Synaptic dimorphism in Onychophoran cephalic ganglia

The taxonomic location of the Onychophora has been controversial because of their phenotypic and genotypic characteristics, related to both annelids and arthropods. We analyzed the ultrastructure of the neurons and their synapses in the cephalic ganglion of a poorly known invertebrate, the velvet worm Peripatus sedgwicki, from the mountainous region of El Valle, Mérida, Venezuela. Cephalic ganglia were dissected, fixed and processed for transmission electron microscopy. The animal has a high degree of neurobiological development, as evidenced by the presence of asymmetric (excitatory) and symmetric (inhibitory) synapses, as well as the existence of glial cell processes in a wide neuropile zone. The postsynaptic terminals were seen to contain subsynaptic cisterns formed by membranes of smooth endoplasmic reticulum beneath the postsynaptic density, whereas the presynaptic terminal showed numerous electron transparent synaptic vesicles. From the neurophylogenetic perspectives, the ultrastructural characteristics of the central nervous tissue of the Onychophora show important evolutionary acquirements, such as the presence of both excitatory and inhibitory synapses, indicating functional synaptic transmission, and the appearance of mature glial cells.

Synaptic dimorphism in Onychophoran cephalic ganglia.

The taxonomic location of the Onychophora has been controversial because of their phenotypic and genotypic characteristics, related to both annelids and arthropods. We analyzed the ultrastructure of the neurons and their synapses in the cephalic ganglion of a poorly known invertebrate, the velvet worm Peripatus sedgwicki, from the mountainous region of El Valle, Mérida, Venezuela. Cephalic ganglia were dissected, fixed and processed for transmission electron microscopy. The animal has a high degree of neurobiological development, as evidenced by the presence of asymmetric (excitatory) and symmetric (inhibitory) synapses, as well as the existence of glial cell processes in a wide neuropile zone. The postsynaptic terminals were seen to contain subsynaptic cisterns formed by membranes of smooth endoplasmic reticulum beneath the postsynaptic density, whereas the presynaptic terminal showed numerous electron transparent synaptic vesicles. From the neurophylogenetic perspectives, the ultrastructural characteristics of the central nervous tissue of the Onychophora show important evolutionary acquirements, such as the presence of both excitatory and inhibitory synapses, indicating functional synaptic transmission, and the appearance of mature glial cells.

Levels of amino acid neurotransmitters during mouse olfactory bulb neurogenesis and in histotypic olfactory bulb cultures.

The developmental changes in the levels of amino acid neurotransmitters were analyzed by high pressure liquid chromatography during mouse olfactory bulb neurogenesis, from embryonic day (E)13 until the young adult age, between postnatal days (P)30 and P40. During the embryonic period, high levels of glutamate, aspartate and GABA were observed, with the values of GABA about 2-fold higher than those of glutamate and aspartate. At P0, the production of these neurotransmitters experienced birth stress as shown by a significant 2-fold reduction in their levels. During the first two postnatal weeks, a progressive increase in the glutamate content was detected diminishing slightly in the adult stage. The aspartate concentrations showed a maximal value at P3 and then decreased gradually until the second postnatal week; in the young adult age, its concentration was comparable with that of glutamate. The postnatal GABA contents increased progressively from birth to maturity, showing maximal levels at P3, P11 and in the adult. Throughout the studied developmental period, the concentration of glycine remained relatively low. With regard to taurine, very low concentrations were detected during the prenatal period but after birth, the taurine content gradually increased with age, and in the adult animal, its concentration was comparable with those of GABA and glutamate. Our data demonstrate the predominance of GABA and glutamate during olfactory bulb synaptogenesis, however, in the adult animal, both glutamate and aspartate exert the same influence in the excitatory synaptic transmission; in the adult inhibitory synaptic transmission, taurine appears to play an important neuromodulatory or neurotransmitter role as that of GABA. To determine the intrinsic neurotransmitter production, primary histotypic olfactory bulb cultures were prepared from mice at P10. The comparative analysis of in vitro neurotransmitter contents with those in in situ adult animal showed higher levels of endogenously produced glutamate, glycine and GABA in the olfactory bulb than the extrinsic ones coming from olfactory nerve axons and higher olfactory brain centers. On the other hand, most of aspartate and taurine neurotransmitters apparently come from extrinsically located neurons.

Levels of amino acid neurotransmitters during mouse cerebellar neurogenesis and in histotypic cerebellar cultures.

The variation in the levels of excitatory (glutamate and aspartate) and inhibitory (GABA, glycine and taurine) neurotransmitters during neurogenesis in mouse cerebellum, from embryonic day (E) 15 until the young adult stage, was analyzed by high-pressure liquid chromatography. Between E15 and E21, high contents of GABA, glutamate and aspartate were detected, with the GABA levels approximately 2- to 3-fold higher than those of glutamate and aspartate. After birth, the levels of GABA remained high during the first 2 postnatal weeks and then reached a plateau comparable to adult values by the third week. The levels of glutamate and aspartate increased gradually from birth to the young adult stage, showing peak values at postnatal days (P) 3 and P11. Glycine and taurine were present at relatively low concentrations during the prenatal period, then rose significantly by about 4-fold after birth; their levels decreased by the end of the first postnatal week but increased gradually thereafter until reaching adult values by the third week. To determine the endogenous neurotransmitter production in the cerebellar cortex, primary histotypic cerebellar cultures prepared at P10 were analyzed and the in vitro transmitter contents were compared with the adult in situ values. The cultures showed about the same levels of glutamate and aspartate; however, their concentrations were lower by half than in vivo, suggesting that both play an equally important role in the excitatory neurotransmission of the cerebellar cortex internal circuitry pathways and that in mature cerebellum, about 50% of the excitatory synaptic inputs derive from the afferent climbing and mossy fibers. The in vitro GABA and glycine contents were comparable with the in vivo levels, whereas the taurine concentrations were about 5-fold lower in vitro than in vivo. These findings indicate that most of the GABA and glycine transmitters are produced intrinsically while a high proportion of taurine in the cerebellum comes from extracerebellar afferents. This study provides data on the changing levels of the amino acid neurotransmitters in developing mouse cerebellum and the relative proportions of neurotransmitter contents that are produced by intrinsic neurons in contrast to those derived from extrinsic afferent fibers.

Levels of monoamine and amino acid neurotransmitters in the developing male mouse hypothalamus and in histotypic hypothalamic cultures.

The variation in the levels of the monoamine and amino acid neurotransmitters was studied during the period of neurogenesis in male mouse hypothalamus, from embryonic day 15 until the age of young adult. The results shown in this study demonstrate that the monoamines appear early in the fetal brain and that the maximum expression of the catecholaminergic system, particularly that of dopamine, occurs during the late neonatal period or mouse infancy, when the role played by the catecholamines on the maturation of the neurosecretory systems is more significant. In relation to the amino acid neurotransmitters, glutamate and taurine seem to be the principal transmitters of the hypothalamus since their concentrations were about five-tenfold higher than the levels of glycine and GABA. Both amino acids had the same pattern of variation during development, showing elevated values during the prenatal, late neonatal and early pubertal period. Increased concentrations of the inhibitory neurotransmitter GABA were observed on the day before birth, at the end of the neonatal period and throughout the prepubertal period, suggesting that the influence of GABA on hypothalamic neurogenesis increases by the time when the hypothalamic nuclei have reached maturity and the local circuits have been established. To determine the intrinsic neurotransmitter production, primary hypothalamic histotypic cultures prepared from mice at postnatal days 8-10 were analyzed for their content of neurotransmitters. The in vitro analysis revealed that the hypothalamic neurons intrinsically produce dopamine, glutamate, taurine and glycine in homologable amounts with those of young adult animals. The comparative analysis also showed that about 50% of the GABA content and less than 5% of the hypothalamic epinephrine level are locally produced, while serotonin comes mainly from extrinsically located neurons.

Hypothalamic synaptogenesis and its relationship with the maturation of hormonal secretion.

1. Information obtained during the last decade has demonstrated that hypothalamic neurons release a wide variety of neuroactive substances, such as neurotransmitters, mostly monoamines and amino acids, and neuromodulators such as the peptides vasopressin (AVP) and oxytocin (OXT) and hypophysial releasing hormones. 2. Synapse formation between hypothalamic neurons was followed at different times within a given nucleus and among different nuclei during development of the mouse hypothalamus. 3. The amounts of various neurotransmitters and hormones were determined at various stages of development. 4. A correlation is presented of the biochemical and ultrastructural features and their functional implications during maturation.

Intradermal implants of histotypic adrenal gland rotary cultures.

The implantation of histotypic adrenal gland cultures is described in the present study. The cultures were prepared from mice at birth and were rotary incubated for 10 days. When the cells reached phenotypic maturation, they were implanted in the dermal bed of the auricular pavilion of young adult mice. This location was ideal for implantation because transillumination enabled daily inspection of the progress of implant survival. Twenty days after implantation, the implants and associated tissues were removed to study the degree of maturation and the viability of the implants. Some cultures showed a moderate percentage of steroidal cells, while others demonstrated a high predominance of the chromaffin cells. All the implanted cultures successfully survived a postoperative period of 20 days. Both adrenergic and noradrenergic cells reached their maximum grade of cytological differentiation and neither inflammatory reactions nor adverse immunological effects were observed. On the contrary, the implants were profusely vascularized by the host tissue, particularly in those implants with a higher content of chromaffin cells.