Quantitative morphological comparison of axon-targeting strategies for gene therapies directed to the nigro-striatal projection.

Cellular targeting of mRNAs and proteins to axons is essential for axon growth during development and is likely to be important for adult maintenance as well. Given the importance and potency of these axon-targeting motifs to the biology of axons, it seems possible that they can be used in the design of transgenes that are intended to enhance axon growth or maintenance, so as to improve potency and minimize off-target effects. To investigate this possibility, it is first essential to assess known motifs for their efficacy. We have therefore evaluated four axon-targeting motifs, using adeno-associated viral vector-mediated gene delivery in the nigro-striatal dopaminergic system, a projection that is predominantly affected in Parkinson's disease. We have tested two mRNA axonal zipcodes, the 3' untranslated region (UTR) of ß-actin and 3' UTR of tau, and two axonal-targeting protein motifs, the palmitoylation signal sequence in GAP-43 and the last 15 amino acids in the amyloid precursor protein, to direct the expression of the fluorescent protein Tomato in axons. These sequences, fused to Tomato, were able to target its expression to dopaminergic axons. Based on quantification of Tomato-positive axons, and the density of striatal innervation, we conclude that the C-terminal of the amyloid precursor protein is the most effective axon-targeting motif.

Impact of Frailty on Gait Speed Improvements in Home Health after Hospital Discharge: Secondary Analysis of Two Randomized Controlled Trials.

More than half of older adults are frail or prefrail in the United States, and hospital-associated deconditioning likely increases this risk. However, the impact of frailty on potential functional improvements after hospital discharge is poorly understood. We sought to identify the influence of baseline frailty on gait speed change in older adults receiving home health physical therapy (PT) after hospital discharge. The severity of frailty was assessed using Cardiovascular Health Study frailty criteria (weakness, slowness, weight loss, physical inactivity, and exhaustion). Gait speed was measured at baseline and 60-days post-hospital discharge. Upon admission to home health rehabilitation services, half of older adults (total N=250) were considered frail, with slowness (90%) and weakness (75%) being the most common characteristics. Older adults, whether pre-frail or frail, demonstrated similar and clinically meaningful improvements in gait speed after receiving home health rehabilitation for 60 days following hospital discharge. These results suggest that clinicians caring for older adults in the hospital can counsel both pre-frail and frail patients that, with home health rehabilitation, clinically significant improvements in function can be expected over the 2 months following discharge. Furthermore, we observed encouraging gait speed improvement with physical therapy following hospitalization in older adults. Results can inform anticipatory guidance on hospital discharge.

Myosin isozymes in normal and cross-reinnervated cat skeletal muscle fibers.

Immunocytochemical characteristics of myosin have been demonstrated directly in normal and cross-reinnervated skeletal muscle fibers whose physiological properties have been defined. Fibers belonging to individual motor units were identified by the glycogen-depletion method, which permits correlation of cytochemical and physiological data on the same fibers. The normal flexor digitorum longus (FDL) of the cat is composed primarily of fast-twitch motor units having muscle fibers with high myosin ATPase activity. These fibers reacted with antibodies specific for the two light chains characteristic of fast myosin, but not with antibodies against slow myosin. Two categories of fast fibers, corresponding to two physiological motor unit types (FF and FR), differed in their immunochemical response, from which it can be concluded that their myosins are distinctive. The soleus (SOL) consists almost entirely of slow-twitch motor units having muscle fibers with low myosin ATPase activity. These fibers reacted with antibodies against slow myosin, but not with antibodies specific for fast myosin. When the FDL muscle was cross-reinnervated by the SOL nerve, twitch contraction times were slowed about twofold, and motor units resembled SOL units in a number of physiological properties. The corresponding muscle fibers had low ATPase activity, and they reacted with antibodies against slow myosin only. The myosin of individual cross-reinnervated FDL muscle units was therefore transformed, apparently completely, to a slow type. In contrast, cross-reinnervation of the SOL muscle by FDL motoneurons did not effect a complete converse transformation. Although cross-reinnervated SOL motor units had faster than normal twitch contraction times (about twofold), other physiological properties characteristic of type S motor units were unchanged. Despite the change in contraction times, cross-reinnervated SOL muscle fibers exhibited no change in ATPase activity. They also continued to react with antibodies against slow myosin, but in contrast to the normal SOL, they now showed a positive response to an antibody specific for one of the light chains of fast myosin. The myosins of both fast and slow muscles were thus converted by cross-reinnervation, but in the SOL, the newly synthesized myosin was not equivalent to that normally present in either the FDL or SOL. This suggests that, in the SOL, alteration of the nerve supply and the associated dynamic activity pattern are not sufficient to completely respecify the type of myosin expressed.

Synaptic activity in motoneurons during natural stimulation of muscle spindles.

Synaptic activity evoked in cat motoneurons during stretch stimulation of muscle spindles in the homonymous muscle has been studied by intracellular recording. A class of miniature excitatory postsynaptic potentials evoked by such physiologic stimulation results from activity in single group la spindle afferent fibers, and the criterion for identification is a predictable pattern of rhythmic occurrence of the synaptic potentials. Statistical examination of the amplitude distributions of this class of miniature synaptic potentials suggests that some group Ia fibers liberate a relatively large number of quantal excitatory postsynaptic potential units per fiber impulse.

Catch property in single mammalian motor units.

The tension output of single motor units in the cat triceps surae muscle was studied during patterned stimulation of the innervating motoneurons. With repetitive stimulation within a rather narrow frequency range, the tension output of slow-twitch (type S) motor units may be quite sensitive to the pattern of stimulus intervals in the train. The presence of only one stimulus interval that is much shorter than the others in the train can cause marked, long-lasting tension enhancement, provided that the stimulus repetition rate in the basic train is within certain limits.

Mammalian motor units: physiological-histochemical correlation in three types in cat gastrocnemius.

The correlation among a variety of physiological properties and the histochemical characteristics of muscle fibers belonging to single motor units in a mixed mammalian muscle is directly demonstrated. The population of motor units making up the cat gastrocnemius was classified into three nonoverlapping groups on the basis of a combination of physiological parameters. The muscle fibers belonging to motor units of each physiological type exhibited a distinctive histochemical profile, such that the three basic histochemical "fiber types" exactly matched the three physiologically defined groups. Within each individual motor unit, the muscle fibers were histochemically uniform.

Primary afferent depolarization evoked by a painful stimulus.

Pulses of intense radiant heat applied to the plantar pad of unanesthetized spinal cats produced negative dorsal root potentials, increased excitability of cutaneous A fibers, and marked activation of ipsilateral flexor motoneurons. The same effects were obtained during cold block of A fiber conduction in the appropriate peripheral nerve. We conclude that adequate noxious activation of cutaneous C fibers depolarizes cutaneous A fibers.

GDNF as a candidate striatal target-derived neurotrophic factor for the development of substantia nigra dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) has been known for many years to protect and restore dopamine neurons of the substantia nigra (SN) in lesion models of parkinsonism, but much less has been known of its normal physiologic role. We have found that GDNF injected into the striatum postnatally suppresses naturally-occurring cell death in SN dopamine neurons, and neutralizing antibodies augments it. Neutralizing antibodies augment cell death during the first phase, which occurs during the first postnatal week, but not during the second phase in the second week. To further explore the possible neurotrophic role of GDNF, we created double transgenic mice which overexpress GDNF exclusively in the target regions of mesencephalic neurons, particularly the striatum. As anticipated for a limiting, target-derived factor, this resulted in an increased surviving number of SN dopamine neurons after the first phase of cell death. However, this increase did not persist into adulthood. We conclude that GDNF is the leading candidate for a target-derived neurotrophic factor for SN dopamine neurons during the first phase of cell death, but that other factors must play an essential role in later development.

Casein and alpha-lactalbumin detection in breast cancer cells by immunocytochemistry.

Casein and alpha-lactalbumin are characteristic proteins produced by normal mammary cells under hormonal stimulation. Specific antisera were used to immunochemically stain for these two proteins in the unstimulated R3230AC transplantable rat mammary tumor as well as in normal lactating rat mammary tissue. Specific staining was observed in the lactating rat alveolar epithelial cells and in the cells lining the intraalveolar ducts with both antisera. In the R3230AC tumor some cells were shown to contain casein or alpha-lactalbumin; the majority of the tumor cells were unstained. These findings indicate that normal mammary cells as well as a small population of cells within the R3230AC tumor are actively synthesizing casein or alpha-lactalbumin. Furthermore, the results suggest that immunocytochemistry may be used to determine the functional heterogeneity of mammary tumors directly.

Lactate dehydrogenase in estrogen-responsive human breast cancer cells.

Lactate dehydrogenase activity (LDH) was measured in the MCF-7 human breast cancer cell line derived at the Michigan Cancer Foundation from a patient with metastatic breast adenocarcinoma. LDH was found in the 46,000 X g supernatant of cell lysates, but not in the culture medium. Only the fifth isozyme (LDH-5) could be demonstrated by cellulose acetate electrophoresis and relative heat inactivation studies. When endogenous steroids were removed from the medium, addition of estrogen to the growth medium for several days elevated LDH 2-fold above controls; LDH was not altered when MCF-7 cells were treated with progesterone, hydrocortisone, prolactin, insulin, or triiodothyronine. A physiological concentration (0.1 nM) of 17beta-estradiol was sufficient to produce a maximal LDH increase. There were no qualitative isozyme changes in response to estrogen. LDH activity may therefore be a useful marker protein for studying hormone action in the MCF-7 human breast cancer cell line.

Horseradish peroxidase study of the spatial and electrotonic distribution of group Ia synapses on type-identified ankle extensor motoneurons in the cat.

Eight functionally identified group Ia muscle afferents from triceps surae or plantaris muscles were labeled intraaxonally with horseradish peroxidase (HRP) in seven adult cats. Subsequently, HRP was injected into two to six homonymous or heteronymous alpha-motoneurons per animal (total = 22), each identified by motor unit type and located near the site of afferent injection. The complete trajectories of labeled afferents were reconstructed, and putative synaptic contacts on HRP-labeled motoneurons were identified at high magnification. Dendritic paths from each contact were also mapped and measured. A total of 24 contact systems (the combination of a group Ia afferent and a postsynaptic motoneuron) were reconstructed, of which 17 were homonymous, and seven were heteronymous. Overall, homonymous contact systems had an average of 9.6 boutons, whereas heteronymous contact systems had an average of 5.9 boutons. The average number of boutons found on type S motoneurons in homonymous contact systems was smaller (6.4, range 3-17) than in systems involving types FF or FR motoneurons (FF: 10.4, range 4-18; FR: 11.3, range 4-32). Neither of these differences were statistically significant. In contrast to earlier reports, a majority (15/24) of contact systems included more than one collateral from the same Ia afferent. The complexity (number of branch points) in the arborization pathway leading to each contact (overall mean 8.4 +/- 3.3) was virtually identical in all contact systems, irrespective of the type of postsynaptic motoneuron. The three-dimensional distribution of group Ia contacts was not coextensive with the radially organized dendrites of motoneurons: Dendrites oriented in the ventromedial to dorsolateral axis had the fewest (8%) contacts, whereas rostrocaudal dendrites had the most (63%) contacts. Nevertheless, contacts were widely distributed on the motoneuron surface, with few on and near the soma (< or = 200 microns radial distance from the soma) or on the most distal parts of the tree (> or = 1,000 microns). The boutons in individual contact systems also showed wide spatial and estimated electrotonic distributions; only 3/24 systems had all contact located within a restricted spatial/electrotonic region. The relations between these anatomical results and existing electrophysiological data on group Ia synaptic potentials are discussed.

Medial gastrocnemius motor nucleus in the rat: age-related changes in the number and size of motoneurons.

The age-related alterations in the number and size of alpha- and gamma-motoneurons were studied in the medial gastrocnemius (MG) motor nuclei in rats at four ages: young (5 months), middle aged (10-13 months), old (26 months), and very old (31 months). Small volumes (0.1-0.5 microliter) of 40% horseradish peroxidase (HRP) solution were injected into the cut MG nerve bilaterally by using glass micropipettes and a pressure injection system. The number, position, and soma size (average soma diameter) of MG motoneurons were determined by using photographic maps of each TMB-stained section. The total number of myelinated axons was counted in seven MG nerves from the same animals. The average soma diameters in each MG nucleus were distributed bimodally; cells with average diameter greater than 21.0-24.0 micron were presumed to be alpha-motoneurons and those with smaller diameters were presumed to be gamma. The mean number of presumed alpha-motoneurons was significantly less in the old and very old groups as compared with the young and middle-aged. In contrast, the number of presumed gamma-motoneurons was the same across age groups. The mean average soma diameter of both alpha- and gamma-motoneurons was smaller in the old animals. The apparent decrease in the total number of labeled motoneurons in old animals was also reflected in a decrease in myelinated axon counts. We conclude that there is a significant decrease in the absolute numbers of motoneurons in rats aged 26 months and older, with most of the decrease occurring among the larger alpha-motoneurons.

The size and dendritic structure of HRP-labeled gamma motoneurons in the cat spinal cord.

We report quantitative data obtained from 60 fully reconstructed dendritic trees belonging to eight gamma-motoneurons (gamma-MNs) and six additional gamma-MNs that were not completely reconstructed. The cells were labeled intracellularly with horseradish peroxidase (HRP). These data are compared to measurements from 79 reconstructed dendrites belonging to seven documented alpha-motoneurons (alpha-MNs), supplemented by a larger sample of alpha-MNs labeled intracellularly or by retrograde transport with HRP. As expected from earlier studies, the soma dimensions and total membrane area of gamma-MNs were smaller than those of alpha-MNs. Although gamma-MN dendrites were, on average, slightly but significantly longer than those of alpha-MNs, the former had, on average, smaller diameter stem dendrites, less membrane area, and less profuse branching, and they tended to branch closer to the soma and to terminate farther from the soma. These differences were evident even when subsets of dendrites with similar stem diameters were compared. Some of the anatomical distinctions suggest that gamma-MNs are qualitatively as well as quantitatively different from alpha-MNs, even though the distributions of many of the morphological variables examined showed no abrupt discontinuities between the two motoneuron groups.

Membrane area and dendritic structure in type-identified triceps surae alpha motoneurons.

The size and branching structure of the dendritic tree were studied in nine type-identified triceps surae alpha-motoneurons that were labeled intracellularly with horseradish peroxidase and reconstructed from serial sections in the light microscope. The average total membrane area (AN) for motoneurons of type S (slow-twitch) motor units was about 22% smaller than AN for cells of type F units (including both FF and FR motor unit types in this category) (480.1 X 10(3) microns 2 vs. 617.7 X 10(3) microns 2, respectively). Systematic correlations were found between stem dendrite diameter and three measures of dendritic size: dendrite membrane area, combined dendritic length, and number of terminations. All of these correlations were significantly different for the dendrites of F and S motoneurons. Power-function relations between stem diameter and dendritic membrane area were used to estimate AN for a sample of 79 type-identified motoneurons. Mean estimated AN values were significantly different for the F and S motoneuron groups, despite a large overlap in AN values between these groups. The branching structure of dendrites of F and S motoneurons also showed clear differences. Type S motoneuron dendrites showed less-profuse branching and a more-even radial distribution of branch points than found in type F cells. Examination of two forms of the "3/2 power rule" for the relation between the diameters of parent and daughter dendritic branches at branch points showed that the dendrites of type S motoneurons conform less well with the anatomical constraints necessary to represent binary branching trees as equivalent cylinders than do dendrites of type F cells. There was no systematic difference between F and S motoneuron dendrites in the degree of asymmetry of first-order daughter trees. The results overall indicate that the dendrites of F and S motoneuron groups are structurally different, giving rise to a systematic difference in AN between these groups. Such structural differences suggest that the F and S groups of alpha-motoneurons can be viewed as intrinsically distinct cell types and not just large vs. small variants of the same cell species.

Three-dimensional architecture of dendritic trees in type-identified alpha-motoneurons.

We have studied the spatial distribution of dendrites of type-identified triceps surae alpha-motoneurons, labeled intracellularly with HRP, using a variety of analytical approaches that were designed to quantify the ways in which dendrites occupy three-dimensional space. All of the methods indicated a strong tendency for motoneuron dendrites to project radially. However, regions dorsal and ventral to the somata contained fewer dendritic elements, and less membrane area, than expected for complete radial symmetry. Individual dendrites projecting into these regions tended to be smaller than those projecting rostrocaudally or mediolaterally. Nevertheless, the center of mass of membrane area for five of six fully analyzed cells was within 100 micron of the soma and, in all six cells, was located in the same dorsoventral plane as the cell soma. Maps of the projection of dendritic branches onto concentric shells at various radial distances from the soma showed that some regions have high concentrations of branches, sometimes with considerable overlap between branches arising from different stem dendrites, while other regions have relatively few branches, or none at all. Each motoneuron exhibited a different pattern of projection and there were no systematic differences between fast-twitch (type F, including both types FF and FR units) and slow-twitch (type S) motoneurons evident in the patterns of dendritic concentration. Assessment of the three-dimensional territories of individual dendrites showed that dendrites with larger numbers of terminal branches tended to have larger spatial territories. Despite considerable scatter, the results suggest that the density of branches tends to be approximately the same in large and small dendrites, and in F and S cell groups. The results are discussed in relation to the spatial location of synaptic input to motoneurons.

Developmental cell death in dopaminergic neurons of the substantia nigra of mice.

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) undergo natural cell death during development in rats. Controversy exists as to the occurrence of this phenomenon in SNpc dopaminergic neurons in the developing mouse. Herein, by using an array of morphologic techniques, we show that many SNpc neurons fulfill the criteria for apoptosis and that the number of apoptotic neurons in the SNpc vary in a time-dependent manner from postnatal day 2 to 32. These dying neurons also show evidence of DNA fragmentation, of activated caspase-3, and of cleavage of beta-actin. Some, but not all of the SNpc apoptotic neurons still express their phenotypic marker tyrosine hydroxylase, confirming their dopaminergic nature. Consistent with the importance of target-derived trophic support in modulating developmental cell death, we demonstrate that destruction of intrinsic striatal neurons by a local injection of quinolinic acid (QA) dramatically enhances the magnitude of SNpc apoptosis and results in a lower number of adult SNpc dopaminergic neurons. Strengthening the apoptotic nature of the observed SNpc developmental cell death, we demonstrate that overexpression of the anti-apoptotic protein Bcl-2 attenuates both natural and QA-induced SNpc apoptosis. The present study provides compelling evidence that developmental neuronal death with a morphology of apoptosis does occur in the SNpc of mice and that this process plays a critical role in regulating the adult number of dopaminergic neurons in the SNpc.

A HRP study of the relation between cell size and motor unit type in cat ankle extensor motoneurons.

The dimensions of the somata and stem dendrites of 57 alpha- and three gamma-motoneurons, identified as to motor unit type and labeled by intracellular injection of horseradish peroxidase, were measured in the triceps surae and plantaris motor pools. The somata of type S motoneurons tended to be smaller (mean diameter 47.9 micrometers) than those of FF and FR units (52.5 and 53.1 micrometer, respectively) but these mean values were not significantly different and the data distributions showed considerable overlap between the unit types. The mean numbers and diameters of stem dendrites exhibited somewhat larger differences related to motor unit type and some of these were statistically significant. The total membrane area (AN) of each cell was estimated from measurements of the soma and stem dendrites, by using recent data and Ulfhake and Kellerth ('81) to calculate the membrane area of a dendritic tree from stem dendrite diameter. Mean AN varied with motor unit type in the sequence FF greater than FR greater than S (average values: 369 X 100(3) micrometers 2, 323 X 100(3) micrometers 2, and 250 X 100(3) micrometers 2, respectively). There was covariation between AN and the conduction velocity of the motor axon as well as with the force output from the muscle unit. Comparison of AN and motoneuron input resistance (RN) in 19 alpha-motoneurons suggested that the specific resistivity of the cell membrane in type S motoneurons was systematically higher than that characteristic of type FF or FR motoneurons.

Tardive dyskinesia: current clinical issues.

Tardive dyskinesia (TD) consists of persistent involuntary movements that are due to antipsychotic drug treatment. Prevention depends on accurate psychiatric diagnosis and use of antipsychotics only for specific indications. Little is known about what antipsychotic drug regimens affect the risk of TD. Clinical subtypes of TD may exist. Tardive dystonia differs from oral choreic TD in its clinical phenomenology, epidemiology, and clinical pharmacology. Another possible subtype, persistent motor restlessness, seems also distinguishable in its phenomenology and epidemiology. Both of these forms of TD can be disabling, whereas typical oral TD often is not. Although TD may spontaneously remit more often than was once believed, it nevertheless is often persistent. No current therapy is universally effective.

Serum trihexyphenidyl levels in the treatment of torsion dystonia.

Using a radioreceptor technique, we assayed serum trihexyphenidyl levels in patients with dystonia being treated chronically with high dosage. We found a significant correlation between total daily dose and the daily lowest (trough) serum levels. There was no relationship between serum levels and therapeutic response or toxicity. Toxicity was more closely related to patient age than to serum level. Although levels may be useful to monitor patient compliance, they cannot be used to judge adequacy of therapy.

Torsion dystonia: a double-blind, prospective trial of high-dosage trihexyphenidyl.

We studied trihexyphenidyl in the treatment of torsion dystonia in a prospective, double-blind crossover protocol. Thirty-one patients completed the protocol. Twenty-two (71%) had a clinically significant response. After a mean follow-up of 2.4 years, 68% of patients continued to take trihexyphenidyl, and 42% continued to show a considerable or dramatic benefit. The 30-mg dose used was generally well tolerated. High-dosage trihexyphenidyl therapy is effective in the management of torsion dystonia.