Gustavo Oscar Zuccolilli

DOPAMINERGIC MESENCEPHALIC SYSTEMS AND BEHAVIORAL PERFORMANCE IN VERY OLD RATS

Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinsons disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4-6 months), old (22-24 months) and senile (30-32 months) Sprague-Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species.

Impact of very old age on hypothalamic dopaminergic neurons in the female rat: A morphometric study

Dopaminergic neurons of the A12 (tuberoinfundibular dopaminergic system) and A14 (periventricular dopaminergic system) hypothalamic areas exert a tonic inhibitory control of prolactin secretion. Tuberoinfundibular dopaminergic system neuron function is known to decline during aging in rats, but little is known about the impact of extreme age on neuron number and morphology in the two systems. We morphometrically assessed the neurons of the tuberoinfundibular dopaminergic system and the periventricular dopaminergic system in female rats 6 (young, Y), 24 (old, O), and 30–32 (senescent, S) months old. Serial coronal sections of fixed hypothalami were immunohistochemically labeled for tyrosine hydroxylase, and immunoreactive perikarya from the A12 and A14 areas were quantitatively characterized and compared among the three age groups. Radioimmunoassay was used to measure serum prolactin. The number of A12 tyrosine hydroxylase–immunoreactive perikarya showed a steady decline with age, whereas the number of A14 tyrosine hydroxylase–immunoreactive perikarya remained stable from young to old age but showed a sharp drop in the senescent rats. In the old rats, tyrosine hydroxylase–immunoreactive neuronal area (A12 = 135.37 and A14 = 158.79 μm2) was significantly higher than that of young (A12 = 72.56 and A14 = 99.7 μm2) and senescent animals (A12 = 95.5 and A14 = 106.5 μm2). Densitometric assessment of median eminence tyrosine hydroxylase immunoreactivity revealed a steady age‐related reduction of tyrosine hydroxylase content in the median eminence. Serum prolactin levels increased steadily with age. We conclude that, in the female rat, aging brings about a progressive loss of both tuberoinfundibular dopaminergic system and periventricular dopaminergic system neurons, which becomes more conspicuous at extreme ages. J. Comp. Neurol. 458:319–325, 2003.

Loss of NeuN immunoreactivity in rat spinal cord neurons during aging

A morphologic study of the impact of aging on neuron marker expression was performed in different segments of the rat spinal cord. Spinal cord specimens from young (5 months), middle-aged (12 months) and senile (32 months) female rats were assessed. We found a complete loss of neuron-specific nuclear protein (NeuN) immunoreactivity in cervical, thoracic and lumbar segments of the senile animals whereas neuron-specific enolase (NSE) immunoreactivity was comparable in young and senile rats. These findings in otherwise morphologically well preserved spinal cord neurons are of interest and reveal that NeuN may not be a reliable marker to identify neurons in the spinal cord of aging rats.

Cognitive impairment and morphological changes in the dorsal hippocampus of very old female rats.

The hippocampus, a medial temporal lobe structure necessary for the formation of spatial memory, is particularly affected by both normal and pathologic aging. In previous studies, we observed a significant age-related increase in dopaminergic neuron loss in the hypothalamus and the substantia nigra of female rats, which becomes more conspicuous at extreme ages. Here, we extend our studies by assessing spatial memory in 4-6 month-old (young), 26-month-old (old) and 29-32-month-old (senile) Sprague-Dawley female rats as well as the age-related histopathological changes in their dorsal hippocampus. Age changes in spatial memory performance were assessed with a modified version of the Barnes maze test. We employed two probe trials (PTs), one and five days after training, respectively, in order to evaluate learning ability as well as short-term and longer-term spatial memory retention. A set of relevant hippocampal cell markers was also quantitated in the animals by means of an unbiased stereological approach. The results revealed that old rats perform better than senile rats in acquisition trials and young rats perform better than both aging groups. However, during short-term PT both aging groups showed a preserved spatial memory while in longer-term PT, spatial memory showed deterioration in both aged groups. Morphological analysis showed a marked decrease (94-97%) in doublecortin neuron number in the dentate gyrus in both aged groups and a reduction in glial fibrillary acidic protein-positive cell number in the stratum radiatum of aging rats. Astroglial process length and branching complexity decreased in aged rats. We conclude that while target-seeking activity and learning ability decrease in aged females, spatial memory only declines in the longer-term tests. The reduction in neuroblast number and astroglial arborescence complexity in the dorsal hippocampus are likely to play a role in the cognitive deficits of aging rats.

Morphometry of cervical segments grey matter in the male rat spinal cord

The cervical portion of the spinal cord is an area frequently affected by alterations of medical and veterinary importance. Since there is scarce quantitative anatomical data on this region, we undertook a morphometric study of the grey matter of all segments of the rat cervical spinal cord of male rats in order to generate reference patterns to be used in future experimental studies. Using image analysis software, the total spinal cord length and grey and white matter area of each segment was recorded. The morphometric characteristics of the neurones populating the laminae of the grey matter of the cervical segments was also recorded. Neurones were classified into small, medium and large sizes for each lamina and statistically compared. The present data fill an anatomical information gap by providing quantitative data about the normal anatomical features of the rat cervical cord. The anatomical data found could be used to better understand the physiological relevance of that region in the rat.

Peripheral and mesencephalic transfer of a synthetic gene for the thymic peptide thymulin.

Thymulin is a thymic peptide with antiinflammatory activity in the brain. We constructed a recombinant adenoviral vector, RAd-FTS, expressing a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin and used it for peripheral and central gene transfer in rats. Thymulin concentration in serum and brain tissue was determined by bioassay. Reporter gene expression in the substantia nigra (SN) was quantitated by enzymohistochemistry or fluorescence microscopy using an appropriate image analysis software. A single intramuscular injection (10(8) plaque forming units (pfu)/animal) of RAd-FTS in thymectomized rats (nondetectable serum thymulin) induced supraphysiologic serum thymulin levels for at least 110 days (123+/-22 fg/ml versus 598+/-144 fg/ml in intact and vector-injected rats, respectively). Stereotaxic intranigral injection of RAd-FTS induced steady expression levels of met-FTS for at least 90 days, whereas expression of adenovirally transferred reporter genes coding for green fluorescent protein fused to HSV thymidine kinase (GFP-TK)(fus) or E.coli beta-galactosidase (beta-gal), declined drastically within a month (% transgene expression in the SN on post-injection day 30 relative to day 2 was: 18, <1 and 125%, for beta-gal, (GFP-TK)(fus) and met-FTS, respectively). We conclude that RAd-FTS constitutes a suitable biotechnological tool for the assessment of peripheral and central thymulin gene therapy in animal models of nigral dopaminergic neurodegeneration induced by pro-inflammatory agents.

Anatomical study of the forearm and hand nerves of the domestic cat ( Felis catus), puma ( Puma concolor) and jaguar ( Panthera onca).

The innervation of the forearm and hand regions of cats has not been well described despite its importance for any surgery or any neurological disorder. It is probably the main area where disorders of peripheral nerves in this species are observed. In felines, the forelimbs facilitate the jump and represent the most important way for capturing prey. The main muscles and nerves involved in this activity are located in the region of the forearm and hand. The aim of the present study was to provide a detailed description of the innervation of the forearm and hand regions of the jaguar and puma, in comparison with that of the domestic cat, contributing thus with the anatomical knowledge of the area for applying it to surgery and pathology. The forearms of three pumas and two jaguars (all of them fixed in formalin) and of six domestic cats (fresh) were dissected. The nerves path and their forearm distribution patterns of all three species were described. The analysed results indicate that the observed variations between species are minimal; thus, the anatomy described for domestic cats can be widely applied to American wild felids.

Neonatal Thymulin Gene Therapy Prevents Ovarian Dysgenesis and Attenuates Reproductive Derangements in Nude Female Mice

Congenitally athymic (nude) female mice show severe ovarian dysgenesis after puberty, which seems to be consequential to a number of neuroendocrine derangements described in these mutants. Thus, considerable evidence suggests that thymulin, a thymic peptide, may be involved in thymus-pituitary communication. In order to clarify the relevance of thymulin for the maturation of the female reproductive system, we assessed at hypothalamic, pituitary, ovarian, and uterine level the preventive action of neonatal thymulin gene therapy (NTGT) on the changes that typically occur after puberty in congenitally athymic female mice. We injected (im) an adenoviral vector harboring a synthetic DNA sequence encoding a biologically active analog of thymulin, methionine-serum thymic factor, in newborn nude mice (which are thymulin deficient) and killed the animals at 70-71 d of age. NTGT in the athymic mice restored the serum thymulin levels. Morphometric analysis revealed that athymic nudes have reduced numbers of brain GnRH neurons and pituitary gonadotropic cells as compared with heterozygous controls. NTGT prevented these changes and also rescued the premature ovarian failure phenotype typically observed in athymic nude mice (marked reduction in the number of antral follicles and corpora lutea, increase in atretic follicles). Serum estrogen, but not progesterone, levels were low in athymic nudes, a reduction that was partially prevented by NTGT. Little to no morphological changes were observed in the endometrium of female nudes. The delay in the age of vaginal opening that occurs in athymic nudes was significantly prevented by NTGT. Our results suggest that thymulin plays a relevant physiologic role in the thymus-hypothalamo-pituitary-gonadal axis.

Distribution of Tyrosine Hydroxylase-Immunoreactive Neurones in the Diencephalon and Mesencephalon of the Coypu (Myocastor coypus)

The aim of the present study was to examine the catecholaminergic neurones located within the midbrain of the coypu, a South American hystricomorph rodent. The neuronal distribution of the catecholaminergic systems and morphological parameters of the immunostained cell bodies and fibres were investigated, using an immunohistochemical method. The brains of five coypu were fixed, immersed in gelatine–glycerol and cut in 40‐μm slices using a freezing microtome. Samples were processed with ultrasound‐based antigen retrieval and stained with labelled antityrosine hydroxylase monoclonal antibody. An image analyser was used to measure the neuronal bodies. The catecholaminergic neurones of the tuberoinfundibular system were mainly observed in the arcuate and periventricular nuclei with their axons projecting towards to the median eminence; they represented 28% of the global population of tyrosine hydroxylase‐immunoreactive cells observed. Significant morphological differences were observed in comparison with the other two studied systems. Fifty per cent of total catecholaminergic neurones were detected in the nigrostriatal system distributed in the reticular and compact substance nigra. Most neuronal bodies had a fusiform aspect. The immunoreactive neurones of the mesolimbic system represented 22% of the total population. They were distributed around the interpeduncular nucleus. Two types of morphologically different catecholaminergic systems of the brain were established: hypothalamic neurones located in the periventricular and arcuate nuclei and mesencephalic neurones located in the substance nigra and interpeduncular nuclei. These systems showed morphological and probably physiological‐pharmacological differences.

GnRH neurones population in the diencephalon of the coypu myocastor coypus

El objetivo del presente estudio fue investigar la distribucion y morfologia de las neuronas que sintetizan y almacenan el factor de liberacion de gonadotrofinas (GnRH), en el diencefalo del coipo ( Myocastor Coypus), roedor sudamericano del Suborden histricomorpha. Para tales fines los encefalos de tres coipos machos, adultos, fueron fijados por perfusion intra-arterial, utilizando una solucion de paraformaldehido y acido picrico. Los bloques del hipotalamo fueron separados del resto del encefalo y seccionados en microtomo de congelacion, obteniendo laminas coronales de 40µm de espesor. Las secciones fueron sometidas a un proceso de recuperacion antigenica por ultrasonido, para desenmascarar los antigenos ocultos, luego las secciones fueron procesadas utilizando una tecnica de inmunohistoquimica para evidenciar las neuronas GnRH, usando un anticuerpo monoclonal (LRH 13). Un grupo de secciones seriadas fue coloreada con violeta de cresilo (tecnica de Nissl), mientras que, las otras secciones fueron coloreadas usando el metodo de Kuver Barrera (azul luxol rapido y violeta de cresilo), para observar los nucleos y tractos nerviosos del hipotalamo, con el microscopio optico. Los analisis morfometrico y cuantitativo de los cuerpos neuronales fue realizado usando un analizador de imagenes. Las neuronas GnRH inmunoreactivas observadas eran bipolares y alargadas. El numero total de cuerpos neuronales para esta especie fue estimado en 1072 ± 27. Todas las celulas fueron localizadas en el hipotalamo rostral, principalmente en el area preoptica. Pocas neuronas fueron observadas en el nucleo de la estria terminal y en el nucleo medial preoptico hipotalamico. Las fibras inmunorreactivas fueron visibles en la capa externa de la eminencia media. De acuerdo a los datos obtenidos, se concluye que la distribucion de las neuronas GnRH en el coipo coincide con la de otros roedores, tales como la rata.