Verification of the seizure liability of compounds based on their in vitro functional activity in cultured rat cortical neurons and co-cultured human iPSC-derived neurons with astrocytes and in vivo extrapolation to cerebrospinal fluid concentration.

Methodical screening of safe and efficient drug candidate compounds is crucial for drug development. A high-throughput and accurate compound evaluation method targeting the central nervous system can be developed using in vitro neural networks. In particular, an evaluation system based on a human-derived neural network that can act as an alternative to animal experiments is desirable to avoid interspecific differences. A microelectrode array (MEA) is one such evaluation system, and can measure in vitro neural activity; however, studies on compound evaluation criteria and in vitro to in vivo extrapolation are scarce. In this study, we identified the parameters that can eliminate the effects of solvents from neural activity data obtained using MEA allow for accurate compound evaluation. Additionally, we resolved the issue associated with compound evaluation criteria during MEA using principal component analysis by considering the neuronal activity exceeding standard deviation (SD) of the solvent as indicator of seizurogenic potential. Overall, 10 seizurogenic compounds and three negative controls were assessed using MEA-based co-cultured human-induced pluripotent stem cell-derived neurons and astrocytes, and primary rat cortical neurons. In addition, we determined rat cerebrospinal fluid (CSF) concentrations during tremor and convulsion in response to exposure to test compounds. To characterize the in vitro to in vivo extrapolation and species differences, we compared the concentrations at which neuronal activity exceeding the SD range of the solvent was detectable using the MEA system and rat CSF concentration.

Trends in the serovar and antimicrobial resistance in clinical isolates of Salmonella enterica from cattle and pigs between 2002 and 2016 in Japan.

AIMS: Given the significance of Salmonella enterica in both human and animal health, and a recent global dissemination of Salmonella 4,[5],12:i:-, changes in the prevalent serovars and antimicrobial resistance in clinical isolates of Salmonella from cattle and pigs were investigated in Japan. METHODS AND RESULTS: The serovars and antimicrobial susceptibilities of 1605 Salmonella enterica isolated from cattle (n = 894) and swine (n = 711) between 2002 and 2016 were examined. The most common serovar among all samples was Salmonella Typhimurium. However, its monophasic variant with antigenic structure S. 4,[5],12:i:-, which was first detected in cattle in 2006 and swine in 2010, has been rapidly increasing in incidence and resistance. Resistance rates to cefotaxime and ciprofloxacin were generally low (<10% in the cattle isolates and <5% in the swine isolates); however, isolates resistant to more than five antimicrobials, which often include these antimicrobials, were recently detected in Salmonella Dublin, S. 4,[5],12:i:-, S. Typhimurium, Salmonella Newport, Salmonella Choleraesuis and Salmonella 6,7:c:-. Among them, two S. 4,[5],12:i:- isolates possessed extended-spectrum ß-lactamase-encoding genes; blaSHV-12 or blaCTX-M-55 , respectively, while all the five S. Typhimurium isolates possessed AmpC-type ß-lactamase gene of blaCMY-2 . CONCLUSIONS: S. 4,[5],12:i:- has been rapidly increasing and exhibiting a remarkable change in antimicrobial resistance in Japan. Considering certain serovars are characterized by multidrug resistance including medically important antimicrobials, continuous monitoring and appropriate measures are required to protect public health and veterinary husbandry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a trend in the serovars and antimicrobial resistance in clinical isolates of Salmonella from cattle and pigs in Japan, and showed that there were certain types of Salmonella serovars depending on the animal origin which needs more attention.

Characterization of mesenchymal progenitor cells in crown and root pulp from human mesiodentes.

OBJECTIVE: Mesiodentes are usually found in the central position of the upper or lower jaw as supernumerary teeth. Here, we obtained 10 mesiodentes and three permanent teeth (PT) and separated the dental pulp (DP) from these into crown and root portions. We then characterized and compared the isolated crown portion-derived cells (crown cells) with root portion-derived cells (root cells) using a range of in vitro assays. MATERIALS AND METHODS: Crown cells and root cells were examined for cell surface marker expression, colony-forming unit-fibroblast (CFU-F), cell proliferation, cell cycle characteristics and markers, and osteogenic and adipogenic differentiation. RESULTS: The proportion of CD105-positive cells (CD105(+) cells) in the crown cells vs the root cells varied among the mesiodentes, but not among the PT. When there were more CD105(+) cells in the root cells than in the crown cells, the root cells showed higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In contrast, when the crown cells contained more CD105(+) cells than the root cells, the crown cells showed the higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In addition, the sorted CD105(+) cells showed higher CFU-F and proliferation capacity than the sorted CD105(-) cells. CONCLUSION: These results indicated that proportion of CD105(+) cells is an effective means of characterizing DP-derived cells in mesiodentes.

Association between atopy and variants of the beta subunit of the high-affinity immunoglobulin E receptor.

The beta-subunit of the high-affinity IgE receptor (Fc epsilon RI-beta) on chromosome 11 is maternally linked to atopy, the state of enhanced IgE responsiveness underlying allergic asthma and rhinitis. We have identified a common variant of Fc epsilon RI-beta, lle181Leu within the 4th transmembrane domain. Leu181 shows significant association with positive IgE responses in a random patient sample. Amongst 60 unrelated nuclear families with allergic asthmatic probands, Leu181 is identified in 10 (17%), is maternally inherited in each, and shows a strong association with atopy. Our data indicate that Fc epsilon RI-beta, subject to maternal modification, may be the atopy-causing locus on chromosome 11q.

Raster plots machine learning to predict the seizure liability of drugs and to identify drugs.

In vitro microelectrode array (MEA) assessment using human induced pluripotent stem cell (iPSC)-derived neurons holds promise as a method of seizure and toxicity evaluation. However, there are still issues surrounding the analysis methods used to predict seizure and toxicity liability as well as drug mechanisms of action. In the present study, we developed an artificial intelligence (AI) capable of predicting the seizure liability of drugs and identifying drugs using deep learning based on raster plots of neural network activity. The seizure liability prediction AI had a prediction accuracy of 98.4% for the drugs used to train it, classifying them correctly based on their responses as either seizure-causing compounds or seizure-free compounds. The AI also made concentration-dependent judgments of the seizure liability of drugs that it was not trained on. In addition, the drug identification AI implemented using the leave-one-sample-out scheme could distinguish among 13 seizure-causing compounds as well as seizure-free compound responses, with a mean accuracy of 99.9 ± 0.1% for all drugs. These AI prediction models are able to identify seizure liability concentration-dependence, rank the level of seizure liability based on the seizure liability probability, and identify the mechanism of the action of compounds. This holds promise for the future of in vitro MEA assessment as a powerful, high-accuracy new seizure liability prediction method.

Efficacy of a "provisional incision" for longitudinal flap division after free-flap surgery.

Fix and flap surgery for severe open limb fractures is already a standard treatment. In cases where the fracture is complicated or accompanied by bone defects, secondary surgery is required for fracture sites covered with a myocutaneous flap after the soft tissue condition has stabilized. We applied the delayed procedure concept used for distant flaps and attempted to prevent postoperative myocutaneous flap necrosis by performing a provisional incision prior to the longitudinal incision of the flap. We report the course of five cases of the longitudinal division of the myocutaneous flap using "provisional incision" after free-flap surgery for severe open fracture and verify its usefulness. In this case series, five patients with severe open limb fractures treated from 2020 to 2021 who underwent longitudinal incision of the myocutaneous flap using provisional incision after free-flap surgery were included. The types of flaps used for soft tissue reconstruction in the acute phase, the reasons for the need for secondary surgery, the period from soft tissue reconstruction to additional surgery, and the healing status of soft tissue after secondary surgery were all investigated retrospectively. The types of flaps used for soft tissue reconstruction were latissimus dorsi myocutaneous flap in four cases and anterolateral thigh flap in one case. The breakdown of secondary surgery was osteosynthesis in one case, plate removal in one case, and bone cement removal and autologous bone grafting in three cases. The period from soft tissue reconstruction to secondary surgery ranged from 6 weeks to 4 months. In all cases, the wound healed without necrosis of the myocutaneous flap. For the treatment of severe open limb fractures, longitudinal division of the myocutaneous flap using "provisional incision" is a safer approach to the necessary secondary surgery and reduces the possibility of necrosis of the flap.

Expressions of PD-L1 and Nectin-4 in urothelial cancer patients treated with pembrolizumab.

OBJECTIVES: Recently, the standard of care for advanced urothelial cancer (UC) has been changed by developing immune-checkpoint inhibitors (ICIs). However, its response rate is limited to 20-30%. The identification of biomarkers to predict the therapeutic effects of ICIs is urgently needed. The present study explored the association between immunohistochemical biomarkers and clinical outcomes in UC patients treated with pembrolizumab. PATIENTS AND METHODS: A total of 85 patients with UC who received pembrolizumab after chemotherapy from January 2018 to May 2020 were retrospectively reviewed. Tumor tissues were obtained for immunohistochemical study from 47 out of 85 patients. The protein expressions of PD-L1, WT1, Nectin-4, CD4, CD8, Foxp3, and CD68 in tumor cells and/or tumor infiltrating lymphocytes were immunohistochemically examined. The associations between protein expressions and overall survival (OS), progression-free survival (PFS), and disease control rate (DCR) were statistically analyzed. RESULTS: Patients with positive PD-L1 in tumor cells showed significantly worse OS (Log-rank test: HR 5.146, p = 0.001, Cox regression analysis: HR 4.331, p = 0.014) and PFS (Log-rank test: HR 3.31. p = 0.022), along with significantly lower DCR (14.3%) compared to the PD-L1 negative patients (67.5%). In addition, patients with strong expression of Nectin-4 in tumor cells showed significantly higher DCR (100%) than the other patients (50%). CONCLUSION: PD-L1 expression in tumor cells was associated with poor prognosis (OS and PFS) and low DCR. Interestingly, the strong expression of Nectin-4 was correlated with high DCR. PD-L1 and Nectin-4 expression in tumor cells could be prognostic biomarkers useful for pembrolizumab in patients with advanced UC.

In vitro and in vivo inhibitory effect of three Cox-2 inhibitors and epithelial-to-mesenchymal transition in human bladder cancer cell lines.

BACKGROUND: Although the anti-tumour effect of cyclooxygenase-2 (Cox-2) inhibitors in invasive bladder cancer has been confirmed, its mechanisms of action are unclear. Recently, the concept of an epithelial-to-mesenchymal transition (EMT) promoting carcinoma progression has been suggested, and a key feature of the EMT is the downregulation of E-cadherin. In this study, we investigated the effect of Cox-2 inhibitors on reversal EMT and tumour growth inhibition in bladder cancer cells. METHODS: We used three Cox-2 inhibitors, etodolac, celecoxib and NS-398 and three human bladder cancer cell lines, T24, 5637 and KK47, in this study. T24 xenograft tumour mouse model was used in the in vivo study. RESULTS: Within the clinical drug concentrations, only etodolac showed the in vitro growth inhibition in T24 not in the other cell lines. Etodolac reduced SNAIL mRNA and vimentin cell surface expression, and induced E-cadherin mRNA and E-cadherin cell surface expression, in T24. Etodolac also most strongly inhibited the cell migration of T24 in vitro and showed the highest tumour growth inhibition in T24 tumour in vivo. CONCLUSION: Etodolac at clinical doses exhibited induced in vitro and in vivo anti-tumour effects and reversal effect of EMT in T24. These results suggest that etodolac is a good candidate for an anti-tumour or chemopreventive reagent for high-grade bladder cancer.

In vivo morphometric analysis of inflammatory condylar changes in rat temporomandibular joint.

OBJECTIVE: Injection of complete Freund's adjuvant (CFA) into the temporomandibular joint (TMJ) causes acute swelling around the joint and subsequent morphological alterations in the condyle. We aimed to evaluate changes in the three-dimensional architecture of the condyle induced with CFA. MATERIALS AND METHODS: The CFA was injected into the unilateral TMJ of rats and morphological changes in the condyle were assessed repeatedly for 14 days by in vivo micro-CT. RESULTS: Osseous abnormalities of condyle were first observed at 3-5 days after CFA injection on the tomographic images, and the condylar deformation became more obvious thereafter. Among 12 condyles examined at 14 days postinjection, osteophytosis was observed in all of the specimens and bone erosion coexisted in five condyles. None of the saline-treated condyles showed architectural changes. Significant changes were detected in the mesiolateral and rostrocaudal widths of the CFA-treated condyles at 10-14 days postinjection (P < 0.01). The extent of both condylar bone formation and resorption was greater in the CFA-injected TMJs than in saline-injected TMJs (P < 0.05). CONCLUSION: These results indicate that CFA causes dynamic morphological changes in the condyle and that our experimental approach will provide new insights into the subacute inflammatory processes in the TMJ.

Principal Component Analysis to Distinguish Seizure Liability of Drugs in Human iPS Cell-Derived Neurons.

Screening for drug discovery targeting the central nervous system requires the establishment of efficient and highly accurate toxicity test methods that can reduce costs and time while maintaining high throughput using the function of an in vitro neural network. In particular, an evaluation system using a human-derived neural network is desirable in terms of species difference. Despite the attention, the microelectrode array (MEA) is attracting among the evaluation systems that can measure in vitro neural activity, an effective analysis method for evaluation of toxicity and mechanism of action has not yet been established. Here we established analytical parameters and multivariate analysis method capable of detecting seizure liability of drugs using MEA measurement of human iPS cell-derived neurons. Using the spike time series data of all drugs, we established periodicity as a new analytical parameter. Periodicity has facilitated the detection of responses to seizurogenic drugs, previously difficult to detect with conventional analytical parameters. By constructing a multivariate analytical method that identifies a parameter set that achieves an arbitrary condition, we found that the parameter set comprising total spikes, maximum frequency (MF), inter- MF interval (IMFI), coefficient of variance of IMFI, and periodicity can uniformly detect the seizure liability of seizurogenic drugs with different mechanisms of action. Seizurogenic drugs were suggested to increase the regularity of the network burst in MEA measurements in human iPS cell-derived neurons.

The inverse association between tuberculin responses and atopic disorder.

Human immune responses are heterogeneous and may involve antagonism between T helper (TH) lymphocyte subsets and their cytokines. Atopy is characterized by immediate immunoglobulin E (IgE)-mediated hypersensitivity to agents such as dust mites and pollen, and it underlies the increasingly prevalent disorder asthma. Among Japanese schoolchildren, there was a strong inverse association between delayed hypersensitivity to Mycobacterium tuberculosis and atopy. Positive tuberculin responses predicted a lower incidence of asthma, lower serum IgE levels, and cytokine profiles biased toward TH1 type. Exposure and response to M. tuberculosis may, by modification of immune profiles, inhibit atopic disorder.

FK506-protective effects against trimethyltin neurotoxicity in rats: hippocampal expression analyses reveal the involvement of periarterial osteopontin.

There is little information on the molecular mechanisms in FK506-mediated neuroprotection. In the present study, we investigated the protective effect of FK506, an immunosuppressant and neuroprotectant, on trimethyltin (TMT)-induced neurotoxicity in the rat hippocampus. Histologically, TMT-induced neuronal damage was partially prevented by FK506 in the hippocampal CA1 region, but not in CA3. FK506 treatment significantly reduced the number of apoptotic cells in CA1, but not in CA3, and also prevented induction of cognitive deficits by TMT. Microarray analysis of the rat hippocampus detected 14 genes with TMT-induced alteration of mRNA expression that was rescued by FK506 treatment. Subsequent quantitative RT-PCR analysis confirmed elevated mRNA levels for four inflammatory genes, glutathione S-transferase, lysozyme, matrix Gla protein, and osteopontin after TMT treatment. Upregulation of these genes was reversed by FK506 treatment at 5 days postgavage. Immunohistochemistry revealed that FK506 reduced osteopontin (OPN) induction by TMT in the periarterial area at 5 days postgavage. Our data suggest that inflammatory gene expression is involved in TMT-induced damage to the hippocampal CA1 region, resulting in apoptosis, and that this process is initiated by periarterial OPN activation, and can be alleviated by FK506.

Mitochondrial DNA determines androgen dependence in prostate cancer cell lines.

Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is very common in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established by inoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatly reduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNA-depleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA may thus play a role in the development of androgen independence, leading to progression of prostate cancers.

Emergence of morphological order in the network formation of Physarum polycephalum.

Emergence in a system appears through the interaction of its components, giving rise to higher order or complexity in the system. We tested for the presence of emergent properties in a biological system using the simplest biological entity of a unicellular organism; the plasmodium of Physarum polycephalum, a giant unicellular amoeboid organism that forms a network-like tubular structure connecting its food sources. We let two plasmodium networks within a single cell interact with each other, and observed how the intracellular interaction affected the morphologenesis of the plasmodium networks. We found that the two networks developed homologous morphology. We further discuss the presence of autonomous and emergent properties in homologous network formation.

Combination of p53-DC vaccine and rAd-p53 gene therapy induced CTLs cytotoxic against p53-deleted human prostate cancer cells in vitro.

Recently, the US FDA approved sipuleucel-T, which is composed of autologous DCs stimulated with a recombinant fusion protein of prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF), as the first immunotherapeutic agent for metastatic castration resistant prostate cancer (mCRPC). However, sipuleucel-T demonstrated only modest efficacy in mCPRC patients. Researchers are now investigating the potential of p53 protein as a tumor-associated antigen (TAA) loaded in DC-based cancer vaccine. Approximately half of all tumors overexpress p53, and up to 20% of prostate cancer cells overexpresses p53. In this study, we evaluated the feasibility of combining p53-DC vaccine and rAd-p53 gene therapy, using the p53-overexpressing and non-expressing prostate cancer cells in vitro. We successfully generated the p53-DC vaccine by culturing autologous DCs infected with rAd-p53. This p53-DC vaccine can differentiate CTLs specifically cytotoxic to p53-overexpressing prostate cancer cells. In addition, rAd-p53 infection can induce overexpression of p53 and thus the cytotoxicity of CTLs differentiated by the p53-DC vaccine in p53 non-expressing prostate cancer cells. These findings suggest that this combination therapy using p53-DC vaccine and rAd-p53 gene therapy together may represent a new paradigm for the treatment of mCRPC.

Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models.

Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed in high levels by osteoblasts. To determine whether the OC promoter mediates cell-specific gene expression in cells of osteoblast lineage, we constructed a recombinant adenovirus, Ad-OC-TK, which contains the OC promoter that drives the expression of herpes simplex virus thymidine kinase (TK). We tested the expression of TK by this virus in osteoblast cell lines as well as in non-osteoblastic cell lines by assessing the enzyme activity of TK in vitro. Whereas the OC promoter failed to drive the expression of the TK gene in several non-osteoblastic cell lines such as WH, a human bladder transitional carcinoma, and NIH 3T3, an embryonic mouse fibroblast cell line, the OC promoter mediated high levels of expression in osteoblast cell lines including murine ROS and human MG-63 cells. The addition of acyclovir (ACV), a pro-drug for the inhibition of cell proliferation, resulted in the induction of osteoblast-specific cell death in vitro. Intratumoral injection of Ad-OC-TK into murine ROS osteosarcoma abolished tumor growth in a host treated with subsequent i.p. ACV injection in vivo. The Ad-OC-TK virus plus ACV treatment appears to be highly selective in blocking the growth of both murine and human osteosarcoma cell lines in vitro and murine osteosarcoma in vivo.

Distinct Excitation to Pulpal Stimuli between Somatosensory and Insular Cortices.

Somatosensory information from the dental pulp is processed in the primary (S1) and secondary somatosensory cortex (S2) and in the insular oral region (IOR). Stimulation of maxillary incisor and molar initially induces excitation in S2/IOR, rostrodorsal to the mandibular incisor and molar pulp-responding regions. Although S1 and S2/IOR play their own roles in nociceptive information processing, the anatomical and physiological differences in the temporal activation kinetics, dependency on stimulation intensity, and additive or summative effects of simultaneous pulpal stimulation are still unknown. This information contributes not only to understanding topographical organization but also to speculating about the roles of S1 and S2/IOR in clinical aspects of pain regulation. In vivo optical imaging enables investigation of the spatiotemporal profiles of cortical excitation with high resolution. We determined the distinct features of optical responses to nociceptive stimulation of dental pulps between S1 and S2/IOR. In comparison to S1, optical signals in S2/IOR showed a larger amplitude with a shorter rise time and a longer decay time responding to maxillary molar pulp stimulation. The latency of excitation in S2/IOR was shorter than in S1. S2/IOR exhibited a lower threshold to evoke optical responses than S1, and the peak amplitude was larger in S2/IOR than in S1. Unexpectedly, the topography of S1 that responded to maxillary and mandibular incisor and molar pulps overlapped with the most ventral sites in S1 that was densely stained with cytochrome oxidase. An additive effect was observed in both S1 and S2/IOR after simultaneous stimulation of bilateral maxillary molar pulps but not after contralateral maxillary and mandibular molar pulp stimulation. These findings suggest that S2/IOR is more sensitive for detecting dental pulp sensation and codes stimulation intensity more precisely than S1. In addition, contra- and ipsilateral dental pulp nociception converges onto spatially closed sites in S1 and S2/IOR.

Cyclic nucleotide phosphodiesterase 3 expression in vivo: evidence for tissue-specific expression of phosphodiesterase 3A or 3B mRNA and activity in the aorta and adipose tissue of atherosclerosis-prone insulin-resistant rats.

With a view of understanding the potential roles of phosphodiesterase (PDE)3 in the acceleration of atherosclerosis in diabetes, we have analyzed the in vivo levels of low Km cAMP PDE3 and PDE4 activities as well as PDE3A and PDE3B mRNA in a relevant animal model. The JCR:LA-cp rat is a unique strain that develops obesity, insulin resistance, and vasculopathy when homozygous for the autosomal recessive cp gene (cp/cp). Lean rats, bred (designated +/?) as a 2:1 mixture of animals that are heterozygous (cp/+) or homozygous normal (+/+), are metabolically normal. We find that PDE3 activity is the major low Km cAMP activity in the aorta of cp/cp rats and is approximately twofold higher than that in lean +/? rats. PDE3A mRNA levels in middle-aged cp/cp rats are also elevated, approximately threefold, compared with those of +/? rats or young 12-week-old cp/cp rats. Thus, in the aorta of atherosclerosis-prone insulin-resistant cp/cp rats, PDE3A gene expression is upregulated, resulting in significantly higher PDE3 activity. This upregulation of PDE3A mRNA levels was a rather unique phenomenon to the aorta of JCR:LA-cp rats compared with that in the aorta of other rat strains. This result is consistent with our hypothesis that an increased PDE3 activity in aortic smooth muscle cells may contribute to accelerated atherosclerosis in diabetes. Furthermore, determination of PDE3 activity and PDE3A and PDE3B mRNA levels in heart and white and brown fat tissues of JCR:LA-cp rats revealed that PDE3B mRNA and activity in white adipose tissue is downregulated in this diabetic animal model, and that PDE3A and PDE3B genes are tissue-specifically expressed and differentially regulated in aorta and adipose tissue, respectively, under hyperinsulinemic conditions.

Maternal phase setting of fetal circadian oscillation underlying the plasma corticosterone rhythm in rats.

We examined the entraining effect of the maternal circadian system on the fetal circadian oscillation during pregnancy. The circadian rhythm of locomotor activity and plasma corticosterone of pregnant rats was abolished by bilateral ablation of the suprachiasmatic nuclei at day 10 of gestation. At term, pups were removed by Cesarean section and were blinded immediately. To avoid possible rhythmic influences of a nursing mother on the pups' circadian rhythms, alternating nursing was imposed on blinded infants. Thus, pups were exchanged every 12 h between two foster mothers, one entrained to a light-dark cycle and the other to dark-light, so that one group of pups were always nursed in the light period and the other in the dark. Both groups of pups showed free-running circadian hormone rhythms with similar phase angles. However, the circadian rhythm of these pups was always phase delayed by about 8 h to that of blinded control pups which were born to unoperated mothers. Furthermore, blinded pups born to and nursed by a suprachiasmatic nuclei lesioned mother developed a circadian hormone rhythm which was phase delayed by 4 h to that of the control. It is concluded that the circadian oscillation underlying the rhythm of corticosterone release in rats has entrained to the maternal circadian system during fetal life. It is further suggested that the entrainment starts before day 10 of gestation.