Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm. The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the beta-actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport.

Experiments were done to determine the form of the input-output relation (i.e. stimulus intensity vs response amplitude) of the corticospinal pathway of the first dorsal interosseous and the tibialis anterior, respectively. Our purpose was to determine from these quantitative relations which input-output parameters would be useful measures in studies dealing with motor cortical task dependence. The motor cortex was excited by focal transcranial magnetic stimuli and the evoked motor response were recorded with surface electromyographic electrodes. In some experiments the discharge probability of single motor units in response to magnetic stimuli of increasing intensity was determined from intramuscular recordings. For both muscles the form of the input-output relation was sigmoidal. The steepness of the relation increased, up to 4-7 times the value at rest, with increasing tonic background activity. The threshold decreased, but only slightly, with increasing tonic background activity. The minimum value of the threshold was reached at activation levels of about 10-20% of the maximum tonic effort, whereas the steepness of the relation reached its maximum at higher activation levels, typically about 30-40% of the maximum tonic effort. These observations imply that these two input-output parameters of the corticospinal pathway - one reflecting the bias level (threshold) and the other the gain (slope) - are determined by different neural mechanisms. The plateau level of the sigmoidal input-output relation was not influenced by the background activation level, except that in some subjects (4/9) it could not be reached when no background motor activity was present. This was probably due, for the most part, to limitation of the maximum stimulator output. Additionally, this finding may reflect a change in the intrinsic excitability of the motor cortex in going from rest to activity, or that convergent inputs from different descending and afferent systems are required for maximal activation of motoneuron pools. Thus, the threshold, steepness and plateau level characterize the input-output parameters of the human corticospinal pathway for a given level of motor activity. In contrast to the nonlinear input-output relation of the corticospinal pathway as whole, which includes the motoneuron pool and any spinal interneuronal relays, the discharge probability of all single motor units was a linearly increasing function of the stimulus strength (r> or =0.9, P<0.01). Thus, the sigmoidal input-output relation of the corticospinal pathway, as a whole, is not due to the input-output properties of single motoneurons. The possible neural mechanisms which underlie the shape and parameters of the input-output relation as well as the methodological implications of the results are considered.

Experiments were done to determine the extent to which the corticospinal tract is linked with the segmental motor circuits controlling ankle flexors and extensors during human walking compared with voluntary motor tasks requiring attention to the level of motor activity. The motor cortex was activated transcranially using a focal magnetic stimulation coil. For each subject, the entire input-output (I-O) curve [i.e., the integral of the motor evoked-potential (MEP) versus stimulus strength] was measured during a prescribed tonic voluntary contraction of either the tibialis anterior (TA) or the soleus. Similarly, I-O curves were measured in the early part of the swing phase, or in the early part of the stance phase of walking. The I-O data points were fitted by the Boltzmann sigmoidal function, which accounted for >/=80% of total data variance. There was no statistically significant difference between the I-O curves of the TA measured during voluntary ankle dorsiflexion or during the swing phase of walking, at matched levels of background electromyographic (EMG) activity. Additionally, there was no significant difference in the relation between the coefficient of variation and the amplitude of the MEPs measured in each task, respectively. In comparison, during the stance phase of walking the soleus MEPs were reduced on average by 26% compared with their size during voluntary ankle plantarflexion. Furthermore, during stance the MEPs in the inactive TA were enhanced relative to their size during voluntary ankle plantarflexion and in four of six subjects the TA MEPs were larger than those of the soleus. Finally, stimulation of the motor cortex at various phases of the step cycle did not reset the cycle. The time of the next step occurred at the expected moment, as determined from the phase-resetting curve. One interpretation of this result is that the motor cortex may not be part of the central neural system involved in timing the motor bursts during the step cycle. We suggest that during walking the corticospinal tract is more closely linked with the segmental motor circuits controlling the flexor, TA, than it is with those controlling the extensor, soleus. However, during voluntary tasks requiring attention to the level of motor activity, it is equally linked with the segmental motor circuits of ankle flexors or extensors.

The topographical relationships between cholinergic neurons, identified by their immunoreactivity for choline acetyltransferase (ChAT) or their staining for beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, and dopaminergic, serotoninergic, noradrenergic, and glutamatergic neurons that occur in the mesopontine tegmentum, were studied in the squirrel monkey (Saimiri sciureus). The ChAT-positive neurons in the pedunculopontine nucleus (PPN) form two distinct subpopulations, one that corresponds to PPN pars compacta (PPNc) and the other to PPN pars dissipata (PPNd). The ChAT-positive neurons in PPNc are clustered along the dorsolateral border of the superior cerebellar peduncle (SP) at trochlear nucleus levels, whereas those in PPNd are scattered along the SP from midmesencephalic to midpontine levels. At levels caudal to the trochlear nucleus, ChAT-positive neurons corresponding to the laterodorsal tegmental nucleus (LDT) lie within the periaqueductal gray and extend caudally as far as locus coeruleus levels. All ChAT-positive neurons in PPN and LDT stain for NADPH-diaphorase; the majority of large neurons in PPN and LDT are cholinergic, but some large neurons devoid of NADPH-diaphorase also occur in these nuclei. Cholinergic neurons in the mesopontine tegmentum form clusters that are largely segregated from raphe serotonin-immunoreactive neurons, as well as from nigral dopaminergic and coeruleal noradrenergic neurons, as revealed by tyrosine hydroxylase immunohistochemistry. Nevertheless, dendrites of cholinergic and noradrenergic neurons are closely intermingled, suggesting the possibility of dendrodendritic contacts. In addition, numerous large and medium-sized glutamate-immunoreactive neurons are intermingled among cholinergic neurons in PPN. Furthermore, at trochlear nucleus levels, about 40% of cholinergic neurons display glutamate immunoreactivity, whereas other neurons express glutamate or ChAT immunoreactivity only. This study demonstrates that 1) cholinergic neurons remain largely segregated from monoaminergic neurons throughout the mesopontine tegmentum and 2) PPN contains cholinergic and glutamatergic neurons as well as neurons coexpressing ChAT and glutamate in primates.

Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 colocalized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can selfassociate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.

The efferent projections of the pedunculopontine nucleus (PPN) to the basal ganglia have been studied in the squirrel monkey (Saimiri sciureus) with [3H]leucine and Phaseolus vulgaris-leucoagglutinin (PHA-L) as anterograde tracers. Following unilateral injections of [3H]leucine or PHA-L in the central portion of the PPN, numerous autoradiographic linear profiles or PHA-L-labeled fibers ascend to the forebrain, both ipsilaterally and contralaterally. These fibers form a compact bundle that courses in the central portion of the mesopontine tegmentum. At rostral mesencephalic levels, this bundle splits into ventromedial and dorsolateral fascicles that arborize in basal ganglia and thalamic nuclei, respectively. The substantia nigra and the subthalamic nucleus are by far the most densely innervated structures of the basal ganglia. In these two nuclei, labeled fibers arborize profusely ipsilaterally and less abundantly contralaterally. The labeled fibers in the substantia nigra are thin and varicose and arborize almost exclusively in the pars compacta, where they closely surround the soma and proximal dendrites of dopaminergic neurons. In the subthalamic nucleus, labeled fibers are also thin and appear to contact more than one neuron along their course. Numerous labeled fibers also occur in the pallidal complex, where they arborize most profusely in the internal segment. Several thick, labeled fibers oriented dorsolaterally in the pallidal complex give rise to thinner fibers that closely surround the soma and proximal dendrites of pallidal neurons. Some labeled fibers are also scattered in the striatum. These fibers abound in the peripallidal and ventral portions of the putamen, are more sparsely distributed in the remaining portion of the putamen as well as in the caudate nucleus, and are virtually absent in the ventral striatum. These results reveal that the PPN gives rise to a massive and highly ordered innervation of the basal ganglia in the squirrel monkey. This nucleus may thus act as an important relay in the basal ganglia circuitry in primates.

A specific antibody raised against 5-hydroxytryptamine (5-HT) conjugated to bovine serum albumin was used to study the serotoninergic innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus). At midbrain level, numerous fine 5-HT-immunoreactive axons were seen to arise from the immunopositive neurons of the dorsal raphe nucleus and less abundantly from those of the nucleus centralis superior. The bulk of these axons formed a rather loosely arranged bundle that arched ventrorostrally through the central portion of the midbrain tegmentum and ascended toward the ventral tegmental area. Several fascicles detached themselves from this bundle to reach the substantia nigra where they arborized into a multitude of heterogeneously distributed 5-HT terminals. The 5-HT innervation was particularly dense in the pars reticulata but much less so in the pars compacta of the substantia nigra. More rostrally other 5-HT fibers swept dorsolaterally and formed a remarkably dense network of varicose fibers within the subthalamic nucleus. A multitude of 5-HT axons continued their ascending course within the lateral hypothalamic area, and many of them swept laterally to invade the lenticular nucleus. At pallidal levels, the 5-HT axons arborized much less profusely in the external segment than in the internal segment, which contained numerous 5-HT varicose fibers and terminals arranged in a typical bandlike pattern. At striatal levels, the 5-HT terminals were particularly abundant in the ventral striatum, including the nucleus accumbens and deep layers of the olfactory tubercle. They also abounded in the ventrolateral region of the putamen and the ventromedial aspect of the caudate nucleus. Overall, the number of 5-HT fibers and terminals decreased progressively along the rostrocaudal axis of the striatum and several large and elongated zones rather devoid of 5-HT immunoreactivity were visualized, particularly in the caudate nucleus and the dorsal putamen. These zones of poor 5-HT immunoreactivity were in register with similar areas devoid of tyrosine hydroxylase immunoreactivity as seen on contiguous sections. These findings reveal that all the core structures of the basal ganglia in primates receive a significant serotoninergic input, but that the densities and patterns of innervation vary markedly from one structure to the other.

The organization of the dopaminergic mesostriatal fibers and their patterns of innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus) were studied immunohistochemically with an antiserum raised against tyrosine hydroxylase (TH). Numerous fibers arose from midbrain TH-positive cell bodies of the substantia nigra pars compacta (group A9), the retrorubral area (group A8), and the lateral portion of the ventral tegmental area (group A10). These fibers accumulated dorsomedially to the rostral pole of the substantia nigra where they formed a massive bundle that coursed through the prerubral field and ascended along the laterodorsal aspect of the medial fore-brain bundle in the lateral hypothalamus. Some ventrally located fibers ran throughout the rostrocaudal extent of the lateral preopticohypothalamic area and could be followed up to the olfactory tubercle, whereas other fibers turned laterodorsally to invade the head of the caudate nucleus. At more dorsal levels in the lateral hypothalamus, many fiber fascicles detached themselves from the main bundle and swept laterally to reach the globus pallidus, the putamen, and the amygdala. Several TH-positive fibers coursed along the dorsal surface of the subthalamic nucleus, and some invaded the dorsomedial third of this structure. The remaining portion of the subthalamic nucleus contained relatively few TH-positive elements. In contrast, the globus pallidus received a dense dopaminergic innervation deriving mostly from two fascicles that coursed backward along the two major output pathways of the pallidum: the lenticular fasciculus caudodorsally and the ansa lenticularis rostroventrally. At the pallidal level, the labeled fibres merged within the medullary laminae and arborized profusely in the internal pallidal segment and less abundantly in the external pallidal segment. However, the caudoventral portion of the external pallidum displayed a dense field of TH-positive axonal varicosities. Other fibers ran through the dorsal two-thirds of the external pallidum en route to the putamen. The striatum contained a multitude of thin axonal varicosities among which a few long and varicosed fibers were scattered. These immunoreactive neuronal profiles were rather uniformly distributed along the rostrocaudal extent of the striatum but appeared slightly more numerous in the ventral striatum than in the dorsal striatum. The pattern of distribution of the TH-positive axonal varicosities in the dorsal striatum was markedly heterogeneous: it consisted of typical zones of poor TH immunoreactivity lying within a matrix of dense terminal labeling.(ABSTRACT TRUNCATED AT 400 WORDS)