Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part I. Venom Extraction, Preparation, and Laboratory Testing.

The redback spider (Latrodectus hasseltii Thorell) reportedly invaded Japan in September 1995. To date, 84 redback spider bite cases have been reported; 7 of these cases employed the antivenom. Antivenom has been imported from Australia in the past, but because of restrictions on exportation it was evident that nearly all of the antivenom present in Japan would expire during 2014. In 2014, a plan was proposed to experimentally manufacture and stockpile a horse antiserum for ourselves, using redback spiders indigenous to Japan. A total of 11,403 female spiders were captured alive: 1,217 from the vicinity of Nishinomiya City, Hyogo prefecture, and 10,186 from Osaka prefecture. Of these, 10,007 females were dissected, and the venom was extracted from the venom glands of individuals and subjected to crude purification to yield 4 lots, of which the majority was α-latrotoxin. Among them, a large amount of single lots with an estimated protein content of 236 mg is subsequently scheduled to be used for immunizing horses. We also determined lethal toxicity of the venom (LD50: 9.17 µg per mouse), and established the assay for the determination of an anti-lethal titer of antivenom in mice.

Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part II. Experimental Production of Equine Antivenom against the Redback Spider.

This is the first report on large-scale experimental production of an equine antivenom against the redback spider (Latrodectus hasseltii) lived in Japan. We captured 10,000 redback spiders in Japan and prepared the toxoids of crude venom extract, mixed the toxoids with a mineral oil adjuvant, and immunized healthy horses repeatedly over a period of several weeks. Thereafter, we separated the horse plasma, purified the γ-globulin fraction, and stocked it as a purified antivenom concentrate. Consequently, we manufactured approximately 6,500 vials of a single-dose freeze-dried test lot from a portion of the purified γ-globulin fraction, equivalent to the extract derived from 520 spiders. This test lot had an antitoxin titer comparable to that of a similar drug commercially available overseas (a liquid preparation), and the other quality met all quality reference specifications based on the Minimum Requirements for Biological Products and other guidelines relevant to existing antivenom drug products in Japan.

Retrospective survey to evaluate the safety and efficacy of Japanese botulinum antitoxin therapy in Japan.

Japanese botulinum antitoxins have been used for more than 50 years; however, their safety and therapeutic efficacy are not clear. In order to analyze the available data on botulinum antitoxin therapy in Japan, we surveyed published reports about botulism cases in which botulinum antitoxins were used, and retrospectively analyzed the safety and efficacy of the therapy. A total of 134 patients administered botulinum antitoxins were identified from published reports. Two cases of side effects (1.5%) were detected after antitoxin administration, both not fatal. The fatality rate was 9.4%, and more than 70% of the patients showed improvement in their symptoms and better clinical conditions than those not treated with antitoxins. These data suggest that the therapy with Japanese antitoxins is safe and highly effective.

The habenulo-interpeduncular and mammillothalamic tracts: early developed fiber tracts in the human fetal diencephalon.

PURPOSE: The habenulo-interpeduncular (HI) and mammillothalamic (MT) tracts are phylogenetically ancient. The clinical relevance of these tracts has recently received attention. In this work, we map the anatomy the developing HI and MT. METHODS: To investigate the topographical anatomy of developing fiber tracts in and around the diencephalon, we examined the horizontal, frontal, and sagittal serial paraffin sections of 28 human fetuses at 8-12 weeks of gestation. RESULTS: In all specimens, eosinophilic early fiber bundles were limited to the bilateral HI and MT tracts in contrast to pale-colored later developing fibers such as the thalamocortical projections and optic tract. The HI and MT tracts ran nearly parallel and sandwiched the thalamus from the dorsal and ventral sides, respectively. The nerve tract course appeared to range from 5-7 mm for the HI tract and 3-5 mm for the MT tract in 15 specimens at 11-12 weeks. The HI tract was embedded in, adjacent to, or distant from the developing parvocellular red nucleus. CONCLUSIONS: In early human fetuses, HI and MT tracts might be limited pathways for primitive cholinergic fiber connections between the ventral midbrain and epithalamic limbic system.

Obstacle clearance and prevention from falling in the bipedally walking Japanese monkey, Macaca fuscata.

BACKGROUND: studies are needed which consider CNS-controlled strategies for accommodating perturbed bipedal (Bp) posture and walking. OBJECTIVE: to demonstrate the suitability of the Japanese monkey, Macaca fuscata, for the above purpose. SETTING AND SUBJECTS: three adult monkeys were operantly trained to use Bp-walking on a moving treadmill belt. On one side of the belt, a rectangular adjustable-height obstacle confronted the ipsilateral leg every 4-6 steps, as determined by belt speed. METHODS: animal posture and walking patterns were captured and digitized by two high-speed video systems. Frame-by-frame analyses of side- and back-view kinematics were obtained. RESULTS: the monkeys learned quickly to proactively clear the in-coming obstacles by use of a flexible hip-knee-ankle flexion strategy. This featured an appropriate postural adjustment and leg trajectory. In cases where a monkey failed to clear the obstacle, it promptly adopted a defensive posture to avoid falling. There was then a quick return to a posture that allowed the resumption of a Bp gait. CONCLUSIONS: when Bp posture and gait are perturbed in a non-human primate model, the prompt adjustment of a flexible hip-knee-ankle flexion strategy and a defensive postural adjustment act together to prevent a fall and enable the speedy resumption of normal Bp posture and gait.

Lumbar commissural interneurons with reticulospinal inputs in the cat: morphology and discharge patterns during fictive locomotion.

The purpose of this study was 1). to characterize the morphology of lumbar commissural neurons (CNs) with reticulospinal inputs and 2). to quantitate their activity during locomotor rhythm generation. Intraaxonal recordings at the L4-7 level of the spinal cord were obtained in 67 neurons in the decerebrate, paralyzed cat. Fourteen of them were subsequently nearly fully visualized following their intraaxonal injection with the tracer neurobiotin. All 14 were CNs with axons projecting across the midline of the spinal cord. Their somata were located mainly in lamina VIII and additionally in laminae VII-VI. Most of the lamina VIII CNs were excited monosynaptically from reticulospinal pathways. They were judged to be interneuronal CNs if they had no, or a short, rostral projection. These CNs commonly gave off multiple axon collaterals in and around their somata's segmental level. They projected mainly to laminae VIII-VII and some additionally to lamina IX. Some laminae VIII and the laminae VII-VI CNs were excited polysynaptically from reticulospinal pathways or were not excited. They were judged to be long propriospinal or ascending tract CNs because they had only an ascending axon. Most lamina VIII CNs discharged rhythmically during fictive locomotion evoked by stimulation of the mesencephalic locomotor region, exhibiting one peak per locomotor cycle. The peak was in phase with neurographic activity of either a left or a right hindlimb extensor nerve. These results suggested that lamina VIII CNs are reciprocally connected bilaterally at each segmental level. Such an arrangement suggests their participation in the generation and coordination of reciprocal and bilateral locomotor activity.

Biomechanical constraints in hindlimb joints during the quadrupedal versus bipedal locomotion of M. fuscata.

The operant-trained Japanese monkey, Macaca fuscata, can walk with both a quadrupedal (Qp) and a bipedal (Bp) gait on the surface of a treadmill belt, which moves at different speeds. The animal can also learn to transform its locomotor pattern from Qp to Bp, and vice versa, without a break in forward walking speed. This nonhuman primate model provides an intriguing opportunity to compare the kinematics of multiple body segments during Qp and Bp walking of the same subject. We found that M. fuscata selects a postural strategy and limb-kinematic parameters appropriate for the execution of both gaits. We propose that the basic locomotor rhythm-generating mechanisms of the brainstem and spinal cord, which are genetically endowed and relatively automatized, are used for the execution of both the Qp and Bp gait. The latter requires in addition, however, some higher-level circuitry which is shaped substantially by motor learning mechanisms.

Reactive and anticipatory control of posture and bipedal locomotion in a nonhuman primate.

Bipedal locomotion is a common daily activity. Despite its apparent simplicity, it is a complex set of movements that requires the integrated neural control of multiple body segments. We have recently shown that the juvenile Japanese monkey, M. fuscata, can be operant-trained to walk bipedally on moving treadmill. It can control the body axis and lower limb movements when confronted by a change in treadmill speed. M. fuscata can also walk bipedally on a slanted treadmill. Furthermore, it can learn to clear an obstacle attached to the treadmill's belt. When failing to clear the obstacle, the monkey stumbles but quickly corrects its posture and the associated movements of multiple motor segments to again resume smooth bipedal walking. These results give indication that in learning to walk bipedally, M. fuscata transforms relevant visual, vestibular, proprioceptive, and exteroceptive sensory inputs into commands that engage both anticipatory and reactive motor mechanisms. Both mechanisms are essential for meeting external demands imposed upon posture and locomotion.

Locomotor role of the corticoreticular-reticulospinal-spinal interneuronal system.

In vertebrates, the descending reticulospinal pathway is the primary means of conveying locomotor command signals from higher motor centers to spinal interneuronal circuits, the latter including the central pattern generators for locomotion. The pathway is morphologically heterogeneous, being composed of various types of inparallel-descending axons, which terminate with different arborization patterns in the spinal cord. Such morphology suggests that this pathway and its target spinal interneurons comprise varying types of functional subunits, which have a wide variety of functional roles, as dictated by command signals from the higher motor centers. Corticoreticular fibers are one of the major output pathways from the motor cortex to the brainstem. They project widely and diffusely within the pontomedullary reticular formation. Such a diffuse projection pattern seems well suited to combining and integrating the function of the various types of reticulospinal neurons, which are widely scattered throughout the pontomedullary reticular formation. The corticoreticular-reticulospinal-spinal interneuronal connections appear to operate as a cohesive, yet flexible, control system for the elaboration of a wide variety of movements, including those that combine goal-directed locomotion with other motor actions.

Integration of multiple motor segments for the elaboration of locomotion: role of the fastigial nucleus of the cerebellum.

This chapter provides a conceptual overview of the role and operation of higher structures of the central nervous system (CNS) in the control of posture and locomotion in the mammal, including the nonhuman primate and the human. Both quadrupedal and bipedal locomotion require the integrated neural control of multiple body segments against gravity. During development, and in selected instances in the adult, motor learning is required, particularly for merging anticipatory and reactive CNS processes, the latter being necessary after tripping and stumbling. We have recently found that the fastigial nucleus (FN) of the cerebellum in the cat plays a particularly important role in the control of locomotion, by virtue of its critical position in uniting the cerebro-cerebellar and the spino-cerebellar loops of neural activity that participate in the integrated control of multiple body segments. Further understanding of the CNS structures that achieve this integration has come from our recent study of an intact nonhuman primate, the Japanese monkey, Macaca fuscata, as it learns to elaborate bipedal locomotion rather than its normal quadrupedal fashion. Based on findings from these two animal species, we now present a model of the overall integrated control of posture and locomotion that features the combined operation of parallel and distributed neural circuitry throughout the CNS.

Ascending and descending brainstem neuronal activity during cystometry in decerebrate cats.

AIMS: This study was undertaken to examine the distribution of pontomedullary neurons related to micturition or urine storage, as well as the connections between the pontine micturition center (PMC), medullary neurons, and the spinal cord. METHODS: In decerebrate cats, extracellular recording of the rostral pontine and rostral medullary neurons was performed. Firing of each neuron was quantitated during cystometry. Connections between the PMC, medullary neurons, and the spinal cord (L1) were also examined electrophysiologically. RESULTS: Ninety-four neurons showed an increase or decrease of the firing rate during micturition. Units with an antidromic response to L1 stimulation and an increased firing rate were located in the nucleus locus coeruleus alpha (LCa; n = 8) corresponding to the PMC, and in the medial reticular formation (MRF) of the medulla (n = 14). Units showing a decreased firing rate were located in the nucleus reticularis pontis oralis (PoO; n = 26) and in the MRF (n = 11). The latencies of antidromic and orthodromic responses of the LCa units were longer than those of the PoO units. MRF neurons responded antidromically and/or orthodromically to stimulation of the PMC or L1. CONCLUSIONS: These results suggest that the pathway concerned with urine storage has a faster spinobulbospinal loop than the micturition reflex pathway and that rostral medullary neurons also play an important role in micturition and urine storage. There may be two descending pathways between the PMC and the spinal cord: both a direct pathway and one by means of medullary neurons.

Acquisition of operant-trained bipedal locomotion in juvenile Japanese monkeys (Macaca fuscata): a longitudinal study.

This study investigated developmental aspects of the acquisition of operant-trained bipedal (Bp) standing and Bp walking in the normally quadrupedal (Qp) juvenile Japanese monkey (M. fuscata). Four male monkeys (age: 1.6 to 2.4 years, body weight: 3.3 to 4.6 kg) were initially operantly trained to stand upright on a smooth floor and a stationary treadmill belt (width = 60 cm, walking length = 150 cm). They were then trained to walk bipedally on the moving treadmill belt (speed: 0.4-0.7 m/s). A regular training program (5 days/week; 30-60 min/day) was given to each monkey for the first 40 to 60 days, followed by less intensive training. After the beginning of locomotor training, upright postural stability and Bp walking capability were assessed kinematically for 592, 534, 526, and 537 days on monkeys A, B, C, and D, respectively. Left side- and back-views of the walking monkey were photographed (10 frames/s) and videotaped (250 frames/s). Stick figures of the head, body, and hindlimbs were drawn with reference to ink-marks positioned in front of the ear and over the pivot points of hindlimb joints. All kinematic data were digitized and analyzed using image-analyzing software. After sufficient physical growth and locomotor training, all the monkeys gradually acquired: (a) a more upright and a more stable posture with a constant body axis orientation during Bp locomotion; (b) a more stable and a stronger functional coupling between the body and hindlimb movements with a less anterior (A)-posterior (P) fluctuation of a body axis; (c) a smaller leftward (Lt)-rightward (Rt) displacement of the midline pelvic position, allowing the monkey to walk along a straight course; (d) a more coordinated relationship among hip-knee, knee-ankle, and ankle-metatarsophalangeal (MTP) joints; and finally (e) the acquisition of well-coordinated Bp walking even at high treadmill belt speeds up to 1.5 m/s. All of these results demonstrated the capability of the physically developing monkey to integrate the neural and musculoskeletal mechanisms required for sufficient coordination of upper (head, neck, trunk) and lower (hindlimbs) motor segments so that Bp standing and Bp walking could be elaborated.