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min every day for 28 days (Meng et al., 2011). In that study, the rat treated by that repeatedly exposed to a set of chronic mild stressors for 4 consecutive weeks was then subjected under a animal model of depression. In addition, the LHb stimulation was conducted in a long-term fashion before behavioral testing. Thus, the difference between these two studies and in comparing the present study, in terms of the effects of LHb stimulation on locomotion, could be attributed to different experimental
protocols used among these studies. A question may be raised in concerning the negative results of LHb stimulation on locomotion in this study. That is, it might be due to the subjects were insensitive to any experimental treatment that is related to brain DA. Accordingly, the same rats were tested in the second part of Experiment 1, which results clearly showed those subjects were significantly affected by
amphetamine. The acute injection of amphetamine increased the locomotor activity is a well established model to test the general motor function modulated by brain DA (e.g. Cole, 1978). Combining the results from the first and second parts of
Experiment 1, it is indicated that the subjects could be sensitively affected by drug treatment agonizing brain DA systems to increase locomotion. Thus, the negative results of LHb stimulation applied in this experiment implied that the intensities of LHb stimulation applied in this study would not affect the general motor function.
However, whether the DA level was altered by the LHb stimulation or not has not been examined in this study, which is essential before making a conclusive remark.
DRL 15-s behavior affected by LHb stimulation
Experiment 2 showed that the LHb stimulation significantly affected DRL 15-s behavior in a frequency-dependent manner. Furthermore, these behavioral changes by LHb stimulation are similar to those induced amphetamine on DRL behavior with interval set in a range of 10 to 20 sec (Liao, 2009). A previous study of this lab
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showed that amphetamine increased the total responses, non-reinforced responses and burst responses, but decreased the reinforced responses and peak time (Cheng and Liao, 2007). With the similarity of DRL 15-s behavioral effects produced by the LHb stimulation and amphetamine treatment, there might be a common mechanism was shared for these two treatments. In terms of neural substrates, with
amphetamine pharmacologically acting as a DA agonist, it was then inferred that the behavioral alterations induced by LHb stimulation was modulated by DA related mechanisms. This was a rationale to carry out Experiment 3 and Experiment 4 (see the relevant discussion below).
The unilateral, but not bilateral, LHb stimulation was applied in the present and induced significant behavioral changes on DRL 15-s behavior. A question may be asked: how did the unilateral LHb stimulation on one side of the brain adequately affect the Hb and then change the behavior? A recent study addressed a strong connectivity between the Hb of left and right hemispheres causes a reliable influence each other via its connecting commissure in terms of anatomy (Kim, 2009). A few recent studies demonstrated significant behavioral effects also induced by the
application of unilateral stimulation (Friedman et al., 2010, 2011; Li et al., 2011; Meng et al., 2011), which may support the aforementioned anatomical argument. So far, most (if not all) studies conducting with LHb stimulation may be due to a technical consideration. That is, the distance between the left and right hemispheres of habenula was about 1.4 mm. This distance was not allowed to make the bilateral implantation of two electrodes in the Hb. Even by the manipulation of unilateral stimulation in the LHb, behavioral changes on DRL 15-s behavior were significantly and reliably observed throughout this study.
The results of DRL 15–s behavioral alterations produced by the present LHb stimulation are worthy to discuss. According to a series of studies done by Hikosaka
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and his associates (2008), low current stimulation in the LHb has been argued to induce an inhibitory effect in DA neurons. Consistent with this hypothesis, Friedman and associates (2011) showed that LHb stimulation attenuated the positive
reward-associated reinforcement as measured by the self-administration of sucrose solution. Also, the DA release was reduced in the subject given that the LHb was activated by electrical stimulation (Lecourtier et al., 2008). These results were not compatible with what Experiment 2 found in this study, given that DA is positively correlated with reward-related processes of behavior or neurochemistry. The difference might be due to stimulation intensity parameters set and electrode used across these studies. The results from a pilot test conducted along with Experiment 1 showed a seizure-like effect was appeared in rats receiving LHb stimulation with the current given over 0.2 mA. Accordingly, the current used for present LHb stimulation was set less than 0.2 mA, where 0.2 mA (or higher amplitude) was applied to the other studies (Friedman et al., 2011; Li et al., 2011). Furthermore, Friedman and associates (2011) used two stainless steel electrodes and 1 mm between cathode and anode. In this study, the stainless steel wire was the same, but the distance between cathode and anode was almost close together. Thus, the electrical
stimulation of the sphere of influence may also contribute to lead a different result in terms of the neuronal effectiveness. It is then possible that the results of Experiment 2 were derived from the LHb neurons suppressed by the present electrical stimulation.
If so, it might cause the DA neurons to be activated and then induced the amphetamine-like behavior on DRL 15-s behavior. Two possibilities can be addressed to elaborate the aforementioned results. First, LHb stimulation may directly activate the soma of dopaminergic neurons located in the VTA and enhance DA release in the striatal areas. Li and associates (2011) administered a retrograde tracer (Alexa Fluor 488) in VTA to target the afferent inputs sent from the LHb.
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Glutamatergic, rather than GABAergic, neurons were further characterized as the LHb efferents projecting to the VTA. Thus, LHb stimulation may directly activate the mesolimbic DA system. Second, if the present LHb stimulation still provokes the LHb neuronal activity, the efferent projection from LHb may have a double-synapse
connection of inhibitory interneuron (e.g. GABA) before connecting to DA neurons.
For instance, LHb efferents connecting to GABAergic neurons located in the
rostromedial tegmentum (RMTg) were demonstrated by Hong and associates (2011), which may synapse to some ultra short GABAergic (inter)neurons within the VTA.
Given this neuroanatomical presumption, the present LHb stimulation could produce a disinhibition on DA neuron that leads to a DA agonism effect. Further study
conducted in a more systemic examination manner is needed for testify these possibilities.
The LHb has been demonstrated as one of brain sites that can be applied by the intracranial self-stimulation (ICSS; Vachon and Miliaressis, 1992). Accordingly, the LHb stimulation can generally produce a rewarding effect. It is, then, likely that the DRL behavior changes produced by the present LHb stimulation are similar to those affected by amphetamine or stimulant drugs. Systemic injection of stimulant drugs is known to affect behavior via drug action with pharmacological property of rewarding.
Does the LHb directly involve in the cores of DRL behavior? One of the key components required for individual to perform on DRL behavior is the timing capability (Skinner, 1938). A previous study showed that the LHb neurons encoded the
multiple time scales of memory (Bromberg-Martin et al., 2010a). It means that the LHb neurons could encode behavioral outcomes of 6 trials prior to the testing trial in the primate subject. In that study, each trial (including its prior inter-trial interval) took approximately 8 sec to complete. Accordingly, in case the LHb neuronal firings to predict a reward and make an operant response, such a behavior may be depended
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on the memory of episodes that occurred in the second-scaled timing. Thus, the LHb might be involved in the function of timing throughout those 6 trials described above. Therefore, the LHb stimulation might affect the timing process where any behavioral performance relies.
Despite an accumulating data in supporting an inhibitory role of the LHb over the midbrain DA neurons (Christoph et al., 1986; Matsumoto and Hikosaka, 2007; Ji and Shepard, 2007; Reisine et al., 1982), some controversial results were reported for DA related behaviors as measured when the LHb had been manipulated by either lesion or stimulation. Wang and associates (2009) reported a negative result of electrolytic lesion on heroin self-administration. Using ICSS model, the reward effectiveness of the brain stimulation in the lateral hypothalamus, VTA, or dorsal raphe nucleus was attenuated by electrolytic stimulation (Morissette and Boye. 2008). More recently, Gifuni and associates (2012) showed that LHb lesion by ibotenate enhanced the locomotion response to amphetamine, but did not alter the reward-potentiating effect of amphetamine on ICSS in the medial forebrain bundle (MFB) or the posterior mesencephalon. All these data indicate the involvement of LHb in modulating the DA related behavior may be more complex at behavioral or system level than it was thought on the basis of in vitro tests. Further, how the LHb affect the DA related behavior may be task dependent. More work is needed before a conclusion can be made for this issue.
DA antagonists reversed the effects of LHb stimulation on DRL 15-s behavior Behavioral alterations induced by LHb stimulation on DRL 15-s were reliably seen in different experiments conducted in this study. Even though it is still not clear about the inhibitory role of LHb stimulation can be play on DA system, one way to help delineating this issue could be approached by pharmacological antagonism tests.
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Thus, with the assumption that behavioral alterations induced by LHb stimulation on DRL 15-s were DA dependent, it is then expected to observe that DA antagonist blocks those behavioral changes by LHb stimulation. These data collected from Experiment 3 indicated that the LHb stimulation effects on DRL 15-s behavior were reversed by DA antagonists. These data first replicated the result of Experiment 2 showing the DRL behavioral changes by LHb stimulation. Further, the SCH23390 and eticlopride treatment administered with the LHb stimulation produced behavioral outcomes returning to the control level. Such an effect indicates the DA antagonist could reverse the behavioral effects produced by LHb stimulation.
Could the results in Experiment 3 be influenced by drug alone? A previous study of this lab showed that SCH23390 in 0.02 mg/kg decreased the total responses, non-reinforced responses, burst responses and peak rate on DRL 10-s behavior (Liao and Cheng, 2007). Compared with Experiment 3, SCH23390 in 0.01 mg/kg was no effect on DRL 15-s behavior. Therefore, the lower dose of DA antagonists could just reverse the effects of LHb stimulation but not affected the DRL behavior.
What is the relationship between the LHb and DA system? The previous studies showed that LHb efferent fibers projected to the VTA and SN. And, in return, the LHb receives afferent fibers from the VTA (Lecourtier et al., 2007, Geisler et al., 2008).
This circuit has been argued to be involved in reward-related behavior
(Bromberg-Martin et al., 2010a). Taking the results of Experiment 3 into account by following this argument, the DA circuit may participate in modulating the effects of LHb stimulation on DRL 15-s behavior. If the LHb stimulation affect behavioral
performance was in along with increasing the level of DA release, the DA antagonists blocking the DA receptors could attenuate the DA-related changes. This inference is confirmed by the effects of LHb stimulation were reversed by DA antagonists as reported here.
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Mixed results of NE antagonists reversing behavioral changes by LHb stimulation on DRL 15-s behavior
These data collected from Experiment 4 indicated that the LHb stimulation effects on DRL 15-s behavior were reversed by propranolol, but not by prozosin or yohimbine.
The LHb stimulation may not rely on the NE system as much as the DA system to affect DRL 15-s behavior. What is the relationship between the LHb and NE system?
The previous study indicated that LHb stimulation in depression model enhanced level of DA, NE and 5-HT in the brain tissue and the blood serum (Meng et al., 2011).
However, the DRL behavior was more like to be as a kind of DA-related behavior (Liao, 2009). It is still possible that the NE level might be increased in the brain tissue and the blood serum by LHb stimulation, but the pharmacological reversal effects couldn’t be detected when these effects were measured by the DRL behavior paradigm. Further tests are needed to verify this issue.
The results of Experiment 4 should be cautiously interpreted if taking the side effects induced by NE antagonists into consideration. Previous studies showed that yohimbine and propranolol, injected via peripheral route, significantly induced the side effect of lowering blood pressure (Okamoto et al., 2012, Richardson et al., 1968).
Thus, the reversal effect of propranolol observed in Experiment 4 might be a result of side effect by this drug. For this reason, the intracerebroventricular (i.c.v.) injection is suggested to overcome this potential confounding effect induced by propranolol given by i.p. injection.
Less degree of influence on DRL 72-s behavior by LHb stimulation
In comparing with DRL 15-s behavior, Experiment 5 tested the effect of LHb stimulation on DRL 72-s behavior. The degree of behavioral changes by LHb
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stimulation on DRL 72-s behavior was less than that observed on DRL 15-s behavior.
Compare with DRL 15-s behavior, the waiting time for the subject to obtain reinforcer was increased to 72 s in DRL 72-s behavioral task. Even though the session time was increased to 60 min (in contrast to 15 min for DRL 15-s task), the response rate in DRL 72-s behavior was lower than that of DRL 15-s behavior. This may be a reason for observing the less effect by LHb stimulation on DRL 72-s behavior, namely the behavioral response was not sensitive. Could the effect of LHb stimulation on DRL 72-s behavior be magnified? Following the idea from a study using the animal model of depression to test LHb stimulation delivered in chronic (Meng et al., 2011), the less degree of LHb stimulation effect as tested on DRL 72-s behavioral task in Experiment 5 may be due to the acute (but not chronic) delivery of stimulation conducted in this study. If the duration time of stimulation would increase to 30 min and conducted over more daily sessions, it might a more significant change effect on DRL 72-s behavior.
The LHb stimulation for 15 min was always conducted right before the behavioral measure. It is, then, more accurate to describe what the present study investigated was engaged in examining the post-stimulation effects of DRL behavior and other behavioral tests. The session time of behavioral test after LHb stimulation might influence the test outcomes (e.g. DRL 15-s vs. DRL 72-s) as described above.
Similarly, a few recent studies demonstrated significant behavioral effects as tested by the after effect of stimulation (Friedman et al., 2010, 2011; Li et al., 2011; Meng et al., 2011). Notice that most of the in vitro studies applied the LHb stimulation almost (if not all) at the same period of time when the neurophysiological or neurochemical test was conducted, which experimental protocols were different from what described above for the behavioral measures.
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No effect of LHb stimulation on discriminability
To verify whether the LHb stimulation would change the ability of discriminating, Experiment 6 was tested on discrimination task. These data of Experiment 6 indicate that the choice of large reward, the omission rate and response latency altered by high frequency of LHb stimulation. But, from statistic tests of simple main effect, it is indicated that the significant change was only seen in the first block out of the four-block consisting test session on the measures of omission rate and response latency. The data of this part was surprising. The previous study showed that flupenthixol decreased choice of the large reward during the last two blocks (St. Onge et al., 2010). Compared with Experiment 6, the effects of LHb stimulation was only in the first block and the choice rate of large reward was 83.6% in high frequency
stimulation. If the LHb stimulation would affect the discrimination task, the effects must be apparent more than just affecting in one block. In the second to the forth block, the three behavior measurements were back to the level of the control
treatment. It is generally believed that the present LHb stimulation did not impair the discrimination ability in the rat.
The application of LHb stimulation on the clinical
One of the clinical applications of electrical stimulation is used to treat the
symptoms of depression. The electrical stimulation of the subgenual cingulate white matter successfully treated depression symptoms in six patients for the first time (Mayberg et al., 2005). Following this study, the electrical stimulation in the treatment of depression has been attended. For example, the treatment of LHb stimulation successfully treated depression symptoms in human patients (Sartorius et al., 2007). Compatible with the animal model, Li and associates (2011)
demonstrated that the LHb stimulation reduced the firing of LHb neurons, and acutely
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reversed helpless behavior in rats. Therefore, with more studies from basic research in tackling the neurobiological mechanisms of LHb stimulation, it will be more
promising to use LHb stimulation as for a clinical treatment of depression and the other psychiatric disorders may as well.
The roles of LHb on different stages of behavior processing
As mentioned above, this study examined the post (LHb) stimulation effects on certain behavioral tests. In this study, the LHb stimulation was always conduced in the subject who performed reliably in a specific behavioral task. Since different neural mechanisms are involved in distinctive stage of behavior from acquisition to expression. It is interested to learn if any behavior effect would be changed by the LHb stimulation given to the subject during the acquisition stage as comparing to the performance of a learned behavior. For instance, to conditioning the subject with a contextual box by brain stimulation, the subjects were stimulated in LHb during the acquisition on a place conditioning task (Friedman et al., 2011), which results showed LHb stimulation produced a conditioned place aversion. Furthermore, in a case of memory processing being involved, the strategy of manipulating the LHb stimulation immediate after the behavioral session or given at the end of learning trial(s). It is possible that the LHb stimulation given right after the behavioral session may change the memory consolidation processing of a specific behavioral task. In order to study the any brain site involved in memory consolidation processing, the striatal stimulation was given for 4 hours right after a behavioral learning session and tested the effect of consolidation in next day (Schumacher et al., 2011). Shumake and associates (2010) reported LHb stimulation given right after the gerbil made a correct avoidance
response significantly impaired the acquisition of an avoidance learning, whereas VTA stimulation could enhance it. To understand the functions of the LHb during the
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different stages of behavior, it is necessary to proceed LHb stimulation conducted with the behavioral task at different stages or timing points.
Conclusion and suggestion for future work
The present study applied the LHb stimulation to test DRL behavior. Significant behavioral alterations by LHb stimulation were revealed in DRL 15-s task, where a less degree of LHb stimulation effect was observed on DRL 72-s task. The former behavioral changes were similar to those induced an amphetamine on DRL behavior.
And, DRL behavioral alterations induced by LHb stimulation were pharmacologically reversed by DA antagonists. These findings of DA dependent behavior change under LHb stimulation were independent to locomotor activity or the discrimination ability changed by the LHb stimulation.
And, DRL behavioral alterations induced by LHb stimulation were pharmacologically reversed by DA antagonists. These findings of DA dependent behavior change under LHb stimulation were independent to locomotor activity or the discrimination ability changed by the LHb stimulation.