Vania Ramirez

vania.ramirez@neuro.ki.se

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Curriculum Vitae

Thesis & Post Doctorate Program

Abstracts

Publications
 
 

Curriculum Vitae

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Born in Rancagua, Chile, the 23rd of January 1967. Resident in Sweden since June 1976. Became Swedish citizen the 14th of February 1990.
Finished medicine studies at the Faculty of Medicine, Karolinska Institutet, 12th of June 1992.
Has been teaching Anatomy at the Karolinska Institutet since 1989,
initially with a position at the former Department of Anatomy, and then at the Department of Neuroscience.
Obtained a passing grade in the examinations at the Faculty of Medicine, Karolinska Institute for a PhD degree in Neuroscience, 25th of April 1997.
Thesis title: Morphological and immunohistochemical studies on the organisation of sacral motoneurons.
Finished internship at Huddinge hospital, 28th of February 1998.
Obtained authorization from the National Swedish Board of Health and Welfare to practise the Medical Profession as a Physician, 7th of April 1998.
Postdoctoral studies at the Department of Anatomy, Institute of Basic Medical Science, Oslo, in collaboration with professor Ole Petter Ottersen, 1999-2000.

The aim of this thesis was to study pre- and postsynaptic elements during postnatal development and aging. Motoneurons from two adjacent sacral motor nuclei in the cat spinal cord were investigated: the motoneurons innervating intrinsic foot-sole muscles (dorsolateral motor nucleus; DLN) and the motoneurons innervating striated perineal muscles (Onuf’s nucleus, ventrolateral motor nucleus; VLN). Most of the work has focused on dendrites, since they constitute >90% of the motoneuron’s receptive domain. The employed techniques have been intracellular tracer labeling, indirect single- and double-staining immunofluorescence, and pre- and post-embedding immunocytochemistry.
The establishment of VLN dendrites in a circumscribed rostro-caudally oriented bundle takes place during the first two postnatal months and appears to be closely linked to the occurrence of dendro-dendritic contacts. During maturation a dramatic drop in the proportion of the dendritic membrane area involved in this type of contacts was seen.
In the DLN, a postnatal remodeling of the dendrites was seen, including a five-fold increase in total dendritic membrane area, changes in branching pattern and a net decrease in number of branches per dendrite. Dendritic growth/remodeling and neo-synaptogenesis were, however, also seen in aged animals (15y), indicating that neurons and their connectivity are continuosly “remodeled”.
Several stigmata of aging were present in the VLN and DLN neuropil of older cats, as the increased frequency of dark-degenerating dendrites, and the occurrence of “aberrant” dendrites with an irregular outline, increased electron density of the dendroplasm and tighter packing of neurotubules.
Regarding presynaptic elements, the distribution and synaptic arrangement of different transmitter identified inputs to the VLN and DLN was examined. In adult animals, the descending bulbospinal serotoninergic pathway to the VLN was of the same size-order as that to the DLN. Experimental lesions (spinal cord transection at the lower thoracic level performed alone or in combination with unilateral dorsal rhizotomy) revealed a dual supraspinal and spinal origin for the substance P input to both nuclei. The contribution of substance P to the innervation of the VLN and DLN was of the same magnitude, although substance P-immunoreactive (IR) fibres of spinal origin were somewhat more frequent in the VLN. In addition, the VLN received also a denser enkephalinergic innervation than the DLN. Thirty-four percent of the boutons apposing VLN dendrites were immunoreactive for glutamate, while fourty-nine percent of all boutons were immunoreactive for GABA and/or glycine. The proportions of boutons immunoreactive to glutamate and GABA and/or glycine were rather similar in the VLN and DLN. However, the proportion of boutons single labeled for glycine was greater in the DLN than in the VLN.
Substance P-IR boutons had a rather even distribution throughout the dendritic trees of the VLN, apposing both thin distal branches and thick proximal dendrites, while TRH and enkephalin were preferentially located in apposition to more proximal dendritic domains. A frequent fining in the VLN was that one axonal bouton could be seen in synaptic contact with 2-3 dendrites. This divergence of the input at the terminal level was commonly seen among substance P-IR, enkephalin-IR and glutamate-IR boutons, as well as among boutons immunoreactive to GABA and/or glycine. A similar arrangement was not observed for TRH, indicating that the supraspinal input to the VLN might rather be a point-to-point system with each axonal bouton acting on a single postsynaptic element.
In the VLN, differences were seen between animals of different ages in the distribution of amino acid inputs, in particular a smaller number of boutons immunoreactive to glutamate was observed in the older animals. Since no such differences were present in the DLN, this may reflect interindividual variations, changes in the endocrine system and/or differences with age.
In the spinal cord of aged rats, axonal profiles showing aging-related morphological changes (accumulation of residual bodies, dystrophy and/or degeneration) were often enriched with immunoreactivity to glutathione (GSH). Since GSH is a scavenger for H2O2, this suggests that oxidative stress may be relevant for axon degeneration during aging. In addition, about sixty-five percent of the GSH enriched axons with aging-related changes contained also glutamate-immunoreactivity. Aging-related degeneration could not be verified for axons containing glycine and/or GABA. This implies that excitatory pathways may be more susceptible to aging-related changes than inhibitory pathways.

Keywords: spinal cord, dendrites, synaptic input, aging.

IBSN 91-628-2403-1.

The ventrolateral nucleus (VLN), considered to be the feline homologue to Onuf’s nucleus in man (Onuf, 1900), is located in the ventral horn at the S1/S2 spinal segment levels. VLN motoneurons innervate striated perineal muscles, as the sphincter urethrae, the sphincter ani externus, the bulbospongiosus and the ischiocavernosus muscles (e.g. Sato et al., 1978; Kuzuhara et al., 1980). Dorsolateral to the VLN is the somatic motor nucleus (dorsolateral nucleus; DLN) whose motoneurons innervate the intrinsic muscles of the foot sole, i.e. the flexors digitorum brevis and longus, and the plantaris muscle (e.g. Egger et al., 1980; Ulfhake and Kellerth, 1983).

In contrast to DLN motoneurons, VLN motoneurons are strongly related with autonomic spinal cord centers, as the sacral parasympathetic nucleus (SPN) that supplies the smooth muscles of the bladder and rectum. The dense aminergic and peptidergic innervation of the VLN is also closer to the innervation pattern of the SPN than to that of the DLN (Blok and Holstege, 1996). The similarities in afferent input between the SPN and VLN may be a means to co-ordinate the activity pattern of somatic and visceral structures in complex functions such as micturition, defecation and copulatory behaviour (Blok and Holstege, 1996).

The predisposition of VLN motoneurons to certain degenerative diseases differs from that seen in adjacent lumbosacral motor nuclei, such as the DLN, and appears instead to be related to the selective vulnerability of autonomic spinal cord centers. Thus, in amyotrophic lateral sclerosis VLN motoneurons are more resistant than nearby located motoneurons (as in the DLN) that degenerate/die. This implicates that incontinence is a rather late symptom among ALS-patients (Mannen et al., 1977). VLN motoneurons are however extensively affected in for example Shy-Drager disease, that affects more selectively the autonomic nervous system (Sung et al., 1979).

Another specific characteristic of the VLN, that has extensively been studied in its homologue in the rat, is the androgen-dependent sexual dimorphism in terms of cells size and number (see Breedlove, 1986; Arnold, 1992). This sexual dimorphism is established during early development and is believed to be due to the fact that VLN motoneurons supply perineal muscles that are of importance in male reproduction, but that are rudimentary/lacking in females.

Against this background, comparative studies between the VLN, a sexually dimorphic motor nucleus that is intimately related to the autonomic nervous system, and the DLN, a presumably non-dimorphic, purely somatic motor nucleus, appear to be of interest.

The aim of this project is to study motoneurons located in two adjacent motor nuclei in the sacral segments of the spinal cord, VLN and DLN. There is both indirect and direct evidence that glutamate, the most important excitatory neurotransmitter in the nervous system, may be involved in the pathology of ALS. It is however not known if the disturbances in glutamatergic signal transmission that have been shown are a part of the primary process or if they represent final stages of injury following several steps that may have caused alterations in neuron metabolism. The described model system (VLN/DLN) appears to be suitable for further studies of, amongst others, the molecular organization and development of glutamatergic synapses. The aims would be to investigate the cause(s) of the relative resistance of VLN motoneurons to ALS, in comparison to the notorious vulnerability of DLN motoneurons.

 One mechanism suggested to induce degenerative changes during aging is cellular accumulation of free radicals (“free radical theory of aging”; e.g. review by Harman, 1981). Thus, aging-related degeneration may, at least in part, be explained by a changed cellular redox status with decreased antioxidant capacity and/or an increased oxidative stress. Since a previous study showed increased glutathione levels in the aged rat lumbar motor nuclei (Ramírez-León et al., 1999), we now examine the distribution of free radical scavengers in the VLN/DLN.

Conventional postembedding immunohistochemistry of freeze-substituted tissue permits a precise localization at the ultrastructural level of antigens otherwise not detectable. We use this technique to study glutamatergic synapses on VLN/DLN motoneurons, looking at the synaptic arrangement and degree of expression of glutamate receptors. In addition, we examine the pre- and postsynaptic distribution of glutathione and superoxide dismutase in the VLN/DLN of young adult and aged animals, as well as the possible presence of these scavengers in glutamatergic pathways. The work is done in collaboration with professor Ole Petter Ottersen, Department of Anatomy, Institute of Basic Medical Sciences, Oslo.

References
Arnold, A.P. (1992) Exp. Gerontol. 27:99-110.
Blok, B.F.M., Holstege, G. (1996) Progress in Brain Res. 107:113-126.
Breedlove, S.M. (1986) J. Neurobiol. 17:157-176.
Egger, M.D. et al. (1980) J. Physiol. 306: 349-363.
Goldstein, L.A., Sengelaub, D.R. (1994) J. Neurobiol. 25:878-892.
Harman, D. (1981) Proc. Natl. Acad. Sci. USA 78:7124-7128.
Hays, T.C. et al. (1996) Dev. Brain Res. 91:20-28.
Kuzuhara, S. et al. (1980) Neurosci. Lett. 16:125-130.
Mannen, T. et al. (1977) J. Neurol. Neurosurg. Psychiatry 40:464-469.
Onuf, B. (1900) Arch. Neurol. Psychopathol. 3:387-412.
Ramírez-León, V. et al. (1999) Europ. J. Neurosci. 11:2935-2948.
Sato, M. et al. (1978) Brain Res. 140:149-154.
Sung, J.H. et al. (1979) J. Neuropathol. Exp. Neurol. 38:353-368.
Ulfhake, B., Kellerth, J.-O. (1983) Brain Res. 264:1-19.