c-Fos immunoreactivity was as described above, i.e., usage of the ABC-Kit followed by an incubation of the series in DAB, and finally dehydrating the slices in ethanol and embedding the slices in Vitro Clud. For analysis of c-Fos expression all subcortical brain regions were qualitatively scanned for c-Fos immunoreactivity after 25 Hz and 130 Hz compared to 0 Hz (sham) stimulation. We then counted the number of c-Fos immunoreactive cells at the stimulation site and in those areas that showed enhanced expression of the groups stimulated with 25 Hz and 130 Hz. The sections used for quantitative analyses were chosen on the basis of the amount of staining and because they could be easily and reproducibly picked up on certain landmarks near the site of expression. For histological analysis a Zeiss Axio Imager Z1m microscope (Go¨ttingen, Germany) equipped with the digital camera SpotTM Persuit and the image analysis software MetaMorph 4.6 (both from Visitron Systems GmbH, Puchheim, Germany) was used. For evaluation of c-Fos immunoreactive neurons in the PPTg the central part of the video frame was placed at the deepest position of the electrode tract and the cells counted in this slice, as well as in the previous and subsequent slice. For evaluation of c-Fos immunoreactivity in the other regions, the coordinates of the area of strongest expression were determined with respect to bregma. Thereafter, all stained neurons were determined ipsilateral to the stimulation by counting all c-Fos immunoreactive neurons in one frame of the video camera at 200 times magnification emerging into focus through the 40 mm sections. Additionally, the cells in the three previous and three subsequent sections (distance between sections 240 mm) were counted, thus covering in total a distance of about 0.8 mm. c-Fos immunoreactive cells in the corresponding contralateral region in the left hemisphere were likewise counted. The mean number of the cells counted in the different slices was used for statistical evaluation. The data were evaluated by a two-way analysis of variance (ANOVA) with lesion (sham-lesion, 6-OHDA lesion) and stimulation intensity (0 Hz, 25 Hz, and 130 Hz) as factors. For post hoc pair wise comparison Tukey’s test was used. All tests were performed two-sided and a p-value of 0.05 was considered to represent a significant effect.
3. Results Only rats with a nearly total loss of tyrosine hydroxylase immunoreactivity in the SNc (i.e., less than 15 TH-positive neurons in the ipsilateral SNc, see Fig. 1) and confined correct placement of
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the electrode in the PPTg were used for statistical analyses. Additionally, rats that did not show c-Fos immunoreactivity in regions with usually high background expression of c-Fos (i.e., the motor cortex or the piriform cortex) were excluded. Thus, 36 of 47 rats were used for final evaluation: 0 Hz stimulation (lesion: n = 6, sham-lesion: n = 6), 25 Hz stimulation (lesion: n = 6, sham-lesion: n = 6), and 130 Hz stimulation (lesion: n = 6, sham-lesion: n = 6). A reconstruction of the electrode placement in the PPTg is shown in Fig. 2A, together with representative histological pictures of the stimulation site for each stimulation group shown in Fig. 2B. In these Nissl-stained sections there was only minor tissue damage around the stimulated site. In only one rat of either group a small number of c-Fos immunoreactive cells were found in regions of the basal ganglia, i.e., in the substantia nigra (SN), the STN, and the zona incerta, but in almost all stimulated rats c-Fos immunoreactivity was shown in the central gray (CG; see Table 1). Besides the stimulation site in the PPTg, different subregions of the CG were therefore used for quantitative analysis. 3.1. PPTg
Fig. 1. Histological pictures of coronal brain slices after immunohistological processing for tyrosine hydroxylase staining of the substantia nigra pars compacta after vehicle-injection (A) and 6-hydroxydopamine-injection; (B) calibration bar: 500 mm.
At the stimulation site the expression of c-Fos was strongest after 25 Hz stimulation, followed by 130 Hz stimulation, but with no difference between 6-OHDA lesioned and sham-lesioned rats (Fig. 3). ANOVA showed an effect for the factor stimulation (F2,35 = 18.692, p < 0.001), a trend for significance for the factor lesion (F1,35 = 3.341, p = 0.078), but no interaction between these factors (F2,35 = 0.334, p = 0.719). Post hoc comparison revealed more expression in rats after 25 Hz stimulation compared to 0 Hz and 130 Hz stimulation (p < 0.001 and p = 0.008, respectively). Additionally, expression after 130 Hz stimulation was stronger compared to 0 Hz sham-stimulation (p = 0.020). See Fig. 3B for
Fig. 2. (A) Schematic drawings (adapted to the atlas of Paxinos and Watson, 1998) of the implantation sites of the electrode tips. Sections on the left site represent shamlesioned (open symbols) and on the right site 6-hydroxydopamine lesioned rats (filled symbols) with 0 Hz stimulation (*,*), 25 Hz stimulation (&/&), and 130 Hz stimulation (~/~) in distance to bregma. (B) Histological photomicrographs showing the electrode tip in the PPTg after 0 Hz stimulation (B1), 25 Hz stimulation (B2), and 130 Hz stimulation (B3; black arrow) with the corresponding electrode tip at higher magnification in B4–6. The tract dorsal to the electrode tip shows tissue damage caused by the cylinder that covered the electrode wires in stimulated and sham-stimulated rats (white arrow). Calibration bar: (B1–3) 500 mm, (B3–6) 100 mm.
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Table 1 Expression of c-Fos in subcortical brain regions of 6-hydroxydopamine-lesioned or sham-lesioned rats after stimulation with 0 Hz, 25 Hz, and 130 Hz. Brain region
PPTg Central gray Substantia nigra Nucleus subthalamicus Zona incerta Hippocampus
0 Hz
25 Hz
130 Hz
Sham
Lesion
Sham
Lesion
Sham
Lesion
3/6 1/6 0/6 0/6 0/6 0/6
4/6 3/6 0/6 0/6 0/6 0/6
6/6 5/6 0/6 0/6 1/6 1/6
6/6 6/6 1/6 1/6 0/6 1/6
6/6 6/6 0/6 0/6 0/6 0/6
6/6 6/6 1/6 1/6 0/6 1/6
Shown is the number of animals expressing c-Fos in relation to the number of stimulated rats.
representative histological pictures of c-Fos expression in the PPTg for all stimulated groups. 3.2. Central gray Ipsilateral to the stimulation site the strongest expression of cFos was found at about 6.8 mm in relation to bregma. This slice was therefore used as the central slice for counting c-Fos immunoreactive neurons in the different subregions (Fig. 4A). cFos expression ipsilateral to the stimulation site was strongest in the dorsolateral CG, followed by the lateral and ventral CG. The expression in the dorsal CG was strongest after 25 Hz stimulation compared to 0 Hz and 130 Hz stimulation, which was similar ipsilateral and contralateral to the stimulation site (Fig. 4B1). No difference was found between 6-OHDA lesioned and shamlesioned rats. Ipsilateral to the lesion ANOVA showed an effect for the factor stimulation (F2,35 = 7.237, p = 0.003), but no effect for the factor lesion (F1,35 = 0.609, p = 0.441) or the interaction between these factors (F2,35 = 0.480, p = 0.624). Post hoc comparison revealed more expression in rats after 25 Hz stimulation compared to 0 Hz and 130 Hz stimulation (p = 0.003 and p = 0.026, respectively), while the effect after 130 Hz stimulation was not different to 0 Hz stimulation (p = 0.624). Fig. 4C shows representative histological pictures of the different stimulation groups. In the dorsal CG contralateral to the lesion ANOVA showed an effect for the factor stimulation (F2,35 = 8.732, p = 0.001) with post hoc testing showing significant more expression after 25 Hz compared to 0 Hz and 130 Hz stimulation (both p < 0.05; Table 2). The factor lesion and the interaction between factors (F1,35 = 2.103, p = 0.157 and F2,35 = 2.022, p = 0.150, respectively) were not significant. For the lateral and ventral CG ipsilateral to the lesion ANOVA also revealed an effect for the factor stimulation (F2,35 = 5.588, p = 0.009 and F2,35 = 3.966, p = 0.030, respectively) with post hoc testing showing more expression in rats after 25 Hz stimulation compared to 0 Hz sham-stimulation (p = 0.006 and p = 0.023; Fig. 4). The factor lesion and the interaction between factors were not significant (all F-values >0.651, all p-values <0.426). In the lateral and ventral CG contralateral to the lesion statistical analysis with ANOVA was only marginally significant for the factor stimulation (F2,35 = 2.866, p = 0.073 and F2,35 = 3.052, p = 0.062, respectively), with no effect for the factor lesion or interaction between factors (all F-values >1.223, all p-values <0.278; Table 2).
4. Discussion Deep brain stimulation of the PPTg induced strong c-Fos expression at the stimulation site and in the CG with 25 Hz stimulation being more effective than 130 Hz stimulation, while expression in the basal ganglia or thalamus was negligible. No
Fig. 3. (A) Number of c-Fos immunoreactive neurons at the stimulation site in the pedunculopontine tegmental nucleus (PPTg) of sham-lesioned and lesioned rats after 0 Hz, 25 Hz and 130 Hz stimulation. Data are presented as means + S.E.M. Significant differences to 0 Hz stimulation are marked by asterisks (*), significant differences between 25 Hz and 130 Hz by circles (8; ANOVA with post hoc Tukey test p < 0.05). (B) Representative photomicrographs of c-Fos immunoreactive neurons after 0 Hz (B1), 25 Hz (B2), and 130 Hz (B3). Calibration bar: (B1–3) 100 mm.
differences were found between 6-OHDA lesioned and shamlesioned rats. Clinical studies have shown that PPN DBS with 25 Hz stimulation appears to be more effective with respect to freezing than frequencies >60 Hz, which are usually used for DBS of the STN and GPi in PD patients (Ferraye et al., 2010; Plaha and Gill, 2005). Experimental studies also found a superior effect of low frequency stimulation compared to high frequency stimulation in nonhuman primates (Jenkinson et al., 2004). While excitotoxic lesions
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Fig. 4. (A) Low-power Nissl-stained coronar section of the CG showing the area of the dorsolateral, lateral and ventral part of the CG used for counting the number of c-Fos immunoreactive neurons. (B) Number of c-Fos immunoreactive neurons in the dorsolateral central gray (CG; B1), the lateral CG (B2), and the ventral CG (C3) of sham-lesioned and lesioned rats after 0 Hz, 25 Hz and 130 Hz stimulation. Data are presented as means + S.E.M. Significant differences to 0 Hz stimulation are marked by asterisks (*), significant differences between 25 Hz and 130 Hz by circles (8; ANOVA with post hoc Tukey test p < 0.05). (C) Representative photomicrographs of c-Fos immunoreactive neurons in the dorsolateral CG after 0 Hz (C1), 25 Hz (C2), and 130 Hz (C3). Calibration bar: (A) 1000 mm, (C1–3) 100 mm.
of the PPN in normal primates cause akinesia, low frequency (2.5– 10 Hz) stimulation or pharmacological disinhibition of the PPN improve motor behaviour in parkinsonian MPTP-lesioned primates (Jenkinson et al., 2004; Nandi et al., 2002). It has been suggested that part of this beneficial effect of STN and GPi
manipulation is achieved by reducing the descending GABA inhibitory influence from these structures to the PPN region (Aziz et al., 1998). Further studies have shown that local injection of GABA antagonist in the PPN reverses akinesia (Aravamuthan et al., 2008; Jenkinson et al., 2004; Nandi et al., 2002). It is assumed that
Table 2 Number of c-Fos immunoreactive neurons contralateral to stimulation in different subregions of the central gray. Brain region
Central gray Dorsolateral Medial Ventral
0 Hz
25 Hz
130 Hz
Sham
Lesion
Sham
Lesion
Sham
Lesion
0.36 0.2 0.31 0.2 0.19 0.2
0.26 0.1 0.86 0.5 6.17 1.6
9.00 3.2*8 5.74 2.0 10.4 3.0
3.62 1.9*8 3.10 1.0 10.9 4.2
1.33 0.6 3.74 3.1 6.98 3.7
1.36 0.4 3.57 1.5 8.33 3.6
Data are presented as means + S.E.M. Significant differences to 0 Hz stimulation are marked by asterisks (*), significant differences between 25 Hz and 130 Hz by circles (8; ANOVA with post hoc Tukey test p < 0.05).
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the modulation of low frequency DBS might disinhibit neurons in the PPN and improve freezing and postural instability. Notably, the c-Fos expression that we found at the stimulation site after DBS does not represent an unspecific response related to tissue damage associated with electrode insertion. After sham stimulation only sporadic induction of c-Fos was observed, which was confined to single cells located along and directly adjacent to the electrode tract. Although the PPTg has strong reciprocal connections to the basal ganglia, in the present study c-Fos expression in the basal ganglia was negligible, both after 25 Hz and 130 Hz stimulation of the PPTg. Expression of c-Fos in the STN was inconsistent and in the entopeduncular nucleus (EPN, i.e., the rat equivalent to the human GPi) no expression was detected. This was unexpected with regard to the connectivity of the PPTg as outlined above. The PPTg receives GABAergic afferents from the SN pars reticulata and the EPN, and excitatory inputs from the STN. In turn it sends ascending excitatory efferents towards the STN and EPN (Matsumura et al., 2000; Steininger et al., 1992). Additionally, our stimulation site was located in the anterior part of the PPTg, where especially strong connections to the SN pars compacta have been demonstrated (Oakman et al., 1995). Physiological studies have also shown that PPTg manipulation affects BG regions. In the rat, the PPTg and the STN are reciprocally connected by excitatory projections. In line with this, in the 6-OHDA rat model both the STN and the PPTg were found to be hyperactive in the 6-OHDA rat model (Breit et al., 2001, 2006). Additionally, lesioning of the PPTg reversed the increased firing rate of STN neurons in the 6-OHDA PD rat model (Breit et al., 2006) and lesions of the anterior PPTg induced locomotor hyperactivity (Alderson et al., 2008). Another study showed that in rats, electrical stimulation of the PPTg increases the firing rates of neurons in the EPN (Scarnati et al., 1988). However, there are limitations of using c-Fos as a marker of neuronal activity. It cannot be used to mark cells with a net inhibitory synaptic or transcriptional drive (Kovacs, 1998). Additionally, the threshold for c-Fos induction throughout the brain is not constant (Dragunov and Faull, 1989). It should also been noted that recent studies support complex modulation of neuronal networks by DBS, such as excitation and inhibition at the stimulation site as well as in projection sites, leading to changes in network synchrony and alterations in oscillatory behaviour of neuronal networks (Hashimoto et al., 2003; Lozano et al., 2002; Vitek, 2002). Similar to lesioning the PPTg, DBS might have reduced activity in the STN (Breit et al., 2006). Also, rather than increasing or decreasing the firing rate, the effects of DBS have been more and more related to its modulatory effect on firing pattern (Hashimoto et al., 2003; Lozano et al., 2002), which might not be represented by enhanced or diminished c-Fos expression. Furthermore, the effects on PPN stimulation on freezing and postural instability have been linked to effects outside the basal ganglia circuitry, in particular when they are refractory to levodopa (Ferraye et al., 2010). In this context it should be noted, that we did not analyze c-Fos expression in brain sites below the PPTg in the brainstem. Enhanced expression was detected in different subregions of the CG in our study. In the dorsolateral CG this effect was strongest after 25 Hz stimulation, but 130 Hz stimulation still had a stronger effect compared to sham-stimulation. In the contralateral hemisphere this effect was similar, but to a lesser extent. In rats, electrical or chemical stimulation in the dorsolateral CG has been shown to enhance locomotor activity, followed by freezing, which is used as a rat model for panic disorders in humans (Bandler et al., 1985, 2000; Brandao et al., 1994; Carrive, 1993). Similarly, in human patients electrical stimulation of the dorsal periaquaeductal gray, which is the equivalent to the dorsomedial CG of the rat, induced an acute and intense stress reaction with autonomous
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responses, similar to that seen during a panic attack (Schenberg et al., 2001). Additionally, after lactate-challenge, which is used to induce panic symptoms in clinical studies, a PET study showed enhanced activity in the dorsal periaquaeductal gray (Boshuisen et al., 2002; Graeff, 1994, 2004; Schenberg et al., 2001). The PPTg has local connections that cross hemispheres (Semba and Fibiger, 1992), and manipulation of this brain region have been shown to affect several nuclei in both hemispheres (Ainge et al., 2004; Gerfen et al., 1982), which could explain the enhanced c-Fos expression in the CG of the contralateral hemisphere. This is interesting, since the question of unilateral versus bilateral stimulation has not been answered yet (Ballanger et al., 2009). Notably, while in non-human primates the PPN has been related to motor function, in rodents mostly non-motor behaviour has been investigated after local manipulation of the PPTg. In rats, the function of the PPTg has been associated with sensorimotor gating, execution of externally cued reward, or place preference context and reinforcement (Dellu et al., 1991; Inglis and Winn, 1995; Koch et al., 1993; Olmstead and Franklin, 1994). Local injection of the excitotoxin ibotenic acid in the PPTg produced deficient sensorimotor gating, increased anxiety and disturbed working memory (Koch et al., 1993; Steiniger and Kretschmer, 2004). Another study has observed a reduction in anxiety-like behaviour after bilateral electrolytic lesion in the PPTg (HomsOrmo et al., 2003). Disturbed motor function, such as akinesia or freezing and postural instability, i.e., deficits related to PD, has not been found after lesioning of the PPTg in rodents, whereas in monkeys or in humans degeneration or lesion of the PPN may cause akinesia or postural disturbance (Aziz, 1997; Kojima et al., 1997; Matsumura and Kojima, 2001; Munro-Davies et al., 1999). In this context it should be noted that to our knowledge, motor function related to freezing and postural control has not been investigated after PPTg manipulation in rats. In a recent study we found an effect of PPTg DBS on motor function in the 6-OHDA rat model (Rauch et al., 2010). In this study, both 25 Hz and 130 Hz stimulation increased locomotor activity and initiation of movement, which was disturbed after 6-OHDA lesion of the nigrostriatal pathway, while motor function of sham-lesioned rats was not affected. Whether the basal ganglia or other brain regions mediated these effects was not investigated in this study. With respect to the present finding, that electrical or chemical stimulation in the dorsolateral CG enhance locomotor activity, which is used as a rat model for panic disorders in humans (Bandler et al., 1985, 2000; Brandao et al., 1994; Carrive, 1993), the enhanced locomotor activity after stimulation of the PPTg may be at least in part related to its effects on neuronal activity in the CG. At the stimulation site and in the CG no difference in c-Fos expression was found between rats with 6-OHDA induced lesions or sham lesions. However, while human PD pathology is characterized by parallel destruction of nigro-striatal dopaminergic neurons and cholinergic neurons in the PPN (Hirsch et al., 1987; Jellinger, 1988; Rinne et al., 2008; Zweig et al., 1989), the 6-OHDA rat model only shows the pathology of DA-ergic destruction in the BG, but no destruction of the PPTg, which may account for the finding that PPTg stimulation had similar effects in sham-lesioned and lesioned rats. On the other hand, the effects on motor function seen after PPTg stimulation were only found in 6-OHDA lesioned rats, but not in sham-lesioned controls (Rauch et al., 2010). The advantage of using anaesthetized rats is that our results are not confounded by changes in rat behaviour induced by the stimulation, e.g., increased or decreased motor activity. With that regard it should be noted that in the present study we used a suprathreshold stimulation of 400 mA, while PPTg DBS induces side effects in freely moving rats already at about 130 mA (Rauch et al., 2010). Notably, Schulte et al. (2006) found induction of c-Fos in
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several basal ganglia regions by high-frequency stimulation of the subthalamic nucleus in anaesthetized rats by using a current intensity of 400 Hz, while the threshold for STN DBS in freely moving rats was similar to that of the PPN in our group. One should be aware that Temel et al. (2004) reported on histological damage after stimulus amplitude of 300 mA due to high current to the target with bipolar high frequency stimulation. However, in this study a concentric electrode with a tip diameter of 200 mm was used, which may have resulted in more unfortunate current-intensity in the STN. The basic function of neuronal control for locomotion and posture has been preserved during evolution, however, the mechanisms of gait in humans obviously is different as compared to quadrupedal animals like rats or cats. There is some evidence that the role of the basal ganglia in locomotor behaviour differs in higher primates and non-primate mammals (Dybdal and Gale, 2000; Murer and Pazo, 1993). Although the anatomical location and morphological structure of the PPN are similar in most mammals, the circuitry distribution of cholinergic, glutamatergic or GABA-ergic neurons in this region and the degree of afferent and efferent fibers vary, which could account for species dependent outcome of behaviour in experimental settings (Alam et al., 2011). Differences may also be related to normal versus parkinsonian states. Experimental studies in the MPTP primate model of PD have found neuronal hypoactivity in the PPN induced by basal ganglia dysfunction (Nandi et al., 2008), whereas rodent studies suggest either excitation of the PPTg (Breit et al., 2001, 2005; Jeon et al., 2003), inhibition of the PPTg (Florio et al., 2007; Gomez-Gallego et al., 2007), or no change in PPTg neuronal activity following unilateral dopamine depletion (Heise and Mitrofanis, 2006). These discrepancies between primate and rodent studies may be due to different functional connectivity and concomitant physiological changes. In rats, the PPTg receives strong input from the SN (Edley and Graybiel, 1983; Steininger et al., 1992), whereas in the monkey, the PPN receives its most extensive afferents from the medial part of the globus pallidus (DeVito et al., 1980; Nandi et al., 2002). One possibility for the effects observed in the study is that fiber pathways of structures nearby the electrodes that project to and receive projections from the CG might have been influenced by stimulation. Additionally, it is important to note, that the effect of DBS might not be mediated by the PPN proper but also by neighbouring structures such as the cuneiforme nucleus (CnF) and sub-CnF nucleus (Alam et al., 2011). 5. Conclusion Our findings show that stimulation of the PPTg did not affect cFos expression in the basal ganglia, but had a strong impact on the CG should elicit interest on possible interferences with other systems than the motor systems in therapeutic DBS. Acknowledgement We wish to thank the Republik Kasachstan for a personal scholarship for Assel Saryyeva References Alam, M., Schwabe, K., Krauss, J.K., 2011. The pedunculopontine nucleus area: critical evaluation of interspecies differences relevant for its use as a target for deep brain stimulation. Brain 134, 11–23. Ainge, J.A., Jenkins, T.A., Winn, P., 2004. Induction of c-fos in specific thalamic nuclei following stimulation of the pedunculopontine tegmental nucleus. Eur. J. Neurosci. 20, 1827–1837. Alderson, H.L., Latimer, M.P., Winn, P., 2008. A functional dissociation of the anterior and posterior pedunculopontine tegmental nucleus: excitotoxic lesions have
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c-Fos expression after deep brain stimulation of the pedunculopontine tegmental nucleus in the rat 6-hydroxydopamine Parkinson model Assel Saryyeva, Makoto Nakamura, Joachim K. Krauss, Kerstin Schwabe * Department of Neurosurgery, Medical University, MHH, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 June 2011 Received in revised form 3 August 2011 Accepted 3 August 2011 Available online 10 August 2011
Deep brain stimulation (DBS) is used to alleviate motor dysfunction in Parkinson’s disease (PD). The pedunculopontine nucleus (PPN) may be a potential target for severe freezing and postural instability with 25 Hz stimulation being considered more effective than 130 Hz stimulation. Here we evaluated the expression of c-Fos after 25 Hz and 130 Hz DBS of the pedunculopontine tegmental nucleus (PPTg, i.e., the rodent equivalent to the human PPN) in the rat 6-hydroxydopamine (6-OHDA) PD model. Anaesthetized male Sprague Dawley rats with unilateral 6-OHDA-induced nigrostriatal lesions were stimulated with 25 Hz, 130 Hz, or 0 Hz sham-stimulation for 4 h by electrodes implanted into the ipsilateral PPTg. Thereafter the distribution and number of neurons expressing the immediate early gene c-Fos, a marker for acute neuronal activity, was assessed. DBS of the PPTg induced strong ipsilateral c-Fos expression at the stimulation site, with 25 Hz having a more marked impact than 130 Hz. Additionally, c-Fos was strongly expressed in the central gray. In the dorsal part expression was stronger after 25 Hz stimulation, while in the medial and ventral part there was no difference between 25 Hz and 130 Hz stimulation. Expression in the basal ganglia was negligible. In the rat 6-OHDA PD model stimulation of the PPTg did not affect c-Fos expression in the basal ganglia, but had a strong impact on other functional circuitries. PPN stimulation in humans might therefore also have an impact on other systems than the motor system. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Basal ganglia Motor activity Central gray
1. Introduction Deep brain stimulation (DBS) of the pedunculopontine nucleus (PPN) is used to treat severe freezing and postural instability, which are refractory to medical treatment in late stage Parkinson’s disease (PD; Alam et al., 2011; Ferraye et al., 2010; Moro et al., 2010; Plaha and Gill, 2005; Stefani et al., 2007). These symptoms may not respond well to dopamine medication or DBS of the subthalamic nucleus (STN) or globus pallidus internus (GPi), i.e., basal ganglia nuclei, which are established targets for the treatment of parkinsonian symptoms in PD (Benabid et al., 2009; Loher et al., 2002; Rodriguez-Oroz et al., 2005; Stefani et al., 2007). The PPN is an important area within the pontomesencephalic locomotor region in the mesopontine tegmentum. It receives direct input from the cerebral cortex, and is reciprocally connected
Abbreviations: ANOVA, analysis of variance; AP, anterior–posterior; BSA, bovine serum albumin; CG, central gray; DAB, diaminobenzidin; DBS, deep brain stimulation; EPN, entopeduncular nucleus; GPi, globus pallidus internus; L, lateral; NGS, normal goat serum; PBS, phosphate buffered saline; PD, Parkinson’s disease; PPN, pedunculopontine nucleus; PPTg, pedunculopontine tegmental nucleus; SN, substantia nigra; STN, subthalamic nucleus; TH, tyrosine hydroxylase; V, ventral. * Corresponding author. Tel.: +49 511 532 2862; fax: +49 511 532 5864. E-mail address: [email protected] (K. Schwabe). 0891-0618/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2011.08.003
mainly with basal ganglia regions, but also some limbic areas, and it also projects to the thalamus (Matsumura et al., 2000; Steininger et al., 1992). It is also further directly connected to several motor nuclei in the brainstem and spinal cord (Garcia-Rill et al., 2001; Takakusaki et al., 2004, 2008). Post mortem studies of patients with advanced PD showed neuronal degeneration in the PPN that correlated with the severity of pre-mortal gait dysfunction (Hirsch et al., 1987; Jellinger, 1988; Lee et al., 2000; Pahapill and Lozano, 2000). While DBS of the STN or GPi is thought to affect the pathological neuronal activity and abnormally patterned oscillations in the basal ganglia caused by the loss of dopamine in the nigrostriatal system (Brown, 2006; Delong and Wichmann, 2007; Hashimoto et al., 2003; Trottenberg et al., 2007), the circuit related consequences of PPN DBS are not well known. Moreover, there is evidence that PPN DBS with 25 Hz is more effective than with 130 Hz, the frequency, which is usually applied in DBS of the STN or GPi (Plaha and Gill, 2005; Ferraye et al., 2010). While the acute effects of DBS are presumably due to immediate electrophysiological alterations, molecular processes are thought to reflect long-term effects. These molecular changes usually start with the expression of immediate early genes. Of these, c-Fos is a marker for acute neuronal and synaptic activity and has been used to study the effects of DBS at cellular levels (Herdegen and Leah, 1998; Schulte et al., 2006).
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We were interested, which brain regions would be activated by stimulation of the pedunculopontine tegmental nucleus (PPTg, i.e., the rat equivalent to the human PPN), whether there would be differences between 25 Hz and 130 Hz stimulation, and whether there would be differences in the normal compared to the pathophysiological state in a PD model. To address these questions, we quantified the number of c-Fos immunoreactive neurons after 0 Hz (sham-stimulation), 25 Hz and after 130 Hz PPTg DBS in 6hydroxydopamine (6-OHDA) lesioned and sham-lesioned rats, a model that has been successfully used in PD research for many years (Deumens et al., 2002). 2. Experimental procedure 2.1. Animals Adult male Sprague-Dawley rats (n = 47; purchased from Charles River Laboratories, Germany, weighing 200 g upon arrival) housed at the Central Animal Facility of the Medical University of Hannover were used for this study. The rats were kept under artificial conditions of light (14 h/10 h light cycle, light on at 7 a.m.), temperature (22 2 8C) and humidity (55 5%). Animals were kept in groups of 2 in standard Macrolon Type III cages (Techniplast, Hohenpeissenberg, Germany). They received tap water and rat chow (altromin, Altromin GmbH & Co. KG, Lage, Germany) ad libitum. Although PD is a bilateral disease, all rats were unilaterally injected with 6-OHDA or vehicle into the medial forebrain bundle for lesion or sham-lesion since we aimed to induce nearly complete dopamine lesions in the Substantia nigra pars compacta (SNc), which, when induced bilaterally, leads to high incidence of death and sever discomfort in rodents (reviewed e.g., in Deumens et al., 2002). Four weeks later an electrode was stereotactically implanted in the PPTg for stimulation or shamstimulation under general anesthesia. All experiments were carried out in accordance with the European Council Directive of 24 November 1986 (86/609/ EEC). 2.2. Stereotactic surgery For 6-OHDA lesion of the SN the rats were intraperitoneally anaesthetized with chloralhydrate (360 mg/kg; Sigma–Aldrich Chemie GmbH, Steinheim, Germany) and mounted in a stereotactic frame. Additionally, the area for surgery was infiltrated with local anesthesia (Xylocain 2%). For microinjection, 6-OHDA (Sigma Chemical Co.) was dissolved in 0.9% saline solution with 0.02% ascorbic acid added. A dose of 3.6 mg/1 ml was used for injection with a microsyringe (5 ml, Hamilton Company, Nevada, USA) at a rate of 1 ml/min. For injection, a burr hole was drilled above the injection site and the tip of the syringe lowered into the target. Rats were injected into the right hemisphere at two targets with coordinates in mm relative to bregma according to the brain atlas of Paxinos and Watson (1998). Three microliters were injected in the medial forebrain bundle at two sites with the following stereotactic coordinates (1) anterior–posterior (AP): 4.0; lateral (L): 0.8; ventral (V): 7.5 from dura level; incisor bar at +3.4, and (2) AP: 4.4; L: 1.2; V: 7.8 from dura level; incisor bar at 2.4. Sham-lesioned rats (vehicle-injection only) served as controls. After injection the cannula was left in place for 3 min before slowly retracting it. Finally, the skin incision was sutured. 2.3. Electrode implantation and deep brain stimulation Four weeks after injection of 6-OHDA stereotactic surgery was carried out for implantation of bipolar electrodes in the PPTg. Again, rats were intraperitoneally anaesthetized with chloral hydrate (360 mg/kg, followed by additional injection with 120 mg/kg each hour) and placed into a stereotactic frame. Additionally, the area for surgery was infiltrated with local anesthesia (Xylocain 2%). The electrodes were made of two parallel insulated platin-iridium electrode wires (90:10), coated with Teflon (un-coated diameter 76 mm, coated diameter 140 mm, A-M Systems, Inc., Carlsborg, USA). The Teflon coated part of the wires was covered in a 15 mm long cylinder (0.45 mm 0.25 mm, Sterican1 Braun Melsungen AG, Germany) and uncovered at their extremities at a length of 500 mm. The distance between the tips of the two uncoated wires was 500 mm for bipolar stimulation. The other end of the wires was welded to a socket. This type of electrodes has been suggested to be especially suitable for stimulation of the rodent STN (Gubellini et al., 2009) and has been reported not to produce tissue damage after stimulation for several days (Gubellini et al., 2006). Electrodes were inserted into the right PPTg at the following coordinates in mm relative to bregma: AP: 7.8; L: 1.8: V: 7.3 from the cranium surface; tooth bar at 3.3. DBS was applied continuously for 4 h on anesthetized rats and delivered by a stimulator (Multichannel systems, Reutlingen, Germany). For electrical stimulation rectangular biphasic pulses were used with duration of 160 ms (change of polarization after 80 ms). The frequency was set at 130 Hz, 25 Hz or 0 Hz sham-stimulation, and the intensity at 400 mA over the stimulation period for all of the stimulated animals.
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2.4. Histological processing After sham-stimulation, respectively immediately after 4 h 130 Hz or 25 Hz stimulation, rats were deeply anesthetized with an overdose of chloralhydrate (720 mg/kg) and transcardially perfused with phosphate buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4). The brains were removed from the skulls and cryoprotected in 30% sucrose for at least 48 h. Each brain was cut in six series of 40 mm coronal sections on a freezing sliding microtome. For histological processing, one series was used for Nissl-staining, one for tyrosine hydroxylase (TH) immunohistochemistry, and one for c-Fos immunohistochemistry. The sections containing the PPTg were Nissl stained with Thionine to check for correct placement of the electrodes. The sections containing the striatum and SN were processed free floating for immunohistochemical staining for TH. All chemicals were purchased from Sigma–Aldrich GmbH, Steinheim, Germany, except as stated otherwise. First the slices were incubated for 10 min in solution with 1 ml methanol, 1 ml 30% H2O2 and 8 ml 0.1 M PBS, followed by a 3-step washing procedure with PBS with each step lasting 5 min. Subsequently, the slices were incubated in the primary antibody solution (1:5000) with IgG mouse anti tyrosine hydroxylase in 1% bovine serum albumin (BSA) in a PBS/Triton X-100 solution over night, followed by a 3-step washing procedure with PBS. Next, the slices were incubated in the secondary antibody solution (1:200) with biotinylated IgG rabbit anti mouse (DakoCytomation, Glostrup, Denmark) in 1% BSA/PBS/Triton X-100 for 60 min, followed by the washing procedure. Thereafter, the slices were incubated for 60 min by using the ABC-Standard-Kit (1:1000; 1 ml A (AvidinH) + 1 ml B (Biotinyl-Peroxidase) in 1 ml PBS (ABC-Kit, Vector Laboratories, Inc., Burlingame, USA), which was again followed by the washing procedure. Finally, a solution of 3,30 -diaminobenzidin (DAB, DakoCytomation, Glostrup, Denmark), used as the chromogen, in a PBS-ammoniumnickelsulfate solution and H2O2 (20 ml 30% H2O2 solved in 580 ml PBS) was used to stain the slices. To stop the staining reaction, the slices were extensively washed with PBS, mounted on a glass slide and coverslipped with Vitro Clud (Langenbrinck, Emmendingen, Germany). The sections were then examined under a light microscope to determine the loss of nigral dopaminergic neurons and the loss of TH-staining in the striatum. To detect c-Fos immunoreactivity one series of sections was incubated for 1 h in a solution of 10% normal goat serum (NGS), 30% H2O2 and 20% Triton to remove endogenous peroxidase activity, and then incubated in primary antibody against cFos (1:10,000; Calbiochem, Germany) in PBS with 0.02% Triton and 10% NGS overnight at room temperature. After rinsing four times in PBS (1 3 min, 1 5 min, 2 15 min), sections were incubated in PBS with 1% bovine serum albumin for 1 h at room temperature. Subsequently, the sections were incubated in biotinylated goat anti-rabbit secondary antibody (1:2000; DakoCytomation, Glostrup, Denmark) in PBS with 1% BSA and Triton for 1 h, and then washed four times in PBS. Visualization of c-Fos immunoreactivity was as described above, i.e., usage of the ABC-Kit followed by an incubation of the series in DAB, and finally dehydrating the slices in ethanol and embedding the slices in Vitro Clud. For analysis of c-Fos expression all subcortical brain regions were qualitatively scanned for c-Fos immunoreactivity after 25 Hz and 130 Hz compared to 0 Hz (sham) stimulation. We then counted the number of c-Fos immunoreactive cells at the stimulation site and in those areas that showed enhanced expression of the groups stimulated with 25 Hz and 130 Hz. The sections used for quantitative analyses were chosen on the basis of the amount of staining and because they could be easily and reproducibly picked up on certain landmarks near the site of expression. For histological analysis a Zeiss Axio Imager Z1m microscope (Go¨ttingen, Germany) equipped with the digital camera SpotTM Persuit and the image analysis software MetaMorph 4.6 (both from Visitron Systems GmbH, Puchheim, Germany) was used. For evaluation of c-Fos immunoreactive neurons in the PPTg the central part of the video frame was placed at the deepest position of the electrode tract and the cells counted in this slice, as well as in the previous and subsequent slice. For evaluation of c-Fos immunoreactivity in the other regions, the coordinates of the area of strongest expression were determined with respect to bregma. Thereafter, all stained neurons were determined ipsilateral to the stimulation by counting all c-Fos immunoreactive neurons in one frame of the video camera at 200 times magnification emerging into focus through the 40 mm sections. Additionally, the cells in the three previous and three subsequent sections (distance between sections 240 mm) were counted, thus covering in total a distance of about 0.8 mm. c-Fos immunoreactive cells in the corresponding contralateral region in the left hemisphere were likewise counted. The mean number of the cells counted in the different slices was used for statistical evaluation. The data were evaluated by a two-way analysis of variance (ANOVA) with lesion (sham-lesion, 6-OHDA lesion) and stimulation intensity (0 Hz, 25 Hz, and 130 Hz) as factors. For post hoc pair wise comparison Tukey’s test was used. All tests were performed two-sided and a p-value of 0.05 was considered to represent a significant effect.
3. Results Only rats with a nearly total loss of tyrosine hydroxylase immunoreactivity in the SNc (i.e., less than 15 TH-positive neurons in the ipsilateral SNc, see Fig. 1) and confined correct placement of
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the electrode in the PPTg were used for statistical analyses. Additionally, rats that did not show c-Fos immunoreactivity in regions with usually high background expression of c-Fos (i.e., the motor cortex or the piriform cortex) were excluded. Thus, 36 of 47 rats were used for final evaluation: 0 Hz stimulation (lesion: n = 6, sham-lesion: n = 6), 25 Hz stimulation (lesion: n = 6, sham-lesion: n = 6), and 130 Hz stimulation (lesion: n = 6, sham-lesion: n = 6). A reconstruction of the electrode placement in the PPTg is shown in Fig. 2A, together with representative histological pictures of the stimulation site for each stimulation group shown in Fig. 2B. In these Nissl-stained sections there was only minor tissue damage around the stimulated site. In only one rat of either group a small number of c-Fos immunoreactive cells were found in regions of the basal ganglia, i.e., in the substantia nigra (SN), the STN, and the zona incerta, but in almost all stimulated rats c-Fos immunoreactivity was shown in the central gray (CG; see Table 1). Besides the stimulation site in the PPTg, different subregions of the CG were therefore used for quantitative analysis. 3.1. PPTg
Fig. 1. Histological pictures of coronal brain slices after immunohistological processing for tyrosine hydroxylase staining of the substantia nigra pars compacta after vehicle-injection (A) and 6-hydroxydopamine-injection; (B) calibration bar: 500 mm.
At the stimulation site the expression of c-Fos was strongest after 25 Hz stimulation, followed by 130 Hz stimulation, but with no difference between 6-OHDA lesioned and sham-lesioned rats (Fig. 3). ANOVA showed an effect for the factor stimulation (F2,35 = 18.692, p < 0.001), a trend for significance for the factor lesion (F1,35 = 3.341, p = 0.078), but no interaction between these factors (F2,35 = 0.334, p = 0.719). Post hoc comparison revealed more expression in rats after 25 Hz stimulation compared to 0 Hz and 130 Hz stimulation (p < 0.001 and p = 0.008, respectively). Additionally, expression after 130 Hz stimulation was stronger compared to 0 Hz sham-stimulation (p = 0.020). See Fig. 3B for
Fig. 2. (A) Schematic drawings (adapted to the atlas of Paxinos and Watson, 1998) of the implantation sites of the electrode tips. Sections on the left site represent shamlesioned (open symbols) and on the right site 6-hydroxydopamine lesioned rats (filled symbols) with 0 Hz stimulation (*,*), 25 Hz stimulation (&/&), and 130 Hz stimulation (~/~) in distance to bregma. (B) Histological photomicrographs showing the electrode tip in the PPTg after 0 Hz stimulation (B1), 25 Hz stimulation (B2), and 130 Hz stimulation (B3; black arrow) with the corresponding electrode tip at higher magnification in B4–6. The tract dorsal to the electrode tip shows tissue damage caused by the cylinder that covered the electrode wires in stimulated and sham-stimulated rats (white arrow). Calibration bar: (B1–3) 500 mm, (B3–6) 100 mm.
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Table 1 Expression of c-Fos in subcortical brain regions of 6-hydroxydopamine-lesioned or sham-lesioned rats after stimulation with 0 Hz, 25 Hz, and 130 Hz. Brain region
PPTg Central gray Substantia nigra Nucleus subthalamicus Zona incerta Hippocampus
0 Hz
25 Hz
130 Hz
Sham
Lesion
Sham
Lesion
Sham
Lesion
3/6 1/6 0/6 0/6 0/6 0/6
4/6 3/6 0/6 0/6 0/6 0/6
6/6 5/6 0/6 0/6 1/6 1/6
6/6 6/6 1/6 1/6 0/6 1/6
6/6 6/6 0/6 0/6 0/6 0/6
6/6 6/6 1/6 1/6 0/6 1/6
Shown is the number of animals expressing c-Fos in relation to the number of stimulated rats.
representative histological pictures of c-Fos expression in the PPTg for all stimulated groups. 3.2. Central gray Ipsilateral to the stimulation site the strongest expression of cFos was found at about 6.8 mm in relation to bregma. This slice was therefore used as the central slice for counting c-Fos immunoreactive neurons in the different subregions (Fig. 4A). cFos expression ipsilateral to the stimulation site was strongest in the dorsolateral CG, followed by the lateral and ventral CG. The expression in the dorsal CG was strongest after 25 Hz stimulation compared to 0 Hz and 130 Hz stimulation, which was similar ipsilateral and contralateral to the stimulation site (Fig. 4B1). No difference was found between 6-OHDA lesioned and shamlesioned rats. Ipsilateral to the lesion ANOVA showed an effect for the factor stimulation (F2,35 = 7.237, p = 0.003), but no effect for the factor lesion (F1,35 = 0.609, p = 0.441) or the interaction between these factors (F2,35 = 0.480, p = 0.624). Post hoc comparison revealed more expression in rats after 25 Hz stimulation compared to 0 Hz and 130 Hz stimulation (p = 0.003 and p = 0.026, respectively), while the effect after 130 Hz stimulation was not different to 0 Hz stimulation (p = 0.624). Fig. 4C shows representative histological pictures of the different stimulation groups. In the dorsal CG contralateral to the lesion ANOVA showed an effect for the factor stimulation (F2,35 = 8.732, p = 0.001) with post hoc testing showing significant more expression after 25 Hz compared to 0 Hz and 130 Hz stimulation (both p < 0.05; Table 2). The factor lesion and the interaction between factors (F1,35 = 2.103, p = 0.157 and F2,35 = 2.022, p = 0.150, respectively) were not significant. For the lateral and ventral CG ipsilateral to the lesion ANOVA also revealed an effect for the factor stimulation (F2,35 = 5.588, p = 0.009 and F2,35 = 3.966, p = 0.030, respectively) with post hoc testing showing more expression in rats after 25 Hz stimulation compared to 0 Hz sham-stimulation (p = 0.006 and p = 0.023; Fig. 4). The factor lesion and the interaction between factors were not significant (all F-values >0.651, all p-values <0.426). In the lateral and ventral CG contralateral to the lesion statistical analysis with ANOVA was only marginally significant for the factor stimulation (F2,35 = 2.866, p = 0.073 and F2,35 = 3.052, p = 0.062, respectively), with no effect for the factor lesion or interaction between factors (all F-values >1.223, all p-values <0.278; Table 2).
4. Discussion Deep brain stimulation of the PPTg induced strong c-Fos expression at the stimulation site and in the CG with 25 Hz stimulation being more effective than 130 Hz stimulation, while expression in the basal ganglia or thalamus was negligible. No
Fig. 3. (A) Number of c-Fos immunoreactive neurons at the stimulation site in the pedunculopontine tegmental nucleus (PPTg) of sham-lesioned and lesioned rats after 0 Hz, 25 Hz and 130 Hz stimulation. Data are presented as means + S.E.M. Significant differences to 0 Hz stimulation are marked by asterisks (*), significant differences between 25 Hz and 130 Hz by circles (8; ANOVA with post hoc Tukey test p < 0.05). (B) Representative photomicrographs of c-Fos immunoreactive neurons after 0 Hz (B1), 25 Hz (B2), and 130 Hz (B3). Calibration bar: (B1–3) 100 mm.
differences were found between 6-OHDA lesioned and shamlesioned rats. Clinical studies have shown that PPN DBS with 25 Hz stimulation appears to be more effective with respect to freezing than frequencies >60 Hz, which are usually used for DBS of the STN and GPi in PD patients (Ferraye et al., 2010; Plaha and Gill, 2005). Experimental studies also found a superior effect of low frequency stimulation compared to high frequency stimulation in nonhuman primates (Jenkinson et al., 2004). While excitotoxic lesions
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Fig. 4. (A) Low-power Nissl-stained coronar section of the CG showing the area of the dorsolateral, lateral and ventral part of the CG used for counting the number of c-Fos immunoreactive neurons. (B) Number of c-Fos immunoreactive neurons in the dorsolateral central gray (CG; B1), the lateral CG (B2), and the ventral CG (C3) of sham-lesioned and lesioned rats after 0 Hz, 25 Hz and 130 Hz stimulation. Data are presented as means + S.E.M. Significant differences to 0 Hz stimulation are marked by asterisks (*), significant differences between 25 Hz and 130 Hz by circles (8; ANOVA with post hoc Tukey test p < 0.05). (C) Representative photomicrographs of c-Fos immunoreactive neurons in the dorsolateral CG after 0 Hz (C1), 25 Hz (C2), and 130 Hz (C3). Calibration bar: (A) 1000 mm, (C1–3) 100 mm.
of the PPN in normal primates cause akinesia, low frequency (2.5– 10 Hz) stimulation or pharmacological disinhibition of the PPN improve motor behaviour in parkinsonian MPTP-lesioned primates (Jenkinson et al., 2004; Nandi et al., 2002). It has been suggested that part of this beneficial effect of STN and GPi
manipulation is achieved by reducing the descending GABA inhibitory influence from these structures to the PPN region (Aziz et al., 1998). Further studies have shown that local injection of GABA antagonist in the PPN reverses akinesia (Aravamuthan et al., 2008; Jenkinson et al., 2004; Nandi et al., 2002). It is assumed that
Table 2 Number of c-Fos immunoreactive neurons contralateral to stimulation in different subregions of the central gray. Brain region
Central gray Dorsolateral Medial Ventral
0 Hz
25 Hz
130 Hz
Sham
Lesion
Sham
Lesion
Sham
Lesion
0.36 0.2 0.31 0.2 0.19 0.2
0.26 0.1 0.86 0.5 6.17 1.6
9.00 3.2*8 5.74 2.0 10.4 3.0
3.62 1.9*8 3.10 1.0 10.9 4.2
1.33 0.6 3.74 3.1 6.98 3.7
1.36 0.4 3.57 1.5 8.33 3.6
Data are presented as means + S.E.M. Significant differences to 0 Hz stimulation are marked by asterisks (*), significant differences between 25 Hz and 130 Hz by circles (8; ANOVA with post hoc Tukey test p < 0.05).
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the modulation of low frequency DBS might disinhibit neurons in the PPN and improve freezing and postural instability. Notably, the c-Fos expression that we found at the stimulation site after DBS does not represent an unspecific response related to tissue damage associated with electrode insertion. After sham stimulation only sporadic induction of c-Fos was observed, which was confined to single cells located along and directly adjacent to the electrode tract. Although the PPTg has strong reciprocal connections to the basal ganglia, in the present study c-Fos expression in the basal ganglia was negligible, both after 25 Hz and 130 Hz stimulation of the PPTg. Expression of c-Fos in the STN was inconsistent and in the entopeduncular nucleus (EPN, i.e., the rat equivalent to the human GPi) no expression was detected. This was unexpected with regard to the connectivity of the PPTg as outlined above. The PPTg receives GABAergic afferents from the SN pars reticulata and the EPN, and excitatory inputs from the STN. In turn it sends ascending excitatory efferents towards the STN and EPN (Matsumura et al., 2000; Steininger et al., 1992). Additionally, our stimulation site was located in the anterior part of the PPTg, where especially strong connections to the SN pars compacta have been demonstrated (Oakman et al., 1995). Physiological studies have also shown that PPTg manipulation affects BG regions. In the rat, the PPTg and the STN are reciprocally connected by excitatory projections. In line with this, in the 6-OHDA rat model both the STN and the PPTg were found to be hyperactive in the 6-OHDA rat model (Breit et al., 2001, 2006). Additionally, lesioning of the PPTg reversed the increased firing rate of STN neurons in the 6-OHDA PD rat model (Breit et al., 2006) and lesions of the anterior PPTg induced locomotor hyperactivity (Alderson et al., 2008). Another study showed that in rats, electrical stimulation of the PPTg increases the firing rates of neurons in the EPN (Scarnati et al., 1988). However, there are limitations of using c-Fos as a marker of neuronal activity. It cannot be used to mark cells with a net inhibitory synaptic or transcriptional drive (Kovacs, 1998). Additionally, the threshold for c-Fos induction throughout the brain is not constant (Dragunov and Faull, 1989). It should also been noted that recent studies support complex modulation of neuronal networks by DBS, such as excitation and inhibition at the stimulation site as well as in projection sites, leading to changes in network synchrony and alterations in oscillatory behaviour of neuronal networks (Hashimoto et al., 2003; Lozano et al., 2002; Vitek, 2002). Similar to lesioning the PPTg, DBS might have reduced activity in the STN (Breit et al., 2006). Also, rather than increasing or decreasing the firing rate, the effects of DBS have been more and more related to its modulatory effect on firing pattern (Hashimoto et al., 2003; Lozano et al., 2002), which might not be represented by enhanced or diminished c-Fos expression. Furthermore, the effects on PPN stimulation on freezing and postural instability have been linked to effects outside the basal ganglia circuitry, in particular when they are refractory to levodopa (Ferraye et al., 2010). In this context it should be noted, that we did not analyze c-Fos expression in brain sites below the PPTg in the brainstem. Enhanced expression was detected in different subregions of the CG in our study. In the dorsolateral CG this effect was strongest after 25 Hz stimulation, but 130 Hz stimulation still had a stronger effect compared to sham-stimulation. In the contralateral hemisphere this effect was similar, but to a lesser extent. In rats, electrical or chemical stimulation in the dorsolateral CG has been shown to enhance locomotor activity, followed by freezing, which is used as a rat model for panic disorders in humans (Bandler et al., 1985, 2000; Brandao et al., 1994; Carrive, 1993). Similarly, in human patients electrical stimulation of the dorsal periaquaeductal gray, which is the equivalent to the dorsomedial CG of the rat, induced an acute and intense stress reaction with autonomous
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responses, similar to that seen during a panic attack (Schenberg et al., 2001). Additionally, after lactate-challenge, which is used to induce panic symptoms in clinical studies, a PET study showed enhanced activity in the dorsal periaquaeductal gray (Boshuisen et al., 2002; Graeff, 1994, 2004; Schenberg et al., 2001). The PPTg has local connections that cross hemispheres (Semba and Fibiger, 1992), and manipulation of this brain region have been shown to affect several nuclei in both hemispheres (Ainge et al., 2004; Gerfen et al., 1982), which could explain the enhanced c-Fos expression in the CG of the contralateral hemisphere. This is interesting, since the question of unilateral versus bilateral stimulation has not been answered yet (Ballanger et al., 2009). Notably, while in non-human primates the PPN has been related to motor function, in rodents mostly non-motor behaviour has been investigated after local manipulation of the PPTg. In rats, the function of the PPTg has been associated with sensorimotor gating, execution of externally cued reward, or place preference context and reinforcement (Dellu et al., 1991; Inglis and Winn, 1995; Koch et al., 1993; Olmstead and Franklin, 1994). Local injection of the excitotoxin ibotenic acid in the PPTg produced deficient sensorimotor gating, increased anxiety and disturbed working memory (Koch et al., 1993; Steiniger and Kretschmer, 2004). Another study has observed a reduction in anxiety-like behaviour after bilateral electrolytic lesion in the PPTg (HomsOrmo et al., 2003). Disturbed motor function, such as akinesia or freezing and postural instability, i.e., deficits related to PD, has not been found after lesioning of the PPTg in rodents, whereas in monkeys or in humans degeneration or lesion of the PPN may cause akinesia or postural disturbance (Aziz, 1997; Kojima et al., 1997; Matsumura and Kojima, 2001; Munro-Davies et al., 1999). In this context it should be noted that to our knowledge, motor function related to freezing and postural control has not been investigated after PPTg manipulation in rats. In a recent study we found an effect of PPTg DBS on motor function in the 6-OHDA rat model (Rauch et al., 2010). In this study, both 25 Hz and 130 Hz stimulation increased locomotor activity and initiation of movement, which was disturbed after 6-OHDA lesion of the nigrostriatal pathway, while motor function of sham-lesioned rats was not affected. Whether the basal ganglia or other brain regions mediated these effects was not investigated in this study. With respect to the present finding, that electrical or chemical stimulation in the dorsolateral CG enhance locomotor activity, which is used as a rat model for panic disorders in humans (Bandler et al., 1985, 2000; Brandao et al., 1994; Carrive, 1993), the enhanced locomotor activity after stimulation of the PPTg may be at least in part related to its effects on neuronal activity in the CG. At the stimulation site and in the CG no difference in c-Fos expression was found between rats with 6-OHDA induced lesions or sham lesions. However, while human PD pathology is characterized by parallel destruction of nigro-striatal dopaminergic neurons and cholinergic neurons in the PPN (Hirsch et al., 1987; Jellinger, 1988; Rinne et al., 2008; Zweig et al., 1989), the 6-OHDA rat model only shows the pathology of DA-ergic destruction in the BG, but no destruction of the PPTg, which may account for the finding that PPTg stimulation had similar effects in sham-lesioned and lesioned rats. On the other hand, the effects on motor function seen after PPTg stimulation were only found in 6-OHDA lesioned rats, but not in sham-lesioned controls (Rauch et al., 2010). The advantage of using anaesthetized rats is that our results are not confounded by changes in rat behaviour induced by the stimulation, e.g., increased or decreased motor activity. With that regard it should be noted that in the present study we used a suprathreshold stimulation of 400 mA, while PPTg DBS induces side effects in freely moving rats already at about 130 mA (Rauch et al., 2010). Notably, Schulte et al. (2006) found induction of c-Fos in
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several basal ganglia regions by high-frequency stimulation of the subthalamic nucleus in anaesthetized rats by using a current intensity of 400 Hz, while the threshold for STN DBS in freely moving rats was similar to that of the PPN in our group. One should be aware that Temel et al. (2004) reported on histological damage after stimulus amplitude of 300 mA due to high current to the target with bipolar high frequency stimulation. However, in this study a concentric electrode with a tip diameter of 200 mm was used, which may have resulted in more unfortunate current-intensity in the STN. The basic function of neuronal control for locomotion and posture has been preserved during evolution, however, the mechanisms of gait in humans obviously is different as compared to quadrupedal animals like rats or cats. There is some evidence that the role of the basal ganglia in locomotor behaviour differs in higher primates and non-primate mammals (Dybdal and Gale, 2000; Murer and Pazo, 1993). Although the anatomical location and morphological structure of the PPN are similar in most mammals, the circuitry distribution of cholinergic, glutamatergic or GABA-ergic neurons in this region and the degree of afferent and efferent fibers vary, which could account for species dependent outcome of behaviour in experimental settings (Alam et al., 2011). Differences may also be related to normal versus parkinsonian states. Experimental studies in the MPTP primate model of PD have found neuronal hypoactivity in the PPN induced by basal ganglia dysfunction (Nandi et al., 2008), whereas rodent studies suggest either excitation of the PPTg (Breit et al., 2001, 2005; Jeon et al., 2003), inhibition of the PPTg (Florio et al., 2007; Gomez-Gallego et al., 2007), or no change in PPTg neuronal activity following unilateral dopamine depletion (Heise and Mitrofanis, 2006). These discrepancies between primate and rodent studies may be due to different functional connectivity and concomitant physiological changes. In rats, the PPTg receives strong input from the SN (Edley and Graybiel, 1983; Steininger et al., 1992), whereas in the monkey, the PPN receives its most extensive afferents from the medial part of the globus pallidus (DeVito et al., 1980; Nandi et al., 2002). One possibility for the effects observed in the study is that fiber pathways of structures nearby the electrodes that project to and receive projections from the CG might have been influenced by stimulation. Additionally, it is important to note, that the effect of DBS might not be mediated by the PPN proper but also by neighbouring structures such as the cuneiforme nucleus (CnF) and sub-CnF nucleus (Alam et al., 2011). 5. Conclusion Our findings show that stimulation of the PPTg did not affect cFos expression in the basal ganglia, but had a strong impact on the CG should elicit interest on possible interferences with other systems than the motor systems in therapeutic DBS. Acknowledgement We wish to thank the Republik Kasachstan for a personal scholarship for Assel Saryyeva References Alam, M., Schwabe, K., Krauss, J.K., 2011. The pedunculopontine nucleus area: critical evaluation of interspecies differences relevant for its use as a target for deep brain stimulation. Brain 134, 11–23. Ainge, J.A., Jenkins, T.A., Winn, P., 2004. Induction of c-fos in specific thalamic nuclei following stimulation of the pedunculopontine tegmental nucleus. Eur. J. Neurosci. 20, 1827–1837. Alderson, H.L., Latimer, M.P., Winn, P., 2008. A functional dissociation of the anterior and posterior pedunculopontine tegmental nucleus: excitotoxic lesions have
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