Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells.

In this study, high-throughput microRNA (miRNA) expression analysis revealed that the expression of miR-140 was associated with chemosensitivity in osteosarcoma tumor xenografts. Tumor cells ectopically transfected with miR-140 were more resistant to methotrexate and 5-fluorouracil (5-FU). Overexpression of miR-140 inhibited cell proliferation in both osteosarcoma U-2 OS (wt-p53) and colon cancer HCT 116 (wt-p53) cell lines, but less so in osteosarcoma MG63 (mut-p53) and colon cancer HCT 116 (null-p53) cell lines. miR-140 induced p53 and p21 expression accompanied with G(1) and G(2) phase arrest only in cell lines containing wild type of p53. Histone deacetylase 4 (HDAC4) was confirmed to be one of the important targets of miR-140. The expression of endogenous miR-140 was significantly elevated in CD133(+hi)CD44(+hi) colon cancer stem-like cells that exhibit slow proliferating rate and chemoresistance. Blocking endogenous miR-140 by locked nucleic acid-modified anti-miR partially sensitized resistant colon cancer stem-like cells to 5-FU treatment. Taken together, our findings indicate that miR-140 is involved in the chemoresistance by reduced cell proliferation through G(1) and G(2) phase arrest mediated in part through the suppression of HDAC4. miR-140 may be a candidate target to develop novel therapeutic strategy to overcome drug resistance.

Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome.

OBJECTIVE: Adult respiratory distress syndrome (ARDS) is responsible for a significant portion of the morbidity and mortality during severe acute pancreatitis. Because inflammatory mediators such as tumor necrosis factor (TNF)-alpha and nitric oxide (NO) produced within the lungs have been implicated in sepsis-induced ARDS, we aimed to determine the role of these mediators in pancreatitis-induced ARDS using a model whereby ascites from animals with pancreatitis is transferred to otherwise healthy animals resulting in pulmonary injury. DESIGN: Prospective, randomized, controlled trial. SETTING: Research laboratory at a university medical school. SUBJECTS: Pathogen-free Sprague-Dawley rats weighing 225-250 g. INTERVENTIONS: Sterile, endotoxin- and cytokine-free pancreatic ascites tested for interleukin (IL)-1beta , TNF-alpha, interferon-gamma, and IL-6 was obtained from rats 18 hrs after the induction of severe, acute pancreatitis. Ascites was subsequently administered intravenously (20 mL/kg) to healthy rats. Sham animals were administered intravenous saline. Healthy animals administered intravenous ascites were randomized to receive a single intraperitoneal injection of the p38 mitogen activated kinase inhibitor CNI-1493 (1 mg/kg) or vehicle. MEASUREMENTS: Pulmonary injury was assessed at 24 hrs by histology and leukocyte and protein concentrations via bronchoalveolar lavage. Pulmonary TNF-alpha protein was detected by immunohistochemistry. Serum nitrite, as a measure of NO production, was measured utilizing the Griess reaction. MAIN RESULTS: After the intravenous administration of pancreatic ascites, the number of leukocytes and the protein concentration within the bronchoalveolar fluid were increased and pulmonary histology was worsened consistent with acute lung injury (all p < .001 vs. sham). Each of these variables of pulmonary injury was lessened in animals receiving CNI-1493 and intravenous ascites (p < .05 vs. vehicle). Pulmonary TNF-alpha protein and serum nitrites were decreased with the administration of CNI-1493 (p < .005 vs. vehicle). CONCLUSIONS: A component of pancreatic ascites other than endotoxin, bacteria, or cytokines (IL-1beta, TNF, interferon-gamma, or IL-6) is capable of inducing ARDS in healthy animals. Inhibition of p38 mitogen activated kinase decreases the pulmonary injury through attenuated production of TNF-alpha and NO suggesting a primary role for these mediators in pancreatitis-induced ARDS.

Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin.

Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of proinflammatory and anti-inflammatory pathways. Endotoxin (lipopolysaccharide), produced by all gram-negative bacteria, activates macrophages to release cytokines that are potentially lethal. The central nervous system regulates systemic inflammatory responses to endotoxin through humoral mechanisms. Activation of afferent vagus nerve fibres by endotoxin or cytokines stimulates hypothalamic-pituitary-adrenal anti-inflammatory responses. However, comparatively little is known about the role of efferent vagus nerve signalling in modulating inflammation. Here, we describe a previously unrecognized, parasympathetic anti-inflammatory pathway by which the brain modulates systemic inflammatory responses to endotoxin. Acetylcholine, the principle vagal neurotransmitter, significantly attenuated the release of cytokines (tumour necrosis factor (TNF), interleukin (IL)-1beta, IL-6 and IL-18), but not the anti-inflammatory cytokine IL-10, in lipopolysaccharide-stimulated human macrophage cultures. Direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxaemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF amounts, and prevented the development of shock.

Evidence of a central role for p38 map kinase induction of tumor necrosis factor alpha in pancreatitis-associated pulmonary injury.

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) has been implicated as an important mediator in acute pancreatitis-associated adult respiratory distress syndrome, but the precise pathogenesis remains unclear. The purpose of this work was to clarify the role of TNF alpha that is produced within the lung parenchyma in the inducement of pancreatitis-related pulmonary injury and to examine 1 of the potential pathways leading to the production of pulmonary TNF alpha. METHODS: Bile salt pancreatitis was induced in rats (n = 40) that were randomized to receive a p38 mitogen-activated protein (MAP) kinase inhibitor or vehicle. A separate group (n = 16) underwent sham operation. Pulmonary capillary permeability was determined with fluorescein isothiocyanate-labeled albumin and Evans blue dye, and lung histologic analysis was performed. TNF alpha protein was measured in bronchoalveolar lavage fluid, and p38 MAP kinase was activity determined by Western blot analysis. RESULTS: The induction of pancreatitis resulted in increased pulmonary capillary leakage and worsened histologic condition (P < .01 vs sham). Effective inhibition of p38 MAP kinase-induced TNF alpha production completely prevented pancreatitis-associated pulmonary injury (P < .01 vs vehicle). CONCLUSIONS: p38 MAP kinase-induced TNF alpha production plays a central role in the development of pulmonary dysfunction, which accompanies severe acute pancreatitis in this rodent model.

Loss of NF-kappaB activity during cerebral ischemia and TNF cytotoxicity.

Recent evidence implicates tumor necrosis factor (TNF), a cytokine with both cytotoxic and cytoprotective activities, in the pathogenesis of cerebral ischemia. The development of TNF cytotoxicity is dependent upon the balance between the activities of intracellular signaling pathways that mediate either apoptotic or anti-apoptotic effects. One critical protective signaling mechanism is the activation of nuclear factor (NF)-kappaB, a ubiquitous transcription factor that regulates expression of anti-apoptotic gene products. Here we show the distribution and kinetics of NF-kappaB activation and the correlation between loss of NF-kappaB activity, TNF up-regulation, and apoptosis in a standardized rat model of focal cerebral ischemia. We observed a rapid and progressive ischemia-induced loss of p65 immunoreactivity within the ischemic core and nearby penumbra. These findings were confirmed by Western blot analysis of nuclear extracts and by electrophoretic mobility shift assay. The anatomical area of suppressed NF-kappaB activity overlapped significantly with the zones of TNF overexpression and apoptosis. Loss of NF-kappaB activity and increased TNF expression preceded the onset of cell death. Direct evidence that loss of NF-kappaB activity can sensitize brain cells to TNF cytotoxicity was obtained in vitro by co-administration of MG-132, an inhibitor of NF-kappaB activation, and TNF to neuronal-like and glial-like cell cultures. Inhibition of NF-kappaB significantly increased the sensitivity of these cultures to TNF cytotoxicity, indicating that the observed loss of neuronal NF-kappaB activity during cerebral ischemia can participate in the development of TNF-induced cytotoxicity.

Cerebral ischemia enhances polyamine oxidation: identification of enzymatically formed 3-aminopropanal as an endogenous mediator of neuronal and glial cell death.

To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after the onset of ischemia in brain homogenates (15.8 +/- 0.9 nmol/h/mg protein) as compared with homogenates prepared from the normally perfused contralateral side (7.4 +/- 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and 3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset of significant cerebral cell damage, and increase in a time-dependent manner with spreading neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis (LD50 = 160 microM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 microM). The tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal-mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that polyamine oxidase-derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.

The tetravalent guanylhydrazone CNI-1493 blocks the toxic effects of interleukin-2 without diminishing antitumor efficacy.

The use of interleukin 2 (IL-2) as an antineoplastic agent has been limited by the serious toxicities that accompany the doses necessary for a tumor response. Elevation of nitric oxide (NO) and tumor necrosis factor (TNF) both have been implicated in IL-2 toxicities. CNI-1493, a tetravalent guanylhydrazone, is an inhibitor of macrophage activation including the synthesis of TNF and other cytokines. Doses of CNI-1493 as low as 1 mg/kg/day conferred complete protection against fatal toxicity of IL-2 with IL-2 doses tenfold higher than the safely tolerated level in Sprague-Dawley rats. Moreover, typical pathologic changes in the lungs, kidneys, and the liver caused by IL-2 infusion were blocked by cotreatment with CNI-1493. When animals bearing established hepatomas were given IL-2 and CNI-1493 combination therapy, 10 of 10 hepatomas regressed from 1 cm3 to <1 mm3. Intracytoplasmic TNF levels were increased in normal tissues from IL-2 treated animals, and treatment with CNI-1493 maintained TNF at control levels. The degree of apoptosis measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining of tumors following IL-2 therapy was not reduced compared with IL-2 cotreated with CNI-1493. In contrast, apoptosis in the liver and lung parenchyma following IL-2 therapy was blocked completely by cotreatment with CNI-1493. Taken together, these data showed that low and infrequent doses of CNI-1493 markedly protected animals from IL-2 systemic toxicities whereas not affecting tumor response to IL-2 therapy. With the protection afforded by CNI-1493 treatment, IL-2 therapy dose levels could be increased to provide significant antitumor effects in animals with established hepatomas.

Tumor necrosis factor is a brain damaging cytokine in cerebral ischemia.

Two contrasting roles, one beneficial and the injurious, have been proposed for tumor necrosis factor (TNF) in the pathogenesis of cerebral ischemia. Reported here are results obtained in a standard model of permanent focal cortical ischemia in rats, in which the volume of cerebral infarction is measured after permanent occlusion of the middle cerebral artery. Administration of neutralizing anti-rat TNF antibodies (P114) into the brain cortex significantly reduced ischemic brain damage (85% reduced infarct volume as compared with preimmune-treated controls). Similar results were achieved by systemic administration of CNI-1493, a recently described tetravalent guanylhydrazone compound, which effectively inhibited endogenous brain TNF synthesis and conferred significant protection against the development of cerebral infarction (80% reduced infarct volume as compared with vehicle controls treated 1 h postischemia with 10 mg/kg). P114 anti-TNF and CNI-1493 were each cerebroprotective when given within a clinically relevant time window for up to 2 h after the onset of ischemia. These findings establish an important, pathophysiological role of TNF in mediating the progression of ischemic brain damage, and suggest that inhibiting TNF with CNI-1493 may be beneficial in the future treatment of stroke.

Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response.

The local production of proinflammatory cytokines mediates the host response to inflammation, infection, and injury, whereas an overexpression of these mediators can injure or kill the host. Recently, we identified a class of multivalent guanylhydrazone compounds that are effective inhibitors of proinflammatory cytokine synthesis in monocytes/macrophages. The structure of one such cationic molecule suggested a molecular mimicry with spermine, a ubiquitous endogenous biogenic amine that increases significantly at sites of inflammation and infection. Here, we addressed the hypothesis that spermine might counterregulate the innate immune response by downregulating the synthesis of potentially injurious cytokines. When spermine was added to cultures of human peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), it effectively inhibited the synthesis of the proinflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, MIP-1alpha, and MIP-1beta. The inhibition of cytokine synthesis was specific and reversible, with significant inhibition of TNF synthesis occurring even when spermine was added after LPS. The mechanism of spermine-mediated cytokine suppression was posttranscriptional and independent of polyamine oxidase activity. Local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carrageenan. These results identify a distinct molecular counterregulatory role for spermine in downregulating the monocyte proinflammatory cytokine response.

Expression of TNF and TNF receptors (p55 and p75) in the rat brain after focal cerebral ischemia.

Cerebral ischemia induces a rapid and dramatic up-regulation of tumor necrosis factor (TNF) protein and mRNA, but the cellular sources of TNF in the ischemic brain have not been defined. The diverse activities of TNF are mediated via ligand interaction with two distinct receptors, p55 and p75, which activate separate intracellular signal transduction pathways, leading to distinct biological effects. Since the effects of cerebral ischemia on TNF receptor (TNFR) expression are unknown, we examined the cellular localization and protein expression of TNF and its two receptors in the rat cerebral cortex in response to permanent middle cerebral artery (MCA) occlusion. The results indicate that focal. cerebral ischemia up-regulates expression of TNF and both TNFRs within the ischemic cortex. The most abundant type of TNF immunoreactivity (IR) was a punctate and filamentous pattern of transected cellular processes; however, cell bodies of neurons, astrocytes, and microglia, as well as infiltrating polymorphonuclear (PMN) leukocytes also showed TNF IR. Brain vasculature displayed TNF IR not only within endothelial cells but also in the perivascular space. MCA occlusion induced significant up-regulation of TNF receptors, with p55 IR appearing within 6 hr, significantly before the appearance of p75 IR at 24 hr after the onset of ischemia. Since p55 has been implicated in transducing cytotoxic signalling of TNF, these results support the proposed injurious role of excessive TNF produced during the acute response to cerebral ischemia.

Transient expression of neuropeptide Y (NPY) immunoreactivity in the developing hamster paraventricular thalamic area is due to apoptosis.

1. A new population of neurons with transient expression of NPY immunoreactivity was described in the developing hamster paraventricular thalamic area. The present study was performed to discover whether this phenomenon is due to programmed cell death or apoptosis. 2. Toward this aim, immunocytochemical and electron microscopic examination of the paraventricular thalamic region, as well as DNA electrophoresis of tissue extracted from the described area, was performed on different stages of embryonic and postnatal development. 3. A sudden increase in neuropeptide Y immunoreactivity (NPY-IR) in the paraventricular thalamic area at embryonic day 14 (E14) was the first symptom of neuronal degeneration. 4. Electron microscopy revealed many neurons with large masses of condensed chromatin within nuclei and extracellular bodies. The affected cells had a convoluted shape and condensed cytoplasm. 5. DNA electrophoresis revealed a ladder of bands between 150 and 1000 bp that is specific for internucleosomal DNA fragmentation. 6. The data strongly suggest that developmental disappearance of NPY-IR neurons within the hamster dorsal thalamic area is due to apoptosis.

Inhibition of noxious stimulus-evoked pain behaviors and neuronal fos-like immunoreactivity in the spinal cord of the rat by supraspinal morphine.

In previous studies, we reported that supraspinally administered DAMGO, a mu-opioid agonist, produces a dose-related, naloxone-reversible inhibition of formalin-evoked pain behaviors and spinal cord Fos-like immunoreactivity (FLI) in the rat spinal cord. Although these results support the hypothesis that activation of supraspinal mu-opioid receptors produces antinociception by increasing the activity of bulbospinal inhibitory pathways, other studies suggest that supraspinal morphine decreases rather than increases descending inhibitory control. In the present study, we specifically examined the effect of intracerebroventricular (i.c.v.) injection of morphine in the rat. Supraspinal morphine produced a dose-related, naloxone-reversible inhibition of both formalin-evoked behaviors nd spinal cord FLI. Although the magnitude of the antinociception produced by i.c.v. morphine in the formalin test was significantly correlated with the numbers of FLI neurons in the spinal cord, the lowest dose of i.c.v. morphine tested (0.70 nmol) produced a significant reduction of FLI in the superficial laminae without producing behavioral antinociception, which is consistent with our hypothesis that noxious stimulus-evoked Fos expression in the superficial laminae is a poor predictor of the magnitude of pain behavior. These data support the hypothesis that the antinociceptive effects of supraspinally administered morphine result from an increase in descending inhibitory control.

Organization of permanent and transient neuropeptide Y-immunoreactive neuron groups and fiber systems in the developing hamster diencephalon.

The development of neuropeptide Y-immunoreactive (NPY-IR) cell and fiber systems in the hamster diencephalon was studied. Eight perinatal groups of NPY-IR neurons develop into 12 distinct sets in nuclei of the adult diencephalon and mesencephalon. NPY-IR neurons of the thalamic precommissural nucleus, nucleus of the optic tract, and olivary pretectal nucleus are derived from the superior group. Those in the adult magnocellular nucleus of the posterior commissure and deep mesencephalic nucleus are from the dorsal group. An arcuate group contributes neurons to the arcuate nucleus and median eminence and a mammillary group transiently exists in the mammillary region. A medial group gives rise to two sets of neurons, one that migrates to the intergeniculate leaflet and another that develops in the medial nucleus reuniens. A very large ventral group provides NPY-IR neurons to the adult medial zona incerta and caudal reticular thalamus. Groups of NPY-IR neurons also appear in the bed nucleus of the stria terminalis and centromedian thalamic nucleus. Superior group neurons may undergo apoptosis. In several groups, neurons become fewer during development, and NPY-IR may disappear. NPY-IR neurons of several groups initially migrate away from the neuroepithelial zone with later emergence of a distinct, persistent set of NPY-IR neurons in the same neuroepithelial region. The data show that neuropeptide content can be used to identify particular sets of neurons early in development, thereby allowing migration patterns to be followed and principles of brain development to be elucidated.

Ontogeny of radial glia, astrocytes and vasoactive intestinal peptide immunoreactive neurons in hamster suprachiasmatic nucleus.

Circadian rhythmicity of rodents is a property of the suprachiasmatic nucleus (SCN). Such rhythmicity can be demonstrated in the prenatal SCN, yet there is little information about the cells in which rhythmicity is generated. The present study was performed to discover the developmental relationships of SCN glial cells and a class of identifiable SCN neurons. Toward this end, vimentin- (VIM), glial fibrillary acidic protein- (GFAP) and vasoactive intestinal peptide- (VIP) immunoreactivity were investigated in SCN radial glia, astrocytes and neurons, respectively. VIP-IR first appears at embryonic day 13 (E13) and is clearly identifiable in neurons at E14. Substantial axon extension begins at E15 and the postnatal day 10 (P10) SCN is adult-like. VIM-IR radial glia fill the SCN region at E13, but by P0, most are absent. On P3, the remaining processes are beaded suggesting degeneration. The first GFAP-IR elements are visible on E15 with a few clear astrocytes present at P0. The number of astrocytes lateral to and in the SCN continues to increase during the postnatal period achieving an adult-like appearance by P21. The data do not support the view that prenatal circadian rhythmicity is derived from astrocytes. VIP-IR neurons are apparently present sufficiently early to be part of the rhythm generating mechanism. These tissues are discussed in the context of development of the SCN.

Specialized neuronal and glial contributions to development of the hamster lateral geniculate complex and circadian visual system.

The intergeniculate leaflet (IGL) is an integral part of the adult circadian visual system. It is characterized by the presence of retinal afferents and peptidergic cells projecting via a geniculohypothalamic tract (GHT) to the suprachiasmatic nucleus (SCN), site of the mammalian circadian clock. The adult IGL also contains abundant reactive astrocytes immunoreactive to GFAP. Because glia have a large role in brain development, we examined the ontogeny of the hamster IGL with respect to both glial and neuronal markers. Neuropeptide Y-immunoreactive (NPY-IR) cells destined for the IGL appear on embryonic day 11 (E11) in a matrix of vimentin (VIM)-IR radial glia. Migratory ellipsoid NPY-IR cells with long leading and trailing processes become oriented between the reticular thalamic neuroepithelial lobule, and the developing IGL. Most NPY-IR cells arrive in the IGL by E14 and extend axons ventrally into the GHT. These penetrate the SCN at P3 and arborize to an adult-like stage by P10. A specialized GFAP-IR radial glial path coinciding with the migratory route of NPY-IR cells appears by E14. As early as E15, cells contributing to this path are found displaced away from the ventricle. As the glial path disappears from the maturing brain, the entire length of the IGL becomes filled with GFAP-IR astrocytes. These features are consistent with translocation and transformation of a specialized set of radial glia into IGL astrocytes. The results demonstrate that the IGL is a large, developmentally important, feature of the lateral geniculate complex that is embryologically distinct from adjacent dorsal and ventrolateral geniculate nuclei.

Development of the hamster serotoninergic system: cell groups and diencephalic projections.

Nuclei of the circadian visual system are extensively innervated by serotoninergic neurons and rhythmicity is modulated by the serotoninergic system. This study investigated the temporal relationships between prenatal origins of serotoninergic cell groups and perinatal innervation of structures in the hamster circadian visual system as well as in the remaining diencephalon. Serotonin-immunoreactive (5-HT-IR) neurons of the B4-B9 complex were first seen on embryonic day 8 (E8). The number of neurons increases sharply by E10 when the first 5-HT-IR cells are evident in the medulla (B1-B3 complex). The distribution of serotoninergic neurons in the hamster brainstem is generally adult-like by E14. Thick 5-HT-IR fibers arch around the mesencephalic flexure at E10 and reach more rostral mesencephalic areas at E11. A branch of the medial forebrain bundle (MFB) projects ventrally toward the retrochiasmatic area; a second branch ascends along the fasciculus retroflexus. Fibers cross the midline in the supraoptic commissure by E12, other arrive in the lateral geniculate region, and a branch of the MFB extends toward the mammillary area. At E13, a periventricular medial thalamic branch of the MFB is seen, axons appear in the supramammillary commissure, and a fine fasciculus between the medial thalamus and intergeniculate leaflet is visible. Lateral, paraventricular, and retrochiasmatic hypothalamic areas and centro- and dorsomedial thalamus are densely innervated at E14. The mammillary area and lateral geniculate body are moderately innervated, and the first fibers appear in the deep laminae of the superior colliculus. The innervation of the suprachiasmatic nuclei, periventricular hypothalamus, and superficial layers of the superior colliculus occurs postnatally. The results are consistent with serotoninergic system development in other species.