Properties and plasticity of paired-pulse depression at a central synapse.

Synaptic depression was studied at the axo-axonic connection between the goldfish Mauthner axon and identified cranial relay interneurons using simultaneous presynaptic and postsynaptic recordings and a paired-pulse stimulus paradigm. We used interstimulus intervals (ISIs) ranging from 10 msec to 1 sec and a cycle time of approximately 5 sec. Depression (Delta EPSP/EPSP1) was maximal at the shorter intervals (80%) and decreased exponentially with a tau approximately 400 msec (360 +/- 107 msec, mean +/- SD). We found the amplitudes of the first and second EPSP were not correlated, indicating the magnitude of depression does not depend on the amount of transmitter released by the conditioning stimulus. At short ISIs, the latency of EPSP2 was 23% longer than that of EPSP1 and recovered to control with tau approximately 400 msec, whereas rise time and decay time were not altered significantly. The latency distribution, which is determined by the timing of the first quantum released each trial, was used to derive alpha(t), the rate of evoked exocytosis after an action potential. alpha(t) was biphasic, and both components were consistently delayed during depression. Presynaptic manipulations of putative intracellular regulatory pathways, such as Ca(2+) and GTPgammaS injections, preferentially affected the amplitude of EPSP1 or EPSP2. These results are not consistent with simple depletion of the available pool of synaptic vesicles as the major mechanism underlying depression. They rather suggest that it is attributable to a modification or refractoriness of the release process and that there may be multiple pathways subserving evoked exocytosis.

Effects of 4-aminopyridine on muscle and motor unit force in canine motor neuron disease.

Hereditary Canine Spinal Muscular Atrophy (HCSMA) is an autosomal dominant disorder of motor neurons that shares features with human motor neuron disease. In animals exhibiting the accelerated phenotype (homozygotes), we demonstrated previously that many motor units exhibit functional deficits that likely reflect underlying deficits in neurotrans-mission. The drug 4-aminopyridine (4AP) blocks voltage-dependent potassium conductances and is capable of increasing neurotransmission by overcoming axonal conduction block or by increasing transmitter release. In this study, we determined whether and to what extent 4AP could enhance muscle force production in HCSMA. Systemic 4AP (1-2 mg/kg) increased nerve-evoked whole muscle twitch force and electromyograms (EMG) to a greater extent in older homozygous animals than in similarly aged, symptomless HCSMA animals or in one younger homozygous animal. The possibility that this difference was caused by the presence of failing motor units in the muscles from homozygotes was tested directly by administering 4AP while recording force produced by failing motor units. The results showed that the twitch force and EMG of failing motor units could be significantly increased by 4AP, whereas no effect was observed in a nonfailing motor unit from a symptomless, aged-matched HCSMA animal. The ability of 4AP to increase force in failing units may be related to the extent of failure. Although 4AP increased peak forces during unit tetanic activation, tetanic force failure was not eliminated. These results demonstrate that the force outputs of failing motor units in HCSMA homozygotes can be increased by 4AP. Possible sites of 4AP action are considered.

Regenerated dorsal root fibers form functional synapses in embryonic spinal cord transplants.

1. The aim of the present study was to determine whether synapses formed by dorsal root afferents that regenerate into intraspinal transplants of fetal spinal cord are functional. Severed L4 or L5 dorsal root stumps were placed at the bottom of dorsal quadrant cavities made in the lumbar spinal cords of adult rats and juxtaposed to embryonic day 14 spinal cord transplants. 2. In animals examined 5-10 weeks later, we recorded extracellularly in transplants from 43 units that fired in response to electrical stimulation of the implanted dorsal root. Latency fluctuations of extracellular firing that increase with stimulus and failure to follow high-frequency and posttetanic potentiation of extracellular firing stimulation suggest that synapses with conventional properties are formed between regenerating afferents and transplant neurons. Limited intracellular recordings confirmed the existence of excitatory postsynaptic potentials in transplant neurons after dorsal root stimulation. 3. In 16 units, extracellular firing occurred in response to single shock stimulation. The remainder of the units required two or more dorsal root shocks to evoke firing; some of these connections also may be monosynaptic. 4. Under the assumption that single shock firing was most likely the result of monosynaptic connections between transplant neurons and regenerated dorsal root fibers, we estimated the conduction velocities of regenerated fibers. These estimates suggest that fibers with conduction velocities in the C, A delta, and A alpha/beta ranges regenerate into transplants of embryonic spinal cord. 5. The results demonstrate that regenerated dorsal root axons establish functional synaptic connections with transplant neurons. The implications for using fetal transplants to help rebuild spinal reflex circuits after spinal cord injury are considered.

Seeing beyond the midline: the role of the contralateral isthmotectal projection in the leopard frog.

The ground level visual field of each eye of the leopard frog includes the entire ipsilateral 180-deg field and approximately 60 deg into the frontal contralateral field. When one eye is covered with an opaque patch, a frog responds to prey stimuli over the entire field of the other eye. Nevertheless, when one optic nerve is cut, the animal responds to prey in the ipsilateral hemifield of the connected eye, but only responds as far as about 30 deg past the frontal midline. If one optic tract is cut, the animal does not respond to prey past the frontal midline. We hypothesized that the responses past the frontal midline might be mediated by input from contralaterally projecting isthmotectal fibers. These fibers originate in the nucleus isthmi, a posterior midbrain structure. We found that when we placed an opaque patch over one eye and either ablated the ipsilateral nucleus isthmi, or cut crossing isthmotectal fibers in the optic chiasm, or blocked input to nucleus isthmi by ablating the ipsilateral tectal lobe, animals did not respond to prey stimuli past the frontal midline. We found that when we placed an opaque patch over one eye and cut crossing optic fibers in the anterior part of the optic chiasm (sparing crossing isthmotectal fibers), animals responded to prey stimuli in the nasal half of the seeing eye's contralateral frontal field. Our results suggest that contralaterally projecting isthmotectal fibers enable the frog to respond to stimuli past the frontal midline. We suggest a one-dimensional model of how nucleus isthmi influences tectal function.

Properties of motor units after self-reinnervation of the cat superior oblique muscle.

1. The mechanical properties of motor units of the cat superior oblique muscle and axonal conduction velocities of trochlear motoneurons have been studied at several postoperative times after intracranial axotomy of the trochlear nerve. 2. Whole muscle twitch forces were generally within the normal range by approximately 4 mo postoperative, indicating that reinnervation is complete at this time. 3. Among animals studied 3.5-4.5 months after trochlear axotomy, average motor-unit tetanic forces were increased by a factor of approximately 2.5 compared with units studied in normal superior oblique muscle. Average motor-unit tetanic forces in animals studied 14.5-23 mo after axotomy were also increased relative to normal, but the difference was not significant. Among all reinnervated motor units, there was a tendency for increased twitch time-to-peak relative to control. Reinnervated motor-unit fatigue properties were similar to normal. 4. Average trochlear motoneuron conduction velocities for animals at all postoperative intervals remained significantly lower than the average conduction velocities from three of four normal animals. 5. Counts of Nissl-stained cell bodies in axotomized and control, contralateral trochlear nuclei showed that some cell loss had occurred, averaging approximately 17% 3.5-4.5 mo postoperative and 24% 14.5-23 mo postoperative. Associated with this loss was an increase (10%) of axotomized motoneuron soma cross-sectional area. 6. Muscle fiber cross-sectional areas (CSA) were measured in reinnervated superior oblique muscles and compared with CSAs from contralateral, control muscles. Average CSA was significantly decreased in all reinnervated muscles, with the relative decreases ranging from approximately 10 to 28%. 7. The results are discussed in terms of factors that determine motor-unit force; muscle fiber CSA, specific force, and innervation ratio. We conclude that the increases of average motor-unit force in short-term reinnervated superior oblique muscles are most likely related to polyneuronal innervation of muscle fibers and that the return of these forces to normal levels in long-term muscles is related to synapse elimination. Our results are compared with those of other self-reinnervation studies, and the potential role played by the time muscle remains denervated in determining the persistence of polyneuronal innervation after reinnervation is considered.

Motor unit behavior in canine motor neuron disease.

Hereditary canine spinal muscular atrophy (HCSMA) is an autosomally dominant disease of motor neurons that shares many pathological features with human motor neuron disease. A particularly striking feature of the affected, accelerated phenotype (homozygous HCSMA) is that profound weakness develops before appreciable motor neuron cell death occurs (Cork et al., 1989a), implying that motor unit functional defects occur initially. The purpose of this study was to identify the site of these defects and characterize their nature. In most young homozygotes (2-3 months postnatal), motor neurons were encountered that could support orthodromic action potential propagation to the muscle but did not activate muscle fibers. The tetanic forces of innervated motor units in young homozygotes tended to be smaller than those in closely age-matched clinically normal animals. In older homozygotes (approximately 4.5 months, postnatal), all motor neurons sampled were capable of activating muscle fibers, but many motor units displayed abnormal behavior including an inability to sustain force output during high frequency activation. Motor units exhibiting tetanic failure also showed proportionately greater twitch potentiation than nonfailing units of similar unpotentiated twitch amplitude. Tetanic failure and large potentiation tended to occur in motor units that possessed the slowest contraction speeds. These results indicate that motor neuron functional defects in HCSMA appear initially in the most distal parts of the motor axon and involve defective neurotransmission. The possible roles of distal nerve degeneration, motor terminal sprouting, and synaptic transmission in causing these deficits are considered.

Regrowth of optic fibers and behavioral recovery after optic chiasm transection.

We studied the effect of optic chiasm midline transection on visually guided behavior and retinotectal fiber regrowth in frogs. After complete transection, frogs do not respond to visually presented prey and looming stimuli. Beginning about 2 months later there is recovery of visual function. However, unlike recovery after optic nerve transection, animals respond as if the stimulus were not at its actual position, but at the symmetric position in the contralateral field. For instance, if a prey stimulus is located 5 cm away from the recovered frog at an eccentricity of 40 degrees to the left of the midline, the animal will respond as if the stimulus were 5 cm away at 40 degrees right. Further, these animals typically respond to looming stimuli not by jumping away from the stimulus, but by either colliding with the stimulus or jumping toward the side from which the stimulus approaches. These behaviors persist throughout the testing period, up to 17.5 months postlesion. Electrophysiological recordings reveal that visual activity in the optic tectum is retinotopically organized but driven primarily by stimuli to the ipsilateral eye. HRP histochemistry reveals that some regenerated retinal fibers are found to cross at the midline of the chiasm. Thus, the midline is not impenetrable to crossing retinal fibers. Frogs with cut of 3/4 of the chiasm respond normally to prey stimuli initially but later respond as if the stimuli are at mirror image locations. In these animals most retinotectal fibers project to the ipsilateral tectum despite the presence of intact contralaterally projecting retinotectal fibers during the recovery period.

Studies on the optic chiasm of the leopard frog. I. Selective loss of visually elicited avoidance behavior after optic chiasm hemisection.

We hemisected either the posterior or anterior portion of the optic chiasm and found that frogs were unresponsive to large looming stimuli anywhere in the visual field. Nonetheless, the animals responded to prey stimuli throughout the visual field. Responses to looming stimuli returned in 1 to 8 weeks post-surgery. After complete transection of the chiasm animals were unresponsive to both prey and large looming stimuli. Frogs responded normally to prey and looming stimuli if less than half the optic chiasm was cut or if the postoptic commissure was cut. Since responses to looming stimuli returned before cut optic fibers could regenerate, these results suggest that visual information concerning prey and large looming objects are mediated by separate optic nerve fiber systems.

Studies on the optic chiasm of the leopard frog. II. Organization of retinotectal fibers in the optic chiasm.

The organization of retinotectal fibers in the optic chiasm was investigated using horseradish peroxidase (HRP) histochemistry and electrophysiological recording. HRP injection into a small region of the tectum led to retrograde staining of labeled fibers in a circumscribed region of the chiasm and staining of labeled ganglion cells in the contralateral retina. In each instance labeled tissue was spread over a greater proportion of the area of a chiasm section than over the flattened retina. Fibers originating in central (older) retina are located in dorsal chiasm. Fibers originating in peripheral (younger) retina are located in ventral chiasm. Viewed with the electron microscope, labeled unmyelinated fibers are admixed with labeled myelinated fibers. Neuronal activity was monitored with an extracellular microelectrode from points in dorsoventral tracks in the chiasm. Multiple units were recorded at each chiasm location. Using visual stimuli, the receptive fields of the units were mapped. The fields were distributed along an arc across the visual field. At ventral chiasm recording sites the arc was in the peripheral part of the visual field. In succeeding dorsal sites the arcs were concentrically arranged so that the more dorsal the chiasm recording site, the more central was the arc in the visual field. Thus, in the optic chiasm, retinal fibers appear to be organized chronotopically but not retinotopically. Fibers of the same age but from different locations in the retina are mixed together.

Relationship between isthmotectal fibers and other tectopetal systems in the leopard frog.

We studied the relationship of isthmotectal input to other tectal afferent fiber systems in three ways. 1) Using horseradish peroxidase (HRP) histochemistry, we determined the nonretinal inputs to the superficial tectum. In different sets of animals we a) applied HRP to the tectal surface; b) inserted HRP crystals into the tectum; c) injected small volumes of HRP solutions into the superficial tectum. N. isthmi accounts for more than 65% of the nonretinal extrinsic input in the superficial tectal layers. One set of fibers from the contralateral n. isthmi projects to the most superficial layer. Fibers from posterior thalamus and tegmentum project to both superficial and deeper layers in the tectum, but not to the most superficial layer. The ipsilaterally projecting isthmotectal fibers terminate in the deeper superficial layers. 2) We investigated the relationship between retinofugal and contralaterally projecting isthmotectal pathways. We orthogradely labelled n. isthmi fibers by unilateral HRP injections into n. isthmi, and we also labelled retinal fibers by injecting tritiated l-proline into both eyes. In such animals contralaterally projecting isthmotectal fibers cross in the dorsal posterior region of the optic chiasm. From the chiasm to the tectum isthmotectal fibers and retinofugal fibers are admixed. 3) We determined whether other fiber systems cross with contralaterally projecting isthmotectal fibers. We cut the posterior part of the optic chiasm and applied HRP crystals to the cut. Only n. isthmi and retina are retrogradely labelled.