Down regulation of the HCN2 channel in rat models of Parkinson s disease Abstract The basal ganglia is a key control centre involved with the regulation of movement. It is therefore a primary interest in research on motor dysfunction diseases such as Parkinson s disease, with much focus directed towards a particular region, the subthalamic nucleus (STN). In diseased states these neurones fire bursts of action potentials which results in the loss of somatotopy between the other nuclei and hence motor dysfunction. The mechanism behind the high frequency bursts was investigated in the STN and a channel known as the HCN (hyperpolarisation activated cyclic nucleotide gated channel) was thought to be involved (1). Physiological data has shown there to be a reduction of the HCN current in Parkinson s disease models (unpublished). The aim of this study was to investigate to see if the HCN channel becomes down regulated in diseased states. A unilateral model of Parkinson s disease was induced in six Sprague Dawley rats by injecting 6 hydroxy-dopamine into the medial forebrain bundle on the right hemisphere, the left hemisphere being left as an internal control. After three weeks the rat brain tissue was fixed by PFA. Immunohistochemistry was applied to the sagitally sliced brain against HCN2 (one of the main subunits of this channel in the STN (unpublished result)). The mean fluorescence intensity was obtained and results were expressed as a relative percentage of the LHS. The results displayed a statistically significant reduction in the mean expression level of the HCN2 channel after dopamine depletion (p<0.01, t test, n=6, mean LHS=100 RHS=77, SD LHS=0 RHS=12.5). The down regulation of this channel in diseased states provides a mechanism for how the burst firing results and therefore could possibly provide a potential route for a treatment of this disease in the future. Introduction Parkinson s disease is characterised by the selective degeneration of the dopaminergic neurones of the substantia nigra pars compacta (SNc) which lies within the midbrain. These neurones make connections with the STN and other nuclei and act to modulate their activity under normal circumstances. In Parkinson s disease therefore, it is the loss of this control which ultimately result in the characteristic symptoms: bradykinesia, rigidity and resting tremor. The STN and GPe fire single spike action potentials in an oscillatory manner when independent of synaptic input [1,2]. These nuclei in vivo are reciprocally connected and they display high somatotopy which is related to movement [3]. In Parkinson s disease, the action potentials become high frequency bursts of activity with a frequency of either 4-10Hz (associated with resting tremor) or 15-30Hz [1]. The burst activity becomes correlated between and within the nuclei and somatotopy is lost, corresponding with motor dysfunction [1]. Although it is a number of basal ganglia nuclei that display this pathological burst firing activity, the STN is of particular interest. One of the reasons for this is that the STN, when a diseased patient is treated with high frequency stimulation improved motor function results [4]. Secondly, there are reciprocal connections between the GPe and the STN which are GABAergic and glutamatergic respectively, the nuclei are therefore capable of maintaining a continuous loop of activity between them [3]. Finally, the cortex
connects to the basal ganglia directly by both the striatum and the STN. It displays slow wave activity which correlates with the low frequency oscillations seen in the STN, yet interestingly these are not present in the striatum [5]. This study focussed on the hyperpolarisation-activated and cyclic nucleotide-gated (HCN) cation channel, which is activated at hyperpolarised membrane potentials. In the basal ganglia the channel is found within the STN, GP, striatum and the SN [6]. A preliminary investigation has shown that the predominant subunit in the HCN channels of the STN to be HCN2. Physiological data has already shown there to be a reduction of the HCN current in Parkinson s disease models (unpublished). This could be due to either a reduced activity or reduced expression of the HCN channel and could be involved in the pathological bursting activity. The latter was investigated in this study by using immunohistochemistry in which the HCN2 subunit was labelled, recording the change in fluorescence intensity between the STN of the two hemispheres of a rat. The left a control side and on the right a model of Parkinson s disease, induced after creating a lesion in the substantia nigra through the injection of 6-hydroxy-dopamine to deplete its store of dopamine. Immunohistochemistry was also carried out against tyrosine hydroxylase (TH) in order to quantify the extent of the lesion and to confirm a reliable model of Parkinson s was induced. Materials and Methods Experimental procedures were carried out on adult Sprague-Dawley rats and were conducted according to the Animals (Scientific Procedures) Act, 1986 (UK). Unilateral lesion of SN dopaminergic neurones Six rats (300g, 7weeks of age) were subject to unilateral lesions in their right cerebral hemisphere. Rats were anesthetised with ketamine (0.1ml/100g) 20mins before the injection which was then maintained throughout. They were also treated with the myod inhibitor xylazine (0.05ml/100g) and the analgesic buprenorphine (0.05ml/100g). To maximize the toxic effects of the 6-OHDA on the dopaminergic neurones, the rats received a dose of pargyline (50mg/kg) and to minimize the effects on other catecholaminergic neurones desipramine (25mg/kg) was also administered. After ensuring the rats were fully anesthetised via a light corneal touch or a tail pinch the rats were placed in a stereotaxic frame. Here a small craniotomy was made directly above the substantia nigra (SN) and the dura mater removed. 2.5ul of a 5mg/ml solution of 6-OHDA was injected into the medial forebrain bundle (adjacent to the SN). This was carried out by a steel cannula attached to a 10ul Hamilton microsyringe, over a period of 5mins and then left for additional 5mins to avoid reflux. (Stereotaxic coordinates: AP= -2.8mm; L=-2.0; Dth=-8.4 from Paxinos and Watson (1996) atlas). Immunohistochemistry Three weeks after the lesion was created, rats were again deeply anesthetised as above before perfusing transcardially with ~200ml of phosphate buffered saline (0.01M PBS ph 7.4). The tissue was then fixed with 250ml of paraformaldehyde (4%) and incubated for 20mins previous to perfusing a second time with ~200ml of 0.1M phosphate buffer (PB; ph 7.4) for ~15mins. The brain was then removed and immersed in 0.01M PBS solution. The brain was sliced in the sagital plane on a vibratome (Leice VT1000s) at a thickness of 70um.
Relative flourescence intensity as a percentage of the control (%)) Slices were washed with 0.01M PBS and then incubated with PBS containing 2% Normal goat serum and 0.5% triton for 1hour at room temperature whilst on a mixer. The tissue was then incubated with the primary antibody against HCN2 (raised in rabbit) or tyrosine hydroxylase (raised in mouse) at a concentration of 1 in 2000 or 1 in 5000 respectively for 60hours. Subsequently the secondary antibody (goat anti rabbit IgG-Alexa Fluor 594 Invitrogen for HCN2 and goat anti-mouse IgG-FITC for TH), which was applied in a concentration of 1 in 500 and 1 in 250 for HCN and TH respectively, was left to incubate for 2hours at room temperature. This was also repeated with a higher concentration of both the primary and secondary antibodies at 1:1000 and 1:100 respectively for the HCN2. Finally slices were mounted on glass slides in PBS and antifade. The HCN2 labelling in the STN was imaged using a fixed stage inverted microscope (Olympus, FV1000), a x60, 1.4 NA oil-immersion lens (Olympus) at an excitation wavelength of 563nm. The TH labelling was visualised using Olympus BX61 Optigrid fluorescence microscope with a 10x lens (exposure time=50ms, gain=34 and offset=6). Results The HCN channel is down regulated after dopamine depletion in a rat model of Parkinson s disease. In each of the six animals the HCN channel was labelled using imunohistochemistry against the HCN2 subunit. The fluorescence intensity was recorded in both hemispheres and a decrease was seen in the RHS showing the HCN channel was down regulated (see figure 1) and this reduction was statistically significant (p<0.01, t test n=6). Figure 1. The mean HCN2 fluorescence intensity decreased after dopamine depletion in the STN. Each individual colour denotes a different animal. The relative fluorescence intensity is expressed as a percentage of the control LHS. 120 100 80 60 40 20 Mean HCN2 flourescence intensity in the STN 0 LHS RHS Electron microscopic images of the STN show the reduction in HCN2 fluorescence. The reduction in fluorescence intensity on the right hemisphere is shown in figure 2 where two similar regions of the STN from the same animal are compared. Table 1 indicates an average reduction of 33% in fluorescence intensity across all six animals.
Mean TH flourescence intensity in the striatum (shown as a ratio to cortex) Figure 2: Immunohistochemistry against the HCN2 subunit displays the down regulation of this channel in the STN after dopamine depletion. The control LHS (a) showing the HCN2 channel arranged in a circular array on the membrane of the cell bodies. The RHS (b) which has undergone dopamine depletion has a reduced overall intensity and a reduction in the number of labelled cell bodies. Both images are taken at 4um below the tissue surface. (c) and (d); average intensity overlay stacks of the left and right hand side respectively lie at 3-6um below the surface and provide a more realistic view of the tissue as a whole. (a) (b) (c) (D) Relative Fluorescence Intensity (%) LHS RHS Mean 100 77.0 SD 0 12.5 Table 1: Mean and standard deviation for the HCN2 relative fluorescence intensities in the STN (n=6). TH quantification indicates a reliable model of Parkinson s disease was induced. The mean TH fluorescence intensity decreased on the RHS by at least 66.4% (see table 2). The greatest decrease being in the first animal which decreased by a value of 83.1%. This shows a great reduction in the level of dopamine in the striatum and therefore a reliable model of Parkinson s disease was induced (see figure 3 for all animals). Figure 3: Mean TH fluorescence intensity decreased on the RHS. 6 5 4 3 2 Mean TH fluorescence intensity in the striatum LHS RHS TH mean fluorescence intensity in the striatum of the LHS and RHS of the four rats. Fluorescence is displayed relative to an internal control (the cortex) since this 1 0 19b5.1 19b5.2 19b5.3 19b5.6 Rat number
Rat number Standard deviation 0f TH % decrease in mean TH fluorescence (3sig fig) fluorescence intensity LHS RHS (%) 19b5.1 1.27 0.180 83.1 19b5.2 0.959 0.111 66.4 19b5.3 0.966 1.99 82.4 19b5.6 0.794 0.0587 80.8 decrease in TH fluorescence intensity from the LHS to the RHS. should remain the same after dopamine depletion. Table 2: Mean values for the TH fluorescence and % Discussion This study has shown that the HCN channel becomes down regulated in the STN of a Parkinson s disease model animal. This has several implications on the effects of the basal ganglia, since the STN is such a significant nucleus in this structure. Under normal circumstances the HCN channel is recruited by inhibitory GABAergic action potentials from the GPe (unpublished). It will then allow the influx of cations, raising the membrane potential acting to oppose the inhibitory input and reduce the de-inactivation of low voltage activated Ca channels. After the loss of dopaminergic modulation caused by the injection of the 6-OHDA the channel was down regulated. With fewer functional channels the membrane potential of the STN neurones would theoretically be more hyperpolarised. The GABAergic input would consequently have a stronger effect and the likelihood of the pathological rebound bursting activity would be increased. Further investigation needs to be carried out in order to see if it is the down regulation of these channels that is the cause of this rebound activity and ultimately the motor symptoms observed in diseased states. This idea could be explored by using transgenic mice lacking the HCN channel and observing to see if any of the Parkinson s like symptoms appear. If so, attempting to insert a functional HCN channel back into the membranes of diseased patients channel could act as a potential treatment for the disease. A further possibility is that the down regulation of the HCN channel is in fact an adaptive response as a result of the loss of dopamine. Parkinson s disease in simple terms is caused by the loss of dopaminergic modulation from the substantia nigra which results in disinhibition of the STN. This ultimately leads to increased inhibitory output from the basal ganglia and reduced motor function (1). As stated earlier, the consequence of the down regulation of the HCN channel increases the effects of the GABAergic inhibitory input. This could be seen to be acting in an adaptive way by trying to reduce the effects of the disinhibition. References 1. Bevan, M.D. et al. (2002) Move to the rhythm: oscillations in the subthalamic nucleusexternal globus pallidus network. TRENDS in Neurosciences. 25, 525-531 2. Bevan, M.D. and Wilson, C.J. (1999) Mechanisms underlying spontaneous oscillation and rhythmic firing in rat subthalamic neurones. J. Neuroscience 19, 7617-7628 3. Jérôme Baufreton et al. (2009) Sparse but selective and potent synaptic transmission from the globus pallidus to the subthalamic nucleus. J. Neurophysiology 102, 532-545 4. Wassilios Meissner et al. (2005) Subthalamic high frequency stimulation resets subthalamic firing and reduces abnormal oscillations. Brain 128, 2372-2382 5. P. J Magill, et al. (2001) Dopamine regulates the impact of the cerebral cortex on the subthalamic nucleus-globus pallidus network. Neuroscience 106, 313-330
6. Santoro et al. (2000). Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. Journal of Neuroscience, 20, 5264-5275. During my summer placement I carried out research on an ion channel which is found in the basal ganglia and is of particular relevance to Parkinson s disease. I found it to be a very fascinating topic and it really stimulated my interest in the subject. I enjoyed carrying out the reading and questioning the theory behind the research. I also feel I have improved my aptitude in reading scientific papers from this which will be very helpful to me in my final year of university. I spent most of my time working alongside a PHD student who was very helpful and welcoming to me. Initially I was shadowing for the majority of my time until the later weeks of the placement. I had the opportunity to learn how to use various microscopes and was given the responsibility of using an epifluorescence microscope independently for the quantification section of the experiment. I also used other pieces of equipment such as a vibratome, learnt how to do immunohistochemistry and observed electrophysiology. I found the placement to be a very valuable experience for me as I developed a number of my personal skills such as communication, analytical skills and my initiative, all of which will be very valuable to me in any career. I also feel have gained a more realistic view of what research is like and I now have a much greater appreciation for the amount of time and dedication that goes into obtaining the results for a paper. I am currently undecided on my career plans but research is still something that I am considering. I think that I would be better suited to a bigger laboratory however as I feel I would be happier working in an environment with more social interaction and collaboration. I therefore think I would need to visit a few other laboratories first in order to make a fully informed decision.