The ossification of the pelvic girdle and leg skeleton of the quail (Coturnix coturnix japonica).

The onset of ossification centres of the pelvic girdle and leg skeleton of the quail in embryos and juvenile birds were studied. Specimens, which were cleared and were stained with Alcian Blue and Alizarin Red S, were examined at the stereomicroscope. The ilium and the pubis began to ossify at the 8th day (E8), whereas the ischium at E9. Perichondral ossification was observed at E6 in the femur, tibia and fibula. A secondary ossification centre was detected in the proximal epiphysis of the tibiotarsus at the 15th post-hatching day (P15). The patella began to ossify at P30. Regarding the tarsal bones tibiale, pre-tibiale and fibulare, ossification was observed at the E15, E12 and E16, respectively. The metatarsals II, III, IV ossified at E7, whereas the metatarsal I at E11. The centres of ossification of the 1st phalanges of all digits were observed at E9. At the same day, the ossification centres of the 2nd phalanx of digits II and III as well as the 3rd phalanx of digit III appeared. At E10, ossification was observed in the 2nd phalanx of digit I, in the 3rd phalanx of digit II and in the 2nd and 3rd phalanx of digit IV. In the 4th phalanx of digit III and in the terminal phalanges of digit IV, ossification was observed at E11. The data presented here provide useful baseline information on the normal sequential pattern of ossification in the pelvic girdle and leg skeleton in this species.

The ossification of the pectoral girdle and wing skeleton of the quail (Coturnix coturnix japonica).

The onset of ossification centres of the pectoral girdle and wing skeleton of the quail (Coturnix coturnix japonica) has been studied in embryos and juvenile birds, which were sampled daily from the 4th day of incubation up to the 12th day after hatching. Specimens, which were cleared and were stained with Alcian Blue and Alizarin Red S, were examined at the stereomicroscope. The furcula ossified with intramembranous ossification at the 7th day of incubation. The first centres of perichondral ossification in the scapula and the coracoid bones were observed at the 7th day of incubation. In the humerus, radius and ulna, perichondral ossification was observed at the 6th day of incubation. The carpal bones were ossified between the 8th and 10th day of the post-hatching period. The 2nd metacarpal bone began to ossify at the 6th day of the post-hatching period, whereas the 3rd and 4th metacarpal bones were ossified at the 7th day of incubation. At the 9th day of incubation, ossification was observed in the 1st phalanx of the alular digit and in the phalanges of the major digit. The 2nd phalanx of the alular digit began to ossify at the 12th, whereas the phalanx of the minor digit at the 14th day of incubation. The data presented here provide useful baseline information on the normal sequential pattern of ossification in the pectoral girdle and wing skeleton in this species.

Natural and lesion-induced apoptosis in the dorsal lateral geniculate nucleus during development.

We investigated natural and lesion-induced apoptosis in the developing rat dorsal lateral geniculate nucleus (dLGN). These lesions involved: i) monocular enucleation, and ii) unilateral ablation of the visual cortex at different postnatal ages before eye opening. We identified dying cells as apoptotic with light and electron microscopy, using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and immunohistochemistry for active caspase-3. In the dLGN of normal animals, TUNEL+cells were detected during the first postnatal week, with a peak at postnatal day (P) 1. Following enucleation at birth or at P7, the frequency of apoptotic cells in the contralateral dLGN increased significantly at postlesion day (PLD) 1 and returned to normal values by PLD7. In contrast to early lesions, enucleation at P14 did not induce significant changes in apoptosis in the dLGN. Cortical lesions performed at P0, P7 or at P14 induced the death of the overwhelming majority of cells in the ipsilateral dLGN, which led to a severe reduction in size of the nucleus by PLD7 and its complete elimination by adulthood. Double labeling with TUNEL and immunofluorescence for neuronal nuclear protein (NeuN) showed that in both normal and lesioned animals, apoptotic cells were mainly neurons. We suggest that: i) apoptosis in the dLGN occurs during the precritical period of neuronal maturation; ii) developing neurons in the dLGN are more dependent on the integrity of their connections with the visual cortex than with the retina for survival; and iii) lesion-induced apoptosis in the dLGN during development depends on the type and extent of the connectivity affected.

Natural and lesion-induced apoptosis in the rat striatum during development.

We evaluated the pattern of apoptosis in the rat striatum during normal development and in two models of lesion-induced cell death. Lesions included i) unilateral ablations of the cerebral cortex at different postnatal ages, and ii) early postnatal lesions of the catecholaminergic afferent systems of the striatum with 6-hydroxydopamine (6-OHDA). Dying cells were identified as apoptotic using the TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling) method at the light and electron microscopic levels. Moreover, we used immunohistochemistry for the apoptotic markers active caspase-3 and fractin. TUNEL+ cells were present in the striatum during the first four postnatal weeks. Their frequency was high during the first postnatal week and peaked at postnatal day (P)5. Cortical lesions at birth, in contrast to those performed at later stages, induced a significant increase in the frequency of TUNEL+ cells in the ipsilateral striatum, which peaked at seven days postlesion. 6-OHDA lesions resulted in a similar and significant increase in the frequency of TUNEL+ cells in the striatum, which also peaked at P7. We also showed that cortical lesions at P0 and 6-OHDA lesions resulted in a reduction in the frequency, as well as in alterations of the morphology of gamma-aminobutyric acid (GABA)-immunoreactive (ir) neurons in the striatum. We suggest that: i) apoptosis in the striatum is temporally coordinated with maturation events in this area and ii) early developmental lesions of major afferent pathways to the striatum affect both the survival and phenotype of striatal neurons.

Postnatal development of the dopaminergic system of the striatum in the rat.

The dopaminergic innervation of the developing caudate-putamen (patches and matrix) and nucleus accumbens (shell and core) of the rat was examined with light and electron microscope immunocytochemistry, using antibodies against dopamine. Light microscopic analysis showed, in accordance with previous studies, that early in life, dopaminergic fibers were relatively thick and present throughout the striatum. Their distribution was heterogeneous, showing dense aggregations, the so-called dopamine islands. The pattern of innervation became more uniform during the third postnatal week with most of the dopamine islands no longer detectable. For electron microscopic analysis, parts of the caudate-putamen containing dopamine islands or matrix, and of the nucleus accumbens, from the shell and the core of the nucleus, were selected. This analysis revealed that symmetrical synapses between immunoreactive profiles and unlabeled dendritic shafts predominated throughout development but, at the late stages, symmetrical axospinous synapses also became a prominent feature. These findings indicate that: (1) although the caudate-putamen and the nucleus accumbens have different connections and functions, they exhibit similar types of dopaminergic synapses, and (2) the relatively late detection of dopaminergic axospinous synapses suggests that the development of the dopaminergic system in the striatum is an active process, which parallels the morphological changes of striatal neurons and may contribute to their maturation.

Distribution and synaptology of dopaminergic fibers in the mature and developing lateral septum of the rat.

The dopamine innervation of the adult and developing lateral septum of the rat was investigated with light and electron microscope immunocytochemistry using anti-dopamine antibodies. Light microscopic analysis showed that the pattern of innervation of the lateral septum exhibited a marked reorganization during the first 2 postnatal weeks, when it acquired features comparable to the adult. Ultrastructural analysis suggested that there may be two different dopamine inputs in the lateral septum. The first develops earlier in life and, through symmetrical axodendritic synapses, affects remote parts of neurons and may cause inhibition. The second develops later and, through asymmetrical axosomatic synapses, affects neuronal somata and may cause excitation. These findings may explain the reported contradictory results concerning the physiological role of dopamine in neurons of the lateral septum.

Activation of the GABAA receptor inhibits the proliferative effects of bFGF in cortical progenitor cells.

Recent studies have localized gamma-aminobutyric acid (GABA)-containing neurons and identified cells that express subunits of the GABAA receptor in the proliferative zone of the developing cerebral cortex and have demonstrated a role for GABA in cortical neurogenesis. We examined here the interactions between a number of neurotrophic factors, known to be involved in cortical cell proliferation and differentiation, and the GABAergic system (GABA and GABAA receptors) in the regulation of cell production in dissociated cortical cell cultures. We found that basic fibroblast growth factor (bFGF) increased the number of cells labelled for the alpha 1 subunit of the GABAA receptor but not for the alpha 2, alpha 3 or alpha 5 subunits. The alpha 1 subunit was expressed by the majority of proliferating neuroepithelial cells as well as by differentiated neurons. We also found that activation of the GABAA receptor by GABA or muscimol inhibited the proliferative effects of bFGF on cortical progenitors, leading to an increased number of differentiated neurons. These results suggest that bFGF stimulates cell proliferation and GABAA receptor expression in cultured progenitor cells of the developing neocortex, and that GABA regulates cell production by providing a feedback signal that terminates cell division.

Multifaceted approach to medication use policy development: the restriction of meperidine.

As institutions continue to expand their drug policy development efforts in order to improve care and reduce cost, the use of multifaceted approaches offer several benefits. Population data on drug use support the need for policy action. The use of institutional outcomes data in conjunction with published evidence augments the process, and the consensus approach to guideline development engenders medical staff support. Such efforts, however, require significant dedication of human resources. Institutions with limited personnel to allocate to drug policy activities may consider increasing the depth of their efforts (using a multifaceted approach) while limiting the breadth of their efforts (only attempting one or two major targets per year, and doing them well).

Interruption of visual deprivation during development restores the normal density of peptidergic neurons in the rat visual cortex.

We have previously shown that dark rearing moderates the normal decline in the number of somatostatin (SRIF) and neuropeptide (NPY) neurons in the developing rat visual cortex. Thus, cortical areas of visually deprived animals at postnatal day (P) 60 contain consistently more SRIF and NPY neurons than do the same areas of rats reared in normal lighting conditions. In present study animals were reared in darkness from birth to P14, P21, P30 or P60 and then placed in ambient light conditions up to the day of sacrifice (P60 or P90). Counting of immunocytochemically identified SRIF and NPY neurons in all cortical visual areas of these and of undeprived animals, showed that interruption of visual deprivation during both the early or the late stages of postnatal development restores the normal density of the above peptidergic neurons.

A veterinary anatomy tutoring system.

A veterinary anatomy tutoring system was developed by using Knowledge Pro, an object-oriented software development tool with hypermedia capabilities, and MS Access, a relational database. Communication between them is facilitated by using the Structured Query Language (SQL). The architecture of the system is based on knowledge sets, each of which covers four different descriptions of an organ, namely gross anatomy (general description), gross anatomy (comparative features), histology, and embryology, which constitute the knowledge units. These knowledge units are linked with three global variables that define the animals, the topographies, and the system to which this organ belongs, creating three data-bases. These three data-bases are interrelated through the organ field in order to establish a relational model. This system allows versatility in the student's navigation through the information space by offering different modes for information location and presentation. These include course mode, review mode, reference mode, dissection mode, and comparison mode. In addition, the system provides a self-evaluation mode.

Cholecystokinin neurons in the developing visual cortex of normal and dark reared rats. Comparison with other peptidergic populations.

The laminar distribution and the density of cholecystokinin-like (CCK) neurons in the developing visual cortical areas (17, 18 and 18 a) were studied in Wistar rats that were reared under normal lighting conditions or in complete darkness. Immunocytochemistry on paraffin sections at postnatal days (P) 7, 14, 21, 30 and 60 showed that the density of CCK neurons in all visual areas of both groups of animals progressively increases from P7 to P21 but declines thereafter, to reach 50-75% of peak densities at P30. From P30 to P60 the density of CCK neurons in the visual cortex remains relatively unchanged in non-deprived animals, but increases slightly again in areas 17 and 18 a of dark reared rats. Fluctuations in the density of CCK neurons reflect changes in the number of immunoreactive neurons mainly present in layers IV-VI. It is concluded that the development of the CCK neurons in the rat visual cortex is minimally affected by light deprivation.

Postnatal development of somatostatin-containing neurons in the visual cortex of normal and dark-reared rats.

The distribution of somatostatin (SRIF)-immunoreactive neurons in the visual cortical areas 17, 18 and 18a of Wistar rats from birth to adulthood was followed in both normal and dark-reared animals. The SRIF neurons show difference in distribution amongst the three cortical areas studied as early as the first postnatal week. Area 17 was distinguished by fewer SRIF cells in the upper layers (I-III), which results in a lower overall density. The SRIF neurons in all areas appeared to increase in numbers up to about 3 weeks and then decline dramatically to adult levels, which were 14-19% of the peak levels. Although this decline was still obvious, it moderated to 25-31% in dark-reared animals. The greatest effect was seen in area 18 where, at 60 days of age, there were twice as many SRIF cells in dark-reared as in normal controls. It is suggested that, under conditions of dark rearing, the overall pattern of development of SRIF neurons, being uninfluenced by extrinsic factors, reveals the cells' genetic potential.

Postnatal development of vasoactive intestinal polypeptide-containing neurons in the visual cortex of normal and dark-reared rats.

The effects of dark rearing on the distribution and density of vasoactive intestinal polypeptide (VIP)-containing neurons in the visual cortical areas (17, 18 and 18a) of rats during postnatal development were examined immunohistochemically. Two groups of Wistar rats, one reared under normal lighting conditions and the other in complete darkness from birth, were used. VIP neurons showed a fairly similar distribution in the three visual areas, being predominantly present in layers II and III. Their pattern of development was found to be similar in the normal and dark-reared animals and was characterized by a marked increase from postnatal day (P) 7 to P21, followed by a gradual diminution to 24-31% of peak densities. Counts of labeled neurons at all ages examined showed that their density was similar in both groups at P7 and P14, but progressively greater in dark-reared animals from P21 and thereafter, so that they only fell to 38-43% of peak densities. Thus, by 60 days of age densities of VIP-labeled neurons in areas 17, 18 and 18a in dark-reared rats were 57%, 49% and 51% higher than in the corresponding areas of the age-matched control rats. These results indicate that the normal decline in the numbers of VIP neurons is not so marked under the conditions of dark rearing.

Postnatal development of neuropeptide Y-containing neurons in the visual cortex of normal- and dark-reared rats.

The postnatal development of neuropeptide Y (NPY)-immunoreactive neurons in the visual cortical areas (17, 18 and 18a) has been studied in Wistar rats reared under normal lighting conditions or in complete darkness. Immunohistochemistry on paraffin sections at postnatal days (P)7, 14, 21, 30 and 60 showed an overall similarity in laminar distribution of NPY neurons in all 3 visual areas of both normal- and dark-reared animals. The pattern of development of NPY neurons was characterized by an increase in their density from P7 to reach a peak at P21 followed by a decline to 37-47% of peak levels at P60. However, this diminution was not so great in dark-reared rats as in the normal, since the density only decreased to 62-78% of peak levels. At P60 the resulting differences in neuron density were marked in areas 17 and 18, where the dark-reared had 75% more cells than normal, and moderate in area 18a (30% more than normal). These results suggest that the normal decline in NPY neurons is not entirely mediated by visual experience since it takes place, albeit to a modified extent, in its total absence.

Coexistence of neuropeptides in the hypothalamic paraventricular nucleus of the sheep.

The chemical organization of the hypothalamic paraventricular nucleus (NPV) of the sheep was studied immunocytochemically by using antisera raised against oxytocin (OXY), ovine corticotropin releasing factor (CRF), somatostatin (SRIF), neurotensin (NT) and vasoactive intestinal polypeptide (VIP). Examination of immunocytochemically stained frozen, 30-40 microns thick, and paraffin serial, 6 microns thick, sections has shown that chemically specified subsets of neurons are not strictly demarcated anatomically and that OXY and SRIF or CRF and VIP are jointly expressed by certain subpopulations of neurons which are different from that producing both OXY and CRF.

Basal forebrain projections to the lower brain stem in the rat.

The projections of the magnocellular nuclei of the basal forebrain to the lower brain stem were studied using the WGA-HRP method. Of these nuclei, the nucleus of the vertical and the nucleus of the horizontal limb of the diagonal band of Broca, the magnocellular preoptic nucleus, and the substantia innominata were found to project bilaterally to the caudal midbrain, pons, and medulla. Labeled cells in the medial septal nucleus and in the nucleus basalis were found ipsilaterally only when injections stained the caudal part of the interpeduncular nucleus. The present findings together with other recent reports reveal a more extensive distribution of basal forebrain efferents than was previously thought.

Distribution of neuropeptides in the infundibular nucleus of the sheep.

The distribution of neurons exhibiting somatostatin (SRIF)-, neuropeptide Y (NPY)-, beta-endorphin- and neurotensin (NT)-like immunoreactivity within the infundibular nucleus (NI) of the sheep, and the extent of coexistence of the above peptides within individual neurons of the NI were investigated with immunocytochemical techniques. Our results show that the above neurochemical types of neurons exhibit specific and largely non-overlapping patterns of distribution within the NI of the sheep. Furthermore, the coexistence of these peptides within neurons of the NI is very limited, as from all possible permutations checked, only SRIF and NPY were found together in a small number of cells.

Neuropeptide Y-like immunoreactive neurons in the hedgehog (Erinaceus europaeus) and the sheep (Ovis aries) brain.

The peroxidase-antiperoxidase immunohistochemical method has been used in order to investigate the distribution of neurons immunoreactive with antiserum against neuropeptide Y in the hedgehog and the sheep brain. Neuropeptide Y-immunoreactive bipolar and multipolar neurons, with a diameter ranging from 10-30 microns, were found in the following regions: cerebral cortex, striatum, Ammon's horn, gyrus dentatus, amygdala, anterior olfactory nucleus, nucleus accumbens, area along the medial forebrain bundle, arcuate nucleus of the hypothalamus and lateral reticular nucleus of both hedgehog and sheep brain. Furthermore, while neuropeptide Y-immunoreactive neurons were also found in the medial and lateral septal nucleus, interstitial nucleus of the stria terminalis, retrochiasmatic area, central gray substance of the midbrain, colliculus caudalis, and nucleus of the solitary tract of the sheep, respective regions of the hedgehog brain appeared to be devoid of immunoreactive perikarya. This distribution pattern is identical to that observed with antiserum against avian pancreatic polypeptide. The differences in the distribution of neuropeptide Y-immunoreactive neurons in the two species studied as well as between our results and those in other species are briefly discussed.