Hisanao Akita

Impaired acquisition of skilled behavior in rotarod task by moderate depletion of striatal dopamine in a pre-symptomatic stage model of Parkinson's disease

In view of recent findings that suggest that the nigrostriatal dopamine (DA) system plays a role in motor control and the acquisition of habits and skills, we hypothesized that the striatum-based function underlying the acquisition of skilled behaviors might be more vulnerable to dopamine depletion than the motor control. To test this hypothesis, we investigated whether impaired acquisition of skilled behaviors occurs in a pre-symptomatic stage model of Parkinsons disease (PD). By using the microdialysis method and the 6-OHDA-technique to destroy dopamine neurons, we confirmed that rats with unilateral partial lesions of the nigral dopamine cells by 6-OHDA are suitable for a pre-symptomatic stage model of Parkinsons disease. The rats in this model exhibited moderate disruption of striatal dopamine release function and relatively intact motor functions. In a rotarod test, the impaired acquisition of skilled behavior occurred in rats with bilateral partial lesions of the nigral dopamine cells by 6-OHDA. These rats displayed intact general motor functions, such as locomotor activity, adjusting steps, equilibrium function and muscle strength. Based on these results, we concluded that the striatum-based function underlying the acquisition of skilled behaviors or sensorimotor learning may be more vulnerable to dopamine depletion than the motor control.

Antisense knockdown of spinal-mGluR1 reduces the sustained phase of formalin-induced nociceptive responses

To examine the role of mGluR1 (a subunit of the group I metabotropic glutamate receptor) in the nociceptive responses of rats following a subcutaneous injection of formalin into the plantar surface of the hind paw, we delivered antisense oligonucleotides (ODNs) against mGluR1 into the rat lumbar spinal cord (L3-L5) intrathecally using an HVJ-liposome-mediated gene transfer method. Rats treated with a single injection of mGluR1 antisense ODNs into the intrathecal space of the lumbar spinal cord showed a marked reduction of the early-sustained phase of formalin-induced nociceptive responses, but not of their acute phase. The reduction of nociceptive behavioral responses became apparent at day 2 after the antisense treatment and lasted for 2 days. This corresponded to a long-lasting down-regulation (46%) of mGluR1 expression in the lumbar cord. This down-regulated mGluR1 was observed at day 2 and persisted until day 4 after the intrathecal infusion of mGluR1 antisense ODN. In contrast, rats treated with mGluR1 sense or mismatch ODNs showed none of these changes. These results suggest that mGluR1 may play a crucial role in the sustained nociception of formalin-induced behavioral responses.

Nigral injection of antisense oligonucleotides to synaptotagmin I using HVJ-liposome vectors causes disruption of dopamine release in the striatum and impaired skill learning.

To produce an animal model of a dopa-responsive motor disorder with depletion of dopamine (DA) release in the striatum by dysfunction of the transmitter release machinery of the nigrostriatal DA system, we performed an intra-nigral injection of an HVJ-liposome gene transfer vector containing antisense oligodeoxynucleotides (ODNs) against synaptotagmin I (SytI), a key regulator of Ca(2+)-dependent exocytosis and endocytosis in adult rats. A unilateral intra-nigral injection of HVJ-liposome vectors containing antisense ODNs against SytI (syt-AS) caused a moderate disruption of methamphetamine-induced release of DA in the treated side of the striatum, while the syt-AS treatment did not affect physiological release of DA in the treated striatum. A bilateral intra-nigral injection of HVJ-liposome vectors containing syt-AS induced an impairment of the striatal DA-mediated acquisition of skilled behavior in a rotarod task without any deficits in general motor functions, such as spontaneous locomotor activity, motor adjusting steps, equilibrium function, or muscle strength. These findings suggest that an intra-nigral treatment with HVJ-liposome vectors containing syt-AS may cause a long-lasting nigral knockdown of SytI which, in turn, leads to a moderate dysfunction of the DA release machinery in the terminals of the nigrostriatal DA system and a subsequent mild depletion of DA release in the striatum.

Activation of the NMDA receptor involved in the alleviating after-effect of repeated stimulation of the subthalamic nucleus on motor deficits in hemiparkinsonian rats.

To test the hypothesis that the cellular mechanism whereby chronic deep brain stimulation of the subthalamic nucleus (STN-DBS) induces the improvement of motor deficits lasting after stimulation in the hemiparkinsonian (hemi-PD) rat involves the NMDA receptor-dependent processes in neurons receiving afferents from the STN, we examined whether the NMDA receptor antagonist prevents the alleviating after-effect of repeated STN-DBS on motor deficits in hemi-PD. The cylinder test was performed before and after repeated STN-DBS over 3 days in hemi-PD that received a unilateral injection of 6-OHDA into the medial forebrain bundle 3 weeks prior to STN-DBS experiments. No significant improvement in the reduced frequency of forelimb use and forelimb-use asymmetry was seen in the cylinder test after the single STN-DBS, while, when the STN-DBS was applied three times at intervals of 24 h, the improvement became apparent and significant only in the reduced frequency of forelimb use (akinesia) after termination of the stimulation, suggesting the alleviating after-effect of chronic stimulation. Then, the effects of intraperitoneal administration of the non-competitive NMDA receptor antagonist MK-801 and the competitive NMDA receptor antagonist CPP on the alleviating after-effect of the STN-DBS were examined in cylinder tests performed before and after repeated STN-DBS for 3 days in hemi-PD. Both MK-801 (0.1 mg/kg) and CPP (0.5 mg/kg) completely prevented the improvement of the akinetic motor deficit after repeated STN-DBS. These results support the hypothesis that activation of the NMDA receptor and subsequent cellular processes in neurons receiving the afferents from the STN may involve in the mechanism underlying the alleviating after-effect of chronic STN-DBS on the akinetic motor deficit in hemi-PD.

The NR2B antagonist, ifenprodil, corrects the l-DOPA-induced deficit of bilateral movement and reduces c-Fos expression in the subthalamic nucleus of hemiparkinsonian rats

The use of NR2B antagonists in Parkinsonism is still controversial. To examine their anti-parkinsonian effects, the NR2B antagonist, ifenprodil, and L-DOPA were administered together and separately in hemiparkinsonian rats (hemi-PD) that were subjected to a cylinder test. Recovery from hypoactivity was achieved by single administration of 3-7 mg/kg of L-DOPA; however, improvement in the deficit of bilateral forelimb use was not observed. When administered alone, ifenprodil had no anti-parkinsonian effects; however, combined administration of ifenprodil and 7 mg/kg of L-DOPA significantly reversed the deficit of bilateral forelimb use without adversely affecting the L-DOPA-induced improvement in motor activity. Next, in order to identify the brain area influenced by L-DOPA and ifenprodil, quantitative analysis of L-DOPA-induced c-Fos immunoreactivity was performed in various brain areas of hemi-PD following administration of L-dopa with and without ifenprodil. Among brain areas with robust c-Fos expression within the motor loop circuit in dopamine-depleted hemispheres, co-administered ifenprodil markedly attenuated L-DOPA-induced c-Fos expression in only the subthalamic nucleus (STN), suggesting that the STN is the primary target for the anti-parkinsonian action of NR2B antagonists.

Characterization of nociceptive response to chemical, mechanical, and thermal stimuli in adolescent rats with neonatal dopamine depletion.

Rats with dopamine depletion caused by 6-hydroxydopamine (6-OHDA) treatment during adulthood and the neonatal period exhibit akinetic motor activity and spontaneous motor hyperactivity during adolescence, respectively, indicating that the behavioral effects of dopamine depletion depend on the period of lesion development. Dopamine depletion during adulthood induces hyperalgesic response to mechanical, thermal, and/or chemical stimuli, whereas the effects of neonatal dopamine depletion on nociceptive response in adolescent rats are yet to be examined. The latter aspect was addressed in this study, and behavioral responses were examined using von-Frey, tail flick, and formalin tests. The formalin test revealed that rats with neonatal dopamine depletion exhibited a significant increase in nociceptive response during interphase (6-15min post formalin injection) and phase 2 (16-75min post formalin injection). This increase in nociceptive response to the formalin injection was not reversed by pretreatment with methamphetamine, which ameliorates motor hyperactivity observed in adolescent rats with neonatal 6-OHDA treatment. The von-Frey filament and tail flick tests failed to reveal significant differences in withdrawal thresholds between neonatal 6-OHDA-treated and vehicle-treated rats. The spinal neuronal response to the formalin injection into the rat hind paw was also examined through immunohistochemical analysis of c-Fos protein. Significantly increased numbers of c-Fos-immunoreactive cells were observed in laminae I-II and V-VI of the ipsilateral spinal cord to the site of the formalin injection in rats with neonatal dopamine depletion compared with vehicle-treated rats. These results suggest that the dopaminergic neural system plays a crucial role in the development of a neural network for tonic pain, including the spinal neural circuit for nociceptive transmission, and that the mechanism underlying hyperalgesia to tonic pain is not always consistent with that of spontaneous motor hyperactivity induced by neonatal dopamine depletion.

Ascending pathways of skin and deep information projecting to the ventral posterior lateral nucleus of thalamus in the cat.

In acupuncture, the typical sensation induced by insertion of a needle into the muscle that called “De Qi” is essential to induce acupuncture analgesia. The effect of acupuncture analgesia may depend on the deep afferents rather than to the cutaneous ones at the acupuncture point. The ascending pathways conveying these skin and deep afferents project to the ventral posterior lateral nucleus (VPL) of thalamus, one of the thalamic relay nuclei projected to the cerebral cortex, were examined by spinal cord lesion at the cervical level with observation of the responses evoked by electrical stimulation applied selectively to the skin and deep tissues.Neuronal discharges were recorded extracellularly from the VPL using the multimicroelectrode technique. After spontaneous neuronal activities were recorded, the receptive fields and characteristics were determined by applying some kinds of mechanical stimulation to the receptive field. The skin and deep structures were then selectively stimulated using a pair of stainless steel needles and insulated needles except for tips. Units were classified for skin units, deep units, and skin-deep units according to the responses to the electrical stimulation. Responses were elicited in some neurons following to both skin and deep stimuli. The latency of response elicited by electrical stimulation applied to the deep structure was slightly shorter than that of the skin structure. The recording sites for deep units were located more rostrally in the dorsal region of the VPL than that for the skin units.The afferents of some skin-deep neurons from both skin and deep structures were ascended through different pathways in the spinal cord. The skin and deep afferents were found to converge at the thalamic level.