Brun Ulfhake

Factors contributing to neuromuscular impairment and sarcopenia during aging.

Motor disturbances and wasting of skeletal muscles (sarcopenia) causes significant impairment of daily life activities and is a major underlying cause for hospitalization in senescence. Herein we review data and present new findings on aging-specific changes in motoneurons, skeletal muscle and the interplay between motoneurons and target muscle fibers. Although many of the changes occurring during aging may be specific to motoneurons and myofibers, respectively, evidence indicates that myofiber regeneration in sarcopenic muscle is halted at the point where reinnervation is critical for the final differentiation into mature myofibers. Combined, evidence suggests that sarcopenia to a significant extent depend on a decreased capacity among motoneurons to innervate regenerating fibers. There are also conspicuous changes in the expression of several cytokines known to play important roles in establishing and maintaining neuromuscular connectivity during development and adulthood. We also present data showing the usefulness of rodent models in studies of successful and unsuccessful patterns of aging. Finally, we show that not only dietary restriction (DR) but also activity and social environment may modulate the pattern of aging.

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Among the hallmarks of aged organisms are an accumulation of misfolded proteins and a reduction in skeletal muscle mass (“sarcopenia”). We have examined the effects of aging and dietary restriction (which retards many age-related changes) on components of the ubiquitin proteasome system (UPS) in muscle. The hindlimb muscles of aged (30 months old) rats showed a marked loss of muscle mass and contained 2–3-fold higher levels of 26S proteasomes than those of adult (4 months old) controls. 26S proteasomes purified from muscles of aged and adult rats showed a similar capacity to degrade peptides, proteins, and an ubiquitylated substrate, but differed in levels of proteasome-associated proteins (e.g. the ubiquitin ligase E6AP and deubiquitylating enzyme USP14). Also, the activities of many other deubiquitylating enzymes were greatly enhanced in the aged muscles. Nevertheless, their content of polyubiquitylated proteins was higher than in adult animals. The aged muscles contained higher levels of the ubiquitin ligase CHIP, involved in eliminating misfolded proteins, and MuRF1, which ubiquitylates myofibrillar proteins. These muscles differed from ones rapidly atrophying due to disease, fasting, or disuse in that Atrogin-1/MAFbx expression was low and not inducible by glucocorticoids. Thus, the muscles of aged rats showed many adaptations indicating enhanced proteolysis by the UPS, which may enhance their capacity to eliminate misfolded proteins and seems to contribute to the sarcopenia. Accordingly, dietary restriction decreased or prevented the aging-associated increases in proteasomes and other UPS components and reduced muscle wasting.

Estrogen receptor‐α and ‐β immunoreactive neurons in the brainstem and spinal cord of male and female mice: Relationships to monoaminergic, cholinergic, and spinal projection systems

For many populations of estrogen‐sensitive neurons it remains unknown how they are associated with central nervous system circuitries that mediate estrogen‐induced modulation of behavioral components. With the use of double‐labeling immunohistochemistry and tracing techniques, the relationships of estrogen receptor (ER)‐α‐ and ER‐β‐immunoreactive (IR) neurons in the mouse brainstem and spinal cord to monoaminergic, cholinergic, and spinal projection systems are explored. Similar distributions of ER‐IR neurons were present in females and males, with differences in labeling intensity of ER‐α immunoreactivity among males and estrogen‐, and oil‐treated females. Barringtons nucleus, the ventrolateral medulla, and the nucleus of the solitary tract contained spinal‐projecting ER‐α‐IR neurons, whereas ER‐α‐IR neurons in the periaqueductal gray, parabrachial nucleus, and catecholaminergic A1 cell group received spinal input. Numerous tyrosine hydroxylase (TH)‐IR ER‐α‐IR neurons were present in the ventral periaqueductal gray, nucleus of the solitary tract, A1 cell group, and lumbosacral cord. The dorsal raphe nucleus contained ER‐α‐IR and ER‐β‐IR neurons that colocalized with serotonin (5HT), and the reticulotegmental nucleus contained 5HT‐IR ER‐α‐IR neurons. Fibers IR for vesicular acetylcholine transporter (VAChT), TH, and 5HT were located among ER‐α‐IR neurons in the dorsal horn and spinal autonomic regions. Robust staining for TH and VAChT, but not 5HT, was present among ER‐α‐IR neurons in the lumbosacral lateral collateral pathway. Possible modulatory actions of estrogen on each of these ER‐IR populations are discussed in the context of their specific function, including micturition, sexual behavior, ejaculation, cardiovascular and respiratory control, tactile and nociceptive sensory processing, anti‐nociception, endocrine regulation, and feeding. J. Comp. Neurol. 488:152–179, 2005.

Motoneurons reinnervate skeletal muscle after ventral root implantation into the spinal cord of the cat

By use of intracellular recording and staining with horseradish peroxidase it was found that alpha and probably also gamma motoneurons were able to reinnervate ventral root implants after an avulsion of ventral roots at the spinal cord surface in the cat. The reinnervation of the implant was achieved after an initial growth of new axons in central nervous system tissue. Reinnervating neurons could be excited or inhibited by segmental reflex activity and their axons could conduct nerve impulses. The character of muscle twitch responses elicited by electrical stimulation of implanted roots strongly indicated that denervated muscles were reinnervated by new motor axons via the implant.

An ultrastructural study of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal boutons in the motor nucleus of spinal cord segments L7-S1 in the adult cat

The distribution and fine structure of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive synaptic boutons and varicosities were studied in the motor nucleus of the spinal cord segments L7-S1 in the cat, using the peroxidase-antiperoxidase immunohistochemical technique and analysis of ultrathin serial sections. The 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive boutons had a similar ultrastructural appearance as judged from serial section analysis. The boutons could be classified into two types on the basis of their vesicular content, with one type containing a large number of small agranular vesicles together with only a few, if any large granular vesicles, while the other type contained a large number of large granular vesicles in addition to small agranular vesicles. The vesicles were spherical or spherical-to-pleomorphic. Postsynaptic dense bodies (Taxi bodies) were occasionally observed in relation to all three types of immunoreactive boutons, which almost invariably formed synaptic junctions with dendrites. Judged by the calibre of the postsynaptic dendrites, the boutons were preferentially distributed to the proximal dendritic domains of motoneurons. In one case, a substance P-immunoreactive bouton formed an axosomatic synaptic contact. In addition to synaptic boutons, 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal varicosities containing a large number of large granular and small agranular vesicles but lacking any form of conventional synaptic contact were observed. Such varicosities were either directly apposing surrounding neuronal elements or separated from the neurons by thin glial processes. The origin of the immunoreactive boutons was not traced, but it was thought likely that the main source of the boutons was neurons with their cell bodies located in the medullary raphe nuclei.

Multiple messengers in descending serotonin neurons: localization and functional implications.

In the present review article we summarize mainly histochemical work dealing with descending bulbospinal serotonin neurons which also express a number of neuropeptides, in particular substance P and thyrotropin releasing hormone. Such neurons have been observed both in rat, cat and monkey, and may preferentially innervate the ventral horns of the spinal cord, whereas the serotonin projections to the dorsal horn seem to lack these coexisting peptides. More recent studies indicate that a small population of medullary raphe serotonin neurons, especially at rostral levels, also synthesize the inhibitory neurotransmitter gamma-amino butyric acid (GABA). Many serotonin neurons contain the glutamate synthesizing enzyme glutaminase and can be labelled with antibodies raised against glutamate, suggesting that one and the same neuron may release several signalling substances, causing a wide spectrum of post- (and pre-) synaptic actions.

Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons

SummaryBy use of fluorescence immunohistochemistry it is shown that sciatic nerve section in cat and rat induces increased levels of immunoreactive calcitonin gene-related peptide (CGRP) in axotomized motoneurons. In the rat, this effect was clearly seen at 2–5 days postoperatively, but could not be demonstrated after 11–21 days. These findings are discussed in relation to previously proposed roles for CGRP in motoneurons.

Distribution of glutamate-, glycine- and GABA-immunoreactive nerve terminals on dendrites in the cat spinal motor nucleus

Abstract The dendritic tree constitutes more than 93% of the receptive membrane area of a spinal motoneuron, yet little is known about its synaptic inputs. In this study we examined the distribution of glutamate-, GABA- and glycine-like immunoreactivity in boutons apposing dendrites in the L7 spinal cord motor nucleus, by use of postembedding immunohistochemistry on serial sections.We examined 799 boutons apposing 401 cross-sectioned dendrites of different calibre (range 0.2–15 µm), and 14 first-order (stem) dendrites. Thirty-five percent (35%) of the boutons were immunopositive for glutamate and 59% for GABA and/or glycine. Among the latter, 30% showed glycine immunoreactivity only and 24% were immunoreactive for both GABA and glycine. Very few were immunoreactive only for GABA (5%). As few as 6% of the boutons were judged as not enriched for any amino acid analysed. The fine structural characteristics of the boutons were in accordance with previous descriptions. The sample of dendrites was arranged in calibre bins in order to facilitate distribution analysis. Stem dendrites differed from the other bins, with a high total bouton covering (61%) and a high bouton density. Sixty-nine percent of the membrane covering was by glycine- and/or GABA-immunoreactive boutons, whereas 18% was covered by boutons enriched in glutamate. For non-stem dendrites, bouton covering fell from 33% to 12% with decreasing calibre. However, bouton apposition length decreased in parallel, yielding a fairly uniform bouton density among dendrites of different calibre. The lack of correlation between packing density and dendrite calibre was also evident when the sample of dendrites was broken down into subsamples based on content of amino acid immunoreactivity. The latter analysis also revealed that both the relative covering and density of boutons containing inhibitory amino acids (57%; glycine and/or GABA) and glutamate (38%), respectively, did not vary systematically with dendrite calibre. Combined, the data indicate that in non-stem dendrites the proportion of excitatory and inhibition inputs does not change systematically throughout the dendritic arborizations of spinal α-motoneurons. Thus, spinal motoneurons can, with respect to the general synaptic architecture, be divided into two main compartments, i.e. the proximal soma-juxtasomatic compartment (including stem dendrites) and the distal dendritc compartment. The proximal domain is under a powerful glycine and/or GABA influence. Finally, based on the data presented here and previously published data, it was calculated that spinal α-motoneurons receive in the range of 50–140×103 synaptic boutons.

Loss of primary sensory neurons in the very old rat: neuron number estimates using the disector method and confocal optical sectioning.

Loss of neurons has been considered to be a prime cause of nervous disturbances that occur with advancing age. However, the notion of a constitutive aging‐related loss of neurons has been challenged recently in several studies that used up‐to‐date methods for counting neurons. In this study, we have applied stereological techniques with the objective of obtaining quantitative data on total neuron numbers and the distribution of neuron cross‐sectional areas in the fifth cervical (C5) and fourth lumbar (L4) dorsal root ganglion (DRG) of 3‐ and 30‐month‐old Sprague‐Dawley rats. Tissue data were recorded on a confocal laser‐scanning microscope with the use of the optical‐disector technique and random, systematic sampling.

Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging.

We have here sought to cross-correlate the expression of immunoreactivities for several neuropeptides, nitric oxide synthase (NOS) and the growth associated protein GAP-43 in subpopulations of dorsal root ganglion (DRG) neurons tagged by the selective markers isolectin B4 and the neurofilament antibody RT97, selective for, respectively, subpopulations of small and large DRG neurons. By use of double- and triple-labeling immunohistochemistry, non-manipulated and sciatic nerve transected young adult rats as well as aged (30-months-old) rats were examined using a confocal microscope equipped with enhanced spectral separation. In young adult rats, the DRG neuron profiles could be divided into three subpopulations (B4 binding (B4+) approximately 50%; RT97-immunoreactive (RT97+) approximately 35%; B4-/RT97- approximately 15%). Calcitonin gene-related peptide (CGRP) is expressed in all three subpopulations. Galanin message-associated peptide (GMAP) colocalize with CGRP (100%) but is not expressed in RT97+ profiles. NOS is present in the RT97- subpopulations and frequently colocalize with CGRP (92%). GAP-43 is expressed in all three DRG subpopulations and colocalize with CGRP (88%), GMAP (38%) and/or NOS (22%). Only very small differences were seen among the young adult rats, implicating that the size of respective subpopulation as well as the expression pattern for neuropeptides, NOS and GAP-43 are fairly stable. Sciatic nerve transection reduced B4-binding but not RT97-like immunoreactivity. Distinct changes in the expression of neuropeptides, NOS and GAP-43 were evident in the DRG subpopulations and, furthermore, the regulatory changes were very similar among the lesioned animals. The relative size of the DRG subpopulations was unaffected by aging, while the expression of neuropeptides was altered showing similarities with the changes induced by axotomy in young adult rats.