Staffan Cullheim

Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons

Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson′s disease (PD), a common age-associated neurodegenerative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. We have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD.

GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury

Glial cell line-derived neurotrophic factor (GDNF) exhibits neurotrophic properties on different types of neurones, including fetal motoneurones and embryonic neurones of sensory ganglia. We demonstrate that chronic injury to the adult rat sciatic nerve induces a rapid up-regulation of GDNF mRNA expression in Schwann cells proximal as well as distal to the injury site, and that expression of this mRNA remains at high levels for at least 5 months after injury. In addition, GDNF mRNA increases and remains high in satellite cells and Schwann cells of the affected L4/L5 DRGs. These findings suggest that GDNF is an important factor in the events that follow upon adult chronic primary sensory neurone injury, and possibly also after adult motoneurone axotomy.

Combined light and electron microscopic tracing of neurons, including axons and synaptic terminals, after intracellular injection of horseradish peroxidase

By using intracellular deposition of horseradish peroxidase (HRP), a method has been designed for combined light and electron microscopic tracing of single fibers and synaptic terminals, belonging to physiologically characterized neurons. This technique has been successfully tested on the axon collateral systems of cat alpha-motoneurons, but should also be applicable to other types of neurons.

Relations between cell body size, axon diameter and axon conduction velocity of cat sciatic α-motoneurons stained with horseradish peroxidase

The relations between cell body size, intramedullary axon diameter and axon conduction velocity were examined in cat sciatic alpha-motoneurons after intracellular staining with horseradish peroxidase. All relations showed significant (P < 0.001) positive linear correlation coefficients.

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.

Large cholinergic nerve terminals on subsets of motoneurons and their relation to muscarinic receptor type 2

The cholinergic C‐bouton is a large nerve terminal found exclusively apposing motoneuron cell somata and proximal dendrites. The origin and function of the C‐bouton is not known. An antiserum against the vesicular acetylcholine transporter was used to identify large cholinergic nerve terminals putatively of the C‐type in close apposition to motoneuron cell somata. This type of nerve terminal was present in the rat spinal cord ventral horn, but only in some cranial motor nuclei. Fluoro‐Gold tracing showed that subsets of spinal motoneuron cell somata were contacted by different numbers of putative C‐boutons. Thus, motoneurons innervating an intrinsic foot muscle were contacted by about half the number of cholinergic terminals found on motoneurons of the predominantly fast‐twitch gastrocnemius muscle. Slow‐twitch soleus motoneurons showed an intermediate innervation. There was a strong correlation between the presence of putative C‐boutons and muscarinic receptor 2 (m2)‐like immunoreactivity (‐LI) within a motor nucleus. By using confocal laser microscopy, the m2‐LI appeared to be confined to the motoneuron cell membrane and strongly enriched beneath the C‐type nerve terminal. Thus, our results suggested a differential distribution of large cholinergic C‐boutons, depending on motoneuron type, and that the presence of this nerve terminal type is associated with m2‐LI in the postsynaptic membrane. J. Comp. Neurol. 460:476–486, 2003.

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.