Complex organization of mouse and rat suprachiasmatic nucleus.

The suprachiasmatic nucleus, site of the dominant mammalian circadian clock, contains a variety of different neurons that tend to form groups within the nucleus. The present investigation used single and multiple label tract tracing and immunofluorescence methods to evaluate the relative locations of the neuron groups and to compare them with the distributions of the three major afferent projections, the retinohypothalamic tract, geniculohypothalamic tract and the serotonergic pathway from the median raphe nucleus. The suprachiasmatic nucleus has a complex order characterized by peptidergic cell groups (vasopressin, gastrin releasing peptide, vasoactive intestinal polypeptide, calbindin, calretinin, corticotrophin releasing factor and enkephalin) that, in most cases, substantially overlap. The retinohypothalamic tract projects bilaterally to virtually all the suprachiasmatic nucleus in both rat (predominantly contralateral) and mouse (symmetric) and its terminal field overlaps that for the geniculohypothalamic tract, but with distinctions visible according to density criteria; neither provides more than sparse innervation of the dorsomedial suprachiasmatic nucleus. In the mouse, the serotonergic terminal field is densest medially and ventrally, but is also distributed elsewhere with varying density. The serotonergic terminal plexus in the rat is densest centromedially and largely, but not completely, overlaps the complete distribution of retinal terminals with density much reduced in the lateral suprachiasmatic nucleus. The locations of vasopressin neurons, retinohypothalamic tract terminals and serotonergic (mouse, rat) or geniculohypothalamic tract (rat) provide evidence for three clear, but not exclusionary, sectors of the suprachiasmatic nucleus. The data, in conjunction with emerging knowledge concerning rhythmically dynamic changes in the size of regions of neuropeptide gene expression in suprachiasmatic nucleus cells, support the view that suprachiasmatic nucleus organization is more complex than a simple "core" and "shell" arrangement. While generalizations about suprachiasmatic nucleus organization can be made with respect to location of cell phenotypes or terminal fields, oversimplification may hinder, rather than facilitate, understanding of suprachiasmatic nucleus structure-function relationships.

Neuromodulator content of hamster intergeniculate leaflet neurons and their projection to the suprachiasmatic nucleus or visual midbrain.

The intergeniculate leaflet (IGL) of the lateral geniculate complex has widespread, bilateral, and reciprocal connections with nuclei in the subcortical visual shell. Its function is poorly understood with respect to its role in visual processing. The most well-known IGL projection, and the only one with a clear function, is the geniculohypothalamic tract (GHT) that terminates in the suprachiasmatic nucleus (SCN), site of the primary circadian clock. The hamster GHT is derived, in part, from IGL neurons containing neuropeptide Y and enkephalin. IGL neurons containing these peptides also project to the pretectal region. The present studies used a combination of immunohistochemical, lesion, and retrograde tracing techniques to study neuron types in the IGL and their projections to hamster SCN and pretectum. Two additional neuromodulators, gamma-aminobutyric acid (GABA) and neurotensin, are shown to be present in IGL neurons. The GABA- and neurotensin-immunoreactive neurons project to the SCN with terminal field patterns very similar to those for neuropeptide Y and enkephalin. IGL neurons of all four types also send projections to the pretectum, but rarely do individual cells project to both the SCN and the pretectum. Nearly all neurotensin is colocalized with neuropeptide Y in IGL neurons, although about half of the neuropeptide Y cells do not contain neurotensin. Otherwise, the extent to which the four neuromodulators are colocalized varies from 6% to 54%. Nearly every SCN neuron appears to contain GABA. In the IGL, the majority of cells studied are not identifiable by GABA immunoreactivity. Putative functions of the various neuromodulator projections from the IGL to pretectum or SCN are discussed.

Forebrain connections of the hamster intergeniculate leaflet: comparison with those of ventral lateral geniculate nucleus and retina.

The hamster intergeniculate leaflet (IGL), part of the circadian rhythm regulatory system, has very extensive interconnections with subcortical visual nuclei. The present investigation describes IGL connections with the hamster diencephalon and telencephalon and compares them with ventral lateral geniculate nucleus (VLG) connections and retinal projections. Connections of the geniculate nuclei were evaluated using anterograde transport of iontophoretically injected Phaseolus vulgaris leucoagglutinin and by retrograde transport of cholera toxin beta fragment. The cholera fragment was also injected intraocularly to trace retinal efferents. The IGL has ipsilateral and contralateral projections to the anterior and posterior hypothalamic nuclei, the ventral preoptic, lateral and dorsal hypothalamic areas, but not to the core ventromedial nucleus and very sparsely to the paraventricular nucleus. There are also IGL projections to the medial and lateral zona incerta, anteroventral, anterodorsal, reuniens, parataenial, paraventricular, centrolateral, central medial, and laterodorsal thalamic nuclei. IGL projections to the telencephalon are found in the horizontal limb of the diagonal band, olfactory tubercle, nucleus of the lateral olfactory tract, posterior bed nucleus of the stria terminalis, ventral pallidum, and in nuclei of the medial amygdala. The only substantial VLG projections are to bed nucleus of the stria terminalis, IGL, medial zona incerta, central medial and laterodorsal thalamic nuclei. Several of the IGL targets, the bed nucleus of the stria terminalis and zona incerta in particular, send projections back to the IGL and VLG. In addition, cells are present in the caudal cingulate cortex that project to both nuclei. Retinal projections are found in many of the regions receiving IGL innervation, including nuclei of the medial basal telencephalon, the posteromedial bed nucleus of the stria terminalis, and nuclei of the hypothalamus. A retinal projection is also visible in the lateral olfactory tract from which it extends rostrally, then medially along the base of the rhinal fissure. Fibers also extend caudally, in a superficial location, to perirhinal cortex. The results further demonstrate the widespread connections of the IGL and support the idea that the IGL modulates olfactory, photic, and circadian rhythm regulation of regulatory physiology and behavior.

Interconnections among nuclei of the subcortical visual shell: the intergeniculate leaflet is a major constituent of the hamster subcortical visual system.

The intergeniculate leaflet (IGL), a major constituent of the circadian visual system, is one of 12 retinorecipient nuclei forming a "subcortical visual shell" overlying the diencephalic-mesencephalic border. The present investigation evaluated IGL connections with nuclei of the subcortical visual shell and determined the extent of interconnectivity between these nuclei. Male hamsters received stereotaxic, iontophoretic injections of the retrograde tracer, cholera toxin beta fragment, or the anterograde tracer, Phaseolus vulgaris-leucoagglutin, into nuclei of the pretectum (medial, commissural, posterior, olivary, anterior, nucleus of the optic tract, posterior limitans), into the superior colliculus, or into the visual thalamic nuclei (lateral posterior, dorsal lateral geniculate, intergeniculate leaflet, ventral lateral geniculate). Retrogradely labeled cell bodies identified nuclei with afferents projecting to the site of injection, whereas the presence of anterogradely labeled fibers with terminals revealed brain nuclei targeted by neurons at the site of injection. The IGL projects bilaterally to all nuclei of the visual shell except the lateral posterior and dorsal lateral geniculate nuclei. The IGL also has afferents from the same set of nuclei, except the nucleus of the optic tract. The extensive bilateral efferent projections distinguish IGL from the ventral lateral geniculate nucleus. The superior colliculus, commissural pretectal, olivary pretectal, and posterior pretectal nuclei also project bilaterally to the majority of subcortical visual nuclei. The IGL has a well-established role in circadian rhythm regulation, but there is as yet no known function for it in the larger context of the subcortical visual system, much of which is involved in oculomotor control.

Neuropeptide Y and enkephalin immunoreactivity in retinorecipient nuclei of the hamster pretectum and thalamus.

This investigation was stimulated by the historical confusion concerning the identity of certain pretectal nuclei and by large differences reported between species with respect to which nuclei receive retinal innervation. Subcortical visual nuclei were studied using immunohistochemistry to identify retinal projections labeled following intraocular injection of cholera toxin, b fragment. In addition, neuropeptide Y (NPY) or enkephalin (ENK) immunoreactive cells and fibers were also evaluated in the retinorecipient pretectal and thalamic areas. The results confirm the established view that the retina directly innervates the nucleus of the optic tract (NOT), posterior (PPT), and olivary pretectal (OPT) nuclei. However, the retina also innervates the hamster medial (MPT) and anterior (APT; dorsal division) pretectal nuclei, results not previously reported in rodents. A commissural pretectal area (CPT) sparsely innervated by retina is also described. The data show for the first time that the posterior limitans nucleus (PLi) receives a moderately dense, direct retinal input. The PLi does not project to the cortex and appears to be a pretectal, rather than thalamic, nucleus. All retinal projections are bilateral, although predominantly contralateral. The PLi contains a moderately dense plexus of NPY- and ENK-IR fibers and terminals. However, peptidergic fibers also traverse the ATP and connect with the dorsomedial pretectium. The OPT contains ENK- and NPY-IR neurons and fibers, but is specifically identifiable by a moderately dense plexus of ENK-IR terminals. Numerous ENK-IR neurons are found in the NOT and PPT. The latter also has moderate numbers of ENK-IR fibers and terminals, but few NPY-IR neurons or fibers. The MPT contains modest numbers of ENK-IR fibers. The APT has no NPY-IR neurons or terminals, but an occasional ENK-IR neuron is seen and there is sparse ENK-IR innervation. Peptidergic innervation of the visual nuclei does not appear to be derived from the retina. The results show a set of retinally innervated, contiguous nuclei extending from the thalamic ventrolateral geniculate nucleus dorsomedially to the midbrain CPT. These nuclei plus the superior colliculus comprise a dorsal "visual shell" embracing a central core of caudal thalamus and rostral midbrain.

The serotonergic projection from the median raphe nucleus to the suprachiasmatic nucleus modulates activity phase onset, but not other circadian rhythm parameters.

The suprachiasmatic nucleus (SCN) is densely innervated by serotonergic fibers originating in the median raphe nucleus (MR). Serotonin (5-HT) specific lesions of the MR alter entrainment and eliminate 5-HT fibers in the SCN, as well as in all other MR-recipient areas. The present study used 5-HT specific lesions of the SCN or the MR to determine the role of 5-HT in the SCN as a regulator of entrainment. Neurotoxic lesions of the MR significantly reduced 5-HT cell bodies in that nucleus and eliminated essentially all 5-HT innervation of the SCN. As previously demonstrated, these anatomical changes were associated with an advance in activity onset, delay in offset and expansion of the activity phase (alpha). Neurotoxin directly applied to the SCN caused an advance in the average activity onset, but had no effect on offset or alpha. About half of the SCN lesion animals had onsets equivalent to the MR lesion group, whereas onsets of the remaining animals were normal. Loss of SCN 5-HT innervation was severe for all SCN lesion animals, but significantly greater for those with advanced activity onsets. These results suggest that although the 5-HT projection to the SCN is likely to be responsible for modulating activity onset, the timing of activity offset appears to be regulated by a MR projection to an area outside the SCN. Furthermore, surprisingly few 5-HT fibers in the SCN are sufficient to maintain the normal phase angle of entrainment.

Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda (Sf-9) insect cells.

Spodoptera frugiperda (Sf-9) insect cells have been grown in serum-free medium in 250-ml spinner flasks. The maximum cell density obtained in these cultures was dependent on the aeration rate of the culture. Similar yields of uninfected cells were obtained when cultures were stirred in spinner flasks at 80 rev min-1 and in a 4-1 stirred-tank bioreactor and the dissolved oxygen in the bioreactor was controlled at 20% of air saturation. Cells were infected with a recombinant baculovirus at different multiplicities of infection: the timing and maximum level of expression of the recombinant protein were dependent on the multiplicity of infection, the cell density at infection, and on the aeration rate of the culture. Oxygen-limited growth resulted in undetectable levels of recombinant protein (< 6 ng recombinant protein 10(-7) cells). Compared with the maximum yields observed in spinner flask cultures, higher levels of recombinant protein were produced when cells were grown and infected in the bioreactor. The level of dissolved oxygen in the bioreactor was controlled at 50% of air saturation.

In vitro tests for the measurement of clostridial toxins, toxoids and antisera. II. Titration of Clostridium perfringens toxins and antitoxins in cell culture.

The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.