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We examined the effects of three benzofurans [1-(Benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB), 1-(Benzofuran-2-yl)-N-methylpropan-2-amine (2-MAPB), and 1-(Benzofuran-5-yl)-N-ethylpropan-2-amine (5-EAPB)] on the extracellular monoamine level in mouse corpus striatum by the microdialysis method and compared them with the effects of psychoactive 3,4-Methylenedioxymethamphetamine (MDMA). The effects of benzofurans on the extracellular monoamine level were qualitatively analogous to that of MDMA, with an increase in serotonin (5-HT) level exceeding dopamine (DA) level. The effects of 2-MAPB and 5-EAPB were almost the same as the effect of MDMA. However, 5-MAPB strongly increased extracellular monoamine level than MDMA. These differences in the potency appear to have a structure-activity relationship. The administration of 5-MAPB (1.6 × 10(-4) mol/kg B.W.) resulted in the death of two-thirds of the mice. The same dose of MDMA did not cause any deaths. The administration of 5-MAPB (1.6 × 10(-4) mol/kg B.W.) produced a 3.41°C ± 0.28°C rise in rectal temperature after 1 hr, whereas the administration of MDMA (1.6 × 10(-4) mol/kg B.W.) produced an approximate 1.85°C ± 0.26°C rise. These results suggest that benzofurans have 5-HT toxicity similar to MDMA, and 5-MAPB has a higher risk of lethal intoxication than MDMA. Furthermore, 5-APB, the metabolic product of 5-MAPB demethylation, may be involved in the acute 5-HT toxicity and may cause lethal intoxication in mice.

The neuro-behavioral observation scorebook that improved the previous observation methods of Irwin was followed, the test material was administered to 5 mice per each group, and the mean value of the obtained score was determined. The behavior of a normal animal was assumed to be point 0, animals showing suppressive behavior were scored in the minus region, and animals that showed excitement behavior were scored in the plus region. Each score was divided into three stages, according to the level of strength of the biological effect. The score of each observation item was totaled, and the level of the strength of the biological effect in the item was judged according to its mean value. These test methods of neuro-behavioral observations we proposed were able to detect the biological effects of a drug simply and promptly, and contributed sufficient data to support an administrative measure aimed at anticipating and improving the prevention of health damage in humans by non-regulated drugs from a scientific perspective. Recently, we developed a method of serial measurement of the quantity of monoamine in the mouse central nervous system by microdialysis, and performed it. The Kanagawa Prefectural Institute of Public Health conducted a study of the biological effect of non-regulated drugs. A characteristic here is what they examined about drug-dependency other than observing the behavior of the animal.

Pael receptor (Pael-R) has been identified as one of the substrates of Parkin, a ubiquitin ligase responsible for autosomal recessive juvenile Parkinsonism (AR-JP). When Parkin is inactivated, unfolded Pael-R accumulates in the endoplasmic reticulum and results in neuronal death by unfolded protein stress, suggesting that Pael-R has an important role in the pathogenesis of AR-JP. Here we report the analyses on Pael-R-deficient (KO) and Pael-R-transgenic (Tg) mice. The striatal dopamine (DA) level of Pael-R KO mice was only 60% of that in normal mice, while in Pael-R Tg mice, striatal 3,4-dihydroxyphenylacetic acid (DOPAC) as well as vesicular DA content increased. Moreover, the nigrostriatal dopaminergic neurons of Pael-R Tg mice are more vulnerable to Parkinson's disease-related neurotoxins while those of Pael-R KO mice are less. These results strongly suggest that the Pael-R signal regulates the amount of DA in the dopaminergic neurons and that excessive Pael-R expression renders dopaminergic neurons susceptible to chronic DA toxicity.

Systemic administration of a dopamine D2 receptor blocker, haloperidol, but not vehicle, significantly increased the number of c-Fos-immunoreactive neurons in the globus pallidus (GP) in rats. Dual immunohistochemistry, a combination of c-Fos immunohistochemistry and retrograde tracing experiments with cholera toxin B (ChB), revealed that a subset of the c-Fos-immunoreactive GP neurons was pallidostriatal feedback neurons. Lesioning of the subthalamic nucleus (STN) by local injection of ibotenic acid inhibited the haloperidol-induced c-Fos expression in the GP neurons, suggesting that the activation of GP neurons is a result of increased excitatory drives from the STN. Therefore, the present findings are evidence of the existence of the subthalamo-pallido-striatal axis as a feedback system in the internal circuits of the basal ganglia.

The regional distribution of c-Fos expression in the brain after the administration of two tremorgenic agents was studied. In both the harmaline- and oxotremorin-treated rats, c-Fos-positive neurons were extensively distributed in the basal ganglia nuclei and the cerebellum. Additionally, in the harmaline-treated rats, numerous c-Fos-positive neurons were also distributed throughout the inferior olivary nucleus. In the oxotremorine-treated rats, while the inferior olive was not involved, c-Fos was strongly expressed in the neurons of the reticular thalamic nucleus, possibly due to the muscarinic effects of oxotremorine. The present study revealed that the inferior olive is selectively activated in the harmaline-administered animals and that the basal ganglia are involved in both harmaline- and oxotremorine-induced tremors.

The present study examined the postural effects of unilaterally local injection of metabotropic glutamate receptor (mGluR) antagonists into the subthalamic nucleus (STN), in rats with haloperidol-induced parkinsonism. In rats which received unilateral microinjections of (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), a selective, subtype-non-specific antagonist of mGluR, but not the vehicle, into the STN, systemic administration of haloperidol induced ipsiversive dystonic posturing. The severity of the dystonic posturing was dose-dependent. However, subtype-specific antagonists of group I, II, or III mGluRs induced no dystonic posturing. The present findings suggest that the activity of the STN under conditions of dopamine blockade is facilitated by blockade of mGluRs in the STN, suggesting that mGluRs exert inhibitory influence on glutamate release in the STN.

The present study examined the postural effects of the local application of glutamatergic antagonists unilaterally into the subthalamic nucleus (STN), on haloperidol-induced akinesia in rats. After intracerebral injections of MK-801, a selective antagonist of N -methyl- d -aspartate (NMDA) receptor, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) disodium, a selective α -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist, or vehicle, unilaterally into the STN, haloperidol was administered systemically and the elicited behaviors were assessed quantitatively. In rats which received injections of MK-801 or CNQX, but not vehicle, unilaterally into the STN, the administration of haloperidol induced contraversive dystonic posturing. The severity of the deviated posturing was dose-dependent. The present findings revealed that the overactivity of the STN under conditions of dopamine blockade is suppressed by interruptions of glutamatergic inputs, mediated via both NMDA or AMPA receptors, to the STN. Therefore, the present study may provide functional evidence in support of a recently proposed hypothesis, that not only disinhibition from the inhibitory globus pallidus efferents but also excitatory glutamatergic inputs to the STN actually contribute to the overactivity of the STN under dopamine-depleted conditions.

This study examined the induction of c-Fos expression in the substantia nigra pars reticulata (SNr) and entopeduncular nucleus (EP) in the rats with a globus pallidus (GP) lesion, following the administration of haloperidol. After a GP lesion was made unilaterally by stereotaxic administration of ibotenic acid, haloperidol was administered systemically, and the number of cells expressing c-Fos was quantitatively assessed. Haloperidol induced a high level of the expression of c-Fos in neurons of the SNr and EP, and the GP lesion significantly decreased the expression of c-Fos in the ipsilateral SNr and EP. Since it has been suggested that c-Fos expression in the SNr/EP is caused by increased excitatory inputs from the subthalamic nucleus (STN), the present results provide functional evidence indicating that neuronal activities of the basal ganglia output nuclei are not increased by GP ablation, unlike D2 receptor blockade, supporting the recently proposed hypothesis that overactivity of the STN resulting from dopamine depletion is not solely a result of disinhibition from inhibitory GP efferents.

c-Fos is one of the transcription factors contributing to the regulation of the downstream gene expression. Administration of dopamine D1 receptor agonist or D2 receptor antagonist have been known to induce c-Fos expressions in striatal projection neurons. We examined the effects of unilateral ablation of the globus pallidus (GP) on apomorphine- or haloperidol-induced expression of c-Fos in the striatum. Haloperidol induced a high level of c-Fos expression in the striatal neurons, predominantly those in the dorsal part, and the unilateral GP lesion caused by ibotenic acid increased the number of neurons exhibiting haloperidol-induced c-Fos expression in the striatum on the side with the GP lesion by about 2- or 3-fold. On the other hand, the unilateral GP lesion had no significant effect on the apomorphine-induced c-Fos expression in the striatal neurons. The present study provides evidence indicating that the activity of GP neurons may have an inhibitory influence on the induction of the immediate early genes by haloperidol in the striatal neurons, suggesting a function of the pallido-striatal feedback loops which have been identified anatomically.

THE pedunculopontine nucleus and its adjacent structure of the mesopontine reticular formation are known as a mesencephalic locomotor region, since either electrical or chemical stimulation of these regions induces locomotion in decerebrate animals. In parkinsonism, it is presumed that the pedunculopontine nucleus is under GABAergic overinhibition from the basal ganglia. To reveal the behavioural effects of GABAergic disinhibition of the mesopontine reticular formation in parkinsonism, picrotoxin, a GABAA antagonist, (5 or 10 ng/ 0.25 microliter) or vehicle was injected unilaterally into the mesopontine reticular formation of rats via implanted cannulae after induction of catalepsy using haloperidol (1.5 mg kg-1, i.p.). Injection of the larger dose of picrotoxin, but not the smaller dose nor the vehicle, abolished the catalepsy with or without spontaneous locomotor activity. The present result suggests that the disinhibition of the brainstem output structures contributes to the recovery of mobility in the cataleptic state induced by blocking the dopaminergic transmission of the basal ganglia.

THE ipsilateral and contralateral corticocortical connections to the fifth somatosensory area (SV) in the feline cortex were determined from the location of retrogradely labelled cells following a single injection of HRP into SV. The injection was made into physiologically defined components of the body representation in SV. After injection of HRP into the face regions of SV, HRP-labelled cells were located ipsilaterally in areas 6 beta, 3b and 1-2 of the primary somatosensory (SI), in the second somatosensory (SII), third somatosensory (SIII), and fourth somatosensory (SIV) areas, along the ansate sulcus, and in areas 5a and 6a beta of the ipsilateral cortex, as well as in area 1-2 of SI and in SV of the contralateral cortex. On the other hand, after HRP had been injected into the trunk/hindlimb area, HRP-labelled cells were located in areas 3a, 1-2 of SI, in area 5, in SII, in SIII and in SIV of the ipsilateral cortex, as well as in area 1-2 of SI, and in SV of the contralateral cortex. The extent of these interconnections suggests that SV receives multiple sensory inputs and may function to integrate this information.

The neuro-behavioral observation scorebook that improved the previous observation methods of Irwin was followed, the test material was administered to 5 mice per each group, and the mean value of the obtained score was determined. The behavior of a normal animal was assumed to be point 0, animals showing suppressive behavior were scored in the minus region, and animals that showed excitement behavior were scored in the plus region. Each score was divided into three stages, according to the level of strength of the biological effect. The score of each observation item was totaled, and the level of the strength of the biological effect in the item was judged according to its mean value. These test methods of neuro-behavioral observations we proposed were able to detect the biological effects of a drug simply and promptly, and contributed sufficient data to support an administrative measure aimed at anticipating and improving the prevention of health damage in humans by non-regulated drugs from a scientific perspective. Recently, we developed a method of serial measurement of the quantity of monoamine in the mouse central nervous system by microdialysis, and performed it. The Kanagawa Prefectural Institute of Public Health conducted a study of the biological effect of non-regulated drugs. A characteristic here is what they examined about drug-dependency other than observing the behavior of the animal.

Forelimb crossed extension reflexes were examined in 22 thalamic cats. These reflexes were elicited either by backward passive movement or by repetitive electrical stimulation of cutaneous and joint afferent nerves in the contralateral forelimb. Single stimulation of the superficial radial nerve evoked two types of reflex responses—early (ER) and late (LR)—from the triceps brachii muscle on the contralateral side. The latencies were about 7 and 16–25 ms, corresponding to the propriospinal (PSR) and spino-bulbo-spinal (SBS) reflexes of the ipsilateral flexor, respectively. Repetitive stimulation of the superficial radial nerve evoked the LR but not the ER. The crossed extension reflex and LR were abolished by lesions of the dorsolateral funiculus of the cervical cord on the side opposite to the recording. The tonic EMG activity, crossed extension reflex and LR in the extensor on the side of lesions were abolished by lesions of the ventrolateral funiculus of the cervical cord. During forelimb stepping, the amplitudes of both ER and LR fluctuated depending on the phase of the step cycle. The ER appeared during a narrow period in the early phase of the stance, whereas the LR was observed during a wide period from the middle of the swing to the middle of the stance. Both responses were absent from the middle of the stance to the middle of the swing. These observations suggest that forelimb crossed extension reflexes involve both spinal and supraspinal (SBS) loop mechanisms, and that these are utilized during stepping, with the latter mechanism in particular playing an important part in the extension phase of the forelimb forward movement.

To analyze the descending pathways of the paralemniscal pontine reticular formation (PLRF), a technique was used for the selective activation of cell bodies by localized injection of putative neurotransmitters in the PLRF. When a small amount (less than 0.1 microliter) of 0.1 M glutamate was injected into the PLRF unilaterally in thalamic cats, the forelimb contralateral (c-forelimb) to the injection was extended, and occasionally the ipsilateral forelimb was flexed. These responses were similar to those obtained by electrical stimulation of the PLRF, but were relatively weaker. Unit spikes of PLRF neurons were increased in frequency following administration of glutamate. The latent periods and durations of increases in spike frequency varied depending on the concentration and quantity of the glutamate solution, and were roughly similar to those of the extensor EMG in the c-forelimb. Since the firing of PLRF neurons preceded the EMG with 11 ms latency, the unit spike of PLRF neurons could be used as a triggering signal to observe a spike triggered averaged EMG response in the extensor muscle of the c-forelimb. Results similar to those with glutamate were observed upon administration of quisqualate, kainate and aspartate. The most effective compound was quisqualate. Application to the PLRF of 1-naphthylacetyl spermine (1-NA-Spm), an analogue of the natural spider toxin JSTX-3 and an antagonist of glutamate, suppressed both the PLRF neuron activity and the extensor EMG of the c-forelimb. These observations suggest that extensor muscles of the forelimb are excited by the contralateral PLRF, perhaps via the crossed reticulospinal tract from the PLRF. PLRF neurons may be activated by glutamate (quisqualate) receptors.

The present study examined the behavioral effects of dopamine receptor agonists, antagonists, or N-methyl- d -aspartate (NMDA) glutamate receptor antagonist in rats with a unilateral excitotoxic lesion of the globus pallidus (GP). After the unilateral GP lesions were made by injections of the ibotenic acid, drugs were systemically given and the elicited behaviors were quantitatively assessed. Systemic administration of haloperidol, but not SCH23390, dose-dependently induced contraversive dystonic posturing in unilateral GP-lesioned rats. On the other hand, systemic administration of quinpirole, but not SKF38393, induced ipsiversive circling. MK-801, only when given at a high dose, caused ipsiversive circling with dystonic posturing. The present results showed that, in unilateral GP-lesioned rat, the D2 receptor agonist and antagonist caused ipsiversive and contraversive posturing or circling, respectively. Since the rotational behavior is induced on the basis of asymmetry of the basal ganglia output activity, there must be a marked difference between the GP ablation and the administration of D2 receptor blockade on the basal ganglia output activity, supporting a speculation that overactivity of the basal ganglia under dopamine depletion is not solely a result of the disinhibition from the inhibitory GP efferents. The present unilateral GP-lesion model appears to be a useful one for the pharmacobehavioral investigation of D2-mediated mechanisms.

Since the development of levodopa therapy, the quality of daily life of patients with Parkinson’s disease (PD) has dramatically improved. However, after several years of levodopa therapy, in most cases, both the efficacy of the levodopa therapy and the level of the patients’ motoricity gradually decrease (Rinne, 1981; Narabayashi, 1993; Marsden and Fahn, 1981; Lieberman et al., 1972; Bonnet et al., 1987). Therefore, it is clinically relevant to investigate why the therapeutic effect of levodopa on akinesia is limited, and why the levodopa effect on akinesia differs from that on other symptoms such as rigidity and tremor. We have no satisfactory answers to these questions, since the exact pathophysiological mechanisms underlying parkinsonian symptoms have not been elucidated.

To analyze changes in the excitability of both the spinal cord and brainstem in thalamic cats stepping on a moving treadmill, we examined the cutaneous propriospinal (PSR) and spino-bulbo-spinal (SBS) reflex responses in 20 adult cats. Tracheal cannulation, spinal transection at the T10 segment, and decerebration at the stereotaxic A12 level were performed under ether anesthesia. Immediately after decerebration, the ether was withdrawn. The head was fixed in a stereotaxic device, the T2 spinal process clamped to a metal frame, and the lumbar region suspended by a hammock, with bilateral forelimb contact on the floor of a treadmill. Electrical stimulation was applied to the superficial radial nerve with a cuff electrode, and two reflex responses (PSR and SBS) were recorded from the biceps brachii muscle in the same forelimb. Shortly before the appearance of forelimb stepping, both PSR and SBS reflex responses were elevated in amplitude. During forelimb stepping, the amplitudes of PSR and SBS reflex responses fluctuated depending on the phase of the step cycle. The PSR response was enhanced in the early phase of the swing, whereas the SBS response was elevated during a wider period from the beginning of the stance to the middle of the swing. The SBS response was completely absent in the late phase of the swing. This period corresponded to the transfer from flexion to extension and the appearance of the EMG of the triceps brachii muscle of the same forelimb. The fluctuation of the SBS response during stepping may be produced at the brainstem level, and not the spinal cord level, because the PSR response was enhanced only during narrow periods. The generation of locomotion thus seems to result in an enhancement of excitability of reflex pathways in the spinal cord and particularly in the brainstem.

Summary We recorded the neuronal activities from, stimulated, and produced lesions in, various regions in the brainstem of thalamic cats. Neuronal activities were recorded during stepping on a motor-driven treadmill, and were averaged by using the flexor EMG as the trigger. In wide regions of the brainstem, an averaged response was obtained with similar latency as the ipsilateral flexor EMG; however, in the paralemniscal pontine reticular formation (PLRF) the averaged response preceded the flexor EMG. When repetitive electrical stimulation was applied to the PLRF on one side, coordinated movements of bilateral forelimbs were elicited. The ipsilateral forelimb was flexed and the contralateral forelimb extended. EMG recordings revealed that on the side ipsilateral to the stimulation, flexor EMGs appeared and increased gradually in amplitude, and the extensor EMG was suppressed, while the extensor EMG was increased and no flexor EMG was obtained on the side contralateral to the stimulation. When pulse train stimulation was alternatingly applied to the PLRF on bilateral sides, alternating forelimb movement, as in stepping, was evoked, depending on each stimulation. A small, unilateral lesion in the PLRF reduced forelimb stepping on a motor-driven treadmill, particularly the flexor muscle contraction in the forelimb ipsilateral to the lesion. Bilateral PLRF lesions abolished forelimb stepping on both sides. Unilateral transection of the ventral quadrant at the cervical cord abolished PLRF-elicited flexor EMGs and associated suppression of extensor EMG ipsilateral to the transection; however, the side contralateral to the transection remained intact. These results suggest that the PLRF exerts a patterned control of the bilateral forelimb muscles and plays a role in initiating and maintaining forelimb stepping.

The paralemniscal pontine reticular neurons (PLRF) exhibited burst discharges when thalamic cats stepped on the moving treadmill, during stepping induced by mesencephalic locomotor region (MLR) stimulation, fictive locomotion by MLR stimulation after immobilization and during MLR stimulation after transection at the C2 level. These results imply that MLR stimulation can evoke burst discharge within the brainstem which may obtain alternating limb movements via descending tract to the cervical cord.

To analyze the roles of joint afferents in relation to initiation of forelimb stepping in thalamic cats, we recorded the unit spikes of the cervical dorsal roots, stimulated the joint afferents, and applied local anesthesia to the joint capsule. Almost all of the joint afferents of the shoulder, elbow, wrist and finger adapted slowly and exhibited alternating firing during forelimb stepping. About 45% of the afferents of each joint showed firings as the limb moved from forward to backward. About 44% of the afferents exhibited discharges as the limb moved from backward to forward. The remaining afferents showed firings as the limb moved in both directions. The application of local anesthesia to joints of the shoulder, elbow or wrist resulted in a marked reduction of forelimb stepping. Forelimb stepping was evoked by electric stimulation of the joint capsule, when excitabilities of flexor motoneurons were increased due to muscle stretching. Impulses originating in the joint afferents of the forelimb entered the spinal cord and ascended to the dorsolateral funiculus of the cervical cord, since forelimb stepping was abolished after bilateral transection of this part. Our results indicate that joint afferents may play an important role in the initiation of forelimb stepping in thalamic cats walking on a motor-driven treadmill.