Plasma pharmacokinetics of vinblastine and the investigational Vinca alkaloid N-(deacetyl-O-4-vinblastoyl-23)-L-ethyl isoleucinate in mice as determined by high-performance liquid chromatography.

The plasma pharmacokinetics of vinblastine and N-(deacetyl-O-4-vinblastoyl-23)-L-ethyl isoleucinate (VileE) in mice have been studied as part of the preclinical investigations of VileE, a new investigational semisynthetic Vinca alkaloid. Groups of animals received the test compounds through i.v. bolus injection at LD10, 0.5 x LD10, and 0.1 x LD10 doses. VileE has also been administered p.o. Drug plasma levels have been analyzed with a sensitive and selective method using liquid-liquid extraction for sample clean-up and high-performance liquid chromatography combined with fluorescence detection for quantification. Following i.v. injection, plasma kinetics of both vinblastine and VileE can be described adequately by a three-compartment open model. VileE demonstrates nonlinear pharmacokinetics with decreasing clearance and increasing terminal half-lives at increasing doses. Comparison of the plasma concentration versus time curves for vinblastine in humans and mice indicates that the toxicity of these compounds may not be directly related to the drug exposure expressed by the area under curve in plasma but by the terminal half-life and the time that a toxic threshold level is attained. Pharmacokinetically guided dose escalation in coming phase I trials of VileE is, therefore, discouraged.

Phase response curve and light-induced fos expression in the suprachiasmatic nucleus and adjacent hypothalamus of Arvicanthis niloticus.

This article describes the phase response curve (PRC), the effect of light on Fos immunoreactivity (Fos-IR) in the suprachiasmatic nucleus (SCN), and the effect of SCN lesions on circadian rhythms in the murid rodent, Arvicanthis niloticus. In this species, all individuals are diurnal when housed without a running wheel, but running in a wheel induces a nocturnal pattern in some individuals. First, the authors characterized the PRC in animals with either the nocturnal or diurnal pattern. Both groups of animals were less affected by light during the middle of the subjective day than during the night and were phase delayed and phase advanced by pulses in the early and late subjective night, respectively. Second, the authors characterized the Fos response to light at circadian times 5, 14, or 22. Light induced an increase in Fos-IR within the SCN during the subjective night but not subjective day; this effect was especially pronounced in the ventral SCN, where retinal inputs are most concentrated, but was also evident in other regions. Both light and time influenced Fos-IR within the lower subparaventricular area. Third, SCN lesions caused animals to become arrhythmic when housed in a light-dark cycle as well as constant darkness. In summary, Arvicanthis appear to be very similar to nocturnal rodents with respect to their PRC, temporal patterns of light-induced Fos expression in the SCN, and the effects of SCN lesions on activity rhythms.

Fos in the suprachiasmatic nucleus of house mouse lines that reveal a different phase-delay response to the same light pulse.

Increased light intensity of a 5-min light pulse is positively correlated with Fos mRNA and Fos protein levels in the suprachiasmatic nucleus (SCN) of hamsters. These findings suggest that the level of Fos activation is proportional to the light intensity and that the magnitude of the phase-shift response depends on the level of Fos activation. However, to what extent different phase-delay responses to the same light pulse are mediated by differential Fos activation is unknown. To elucidate this, the authors used selected house mouse lines that reveal an almost threefold difference in phase-delay responses in constant darkness (DD) between circadian time (CT) 16 and CT 20 to the same light pulse. The authors measured wheel-running activity and subjected male mice of these lines to a 15-min light pulse at CT 16 after 2 weeks in DD. The behavioral response was measured and 10 to 12 days later the animals were again subjected to the same light pulse at CT 16. One hour after the start of the second light pulse, the animals were sacrificed for Fos immunocytochemistry. Results indicate a significant difference between the lines in the phase-delay response (F2,26 = 5.112, p < 0.017) and the level of Fos activation (F2,26 = 27.15, p < 0.0001) after a 15-min light pulse at CT 16. These findings support the hypothesis that the magnitude of the phase-delay response is proportional to the number of cells in the SCN that exhibit Fos induction after the same 15-min light pulse at CT 16 in DD. It also indicates a possible difference in the input pathways among the lines.

Daily rhythms of Fos expression in hypothalamic targets of the suprachiasmatic nucleus in diurnal and nocturnal rodents.

Little is known about the differences in the neural substrates of circadian rhythms that are responsible for the maintenance of differences between diurnal and nocturnal patterns of activity in mammals. In both groups of animals, the suprachiasmatic nucleus (SCN) functions as the principal circadian pacemaker, and surprisingly, several correlates of neuronal activity in the SCN show similar daily patterns in diurnal and nocturnal species. In this study, immunocytochemistry was used to monitor daily fluctuations in the expression of the nuclear phosphoprotein Fos in the SCN and in hypothalamic targets of the SCN axonal outputs in the nocturnal laboratory rat and in the diurnal murid rodent, Arvicanthis niloticus. The daily patterns of Fos expression in the SCN were very similar across the two species. However, clear species differences were seen in regions of the hypothalamus that receive inputs from the SCN including the subparaventricular zone. These results indicate that differences in the circadian system found downstream from the SCN contribute to the emergence of a diurnal or nocturnal profile in mammals.

Penetration of zidovudine into the cerebrospinal fluid of patients infected with HIV.

OBJECTIVE: To investigate penetration of zidovudine (ZDV) into the cerebrospinal fluid (CSF) of HIV-infected patients for whom a lumbar puncture was indicated. DESIGN: A prospective study. SETTING: General 525-bed hospital with special funding for treatment and research of HIV-infected patients. PATIENTS, PARTICIPANTS: Thirty-nine patients with a medical indication for lumbar puncture who used ZDV chronically were included in this study (50 samples in total). MAIN OUTCOME MEASURE: Determination of ZDV and proteins in CSF and plasma samples. RESULTS: CSF concentrations of ZDV showed little fluctuation 1-8 h after the last ingestion of ZDV. In contrast, plasma levels displayed large variability in this period and decreased exponentially over time. As a result, the CSF/plasma ratio increased linearly over time. No significant relation between the ZDV dose, neither the medical indication for lumbar puncture nor the protein ratio (as a measure for the integrity of the blood-brain barrier), and CSF levels of ZDV was found. The CSF/plasma ratio of ZDV did not give essential information on drug distribution into CSF. CONCLUSIONS: Penetration of ZDV into the CSF appears to be independent of the dose (range, 200-1250 mg daily), which may be an explanation for the efficacy of low doses of ZDV in the prevention and treatment of HIV-related neurological diseases. ZDV levels were at steady-state during the first 6 h after ingestion. The CSF/plasma ratio of ZDV concentrations is not an appropriate marker for drug penetration into CSF.

Pharmacokinetic variability of zidovudine in HIV-infected individuals: subgroup analysis and drug interactions.

OBJECTIVE: To investigate determinants of inter- and intraindividual variability of zidovudine (ZDV) pharmacokinetics in HIV-infected patients. DESIGN: A prospective study in a general 525-bed hospital with special funding for treatment and research of HIV-infected patients. METHODS: Serial blood samples were collected from 68 HIV-infected individuals providing a total of 95 pharmacokinetic curves. ZDV was measured with high-performance liquid chromatography and radioimmunoassay. Pharmacokinetic parameters were calculated by non-compartmental analysis. Patient characteristics were investigated by multivariate analysis for an influence on ZDV pharmacokinetics. RESULTS: Apparent ZDV clearance was significantly lower in patients with a lower body weight, in women, and in patients with a more advanced stage of HIV disease. Co-administration of methadone with ZDV resulted in higher plasma concentrations of ZDV, while rifampin and ganciclovir increased apparent ZDV clearance. Age, the duration of ZDV use, CD4+ cell count, creatinine clearance, elevated serum liver enzyme levels, and the use of 11 other co-administered medications were not independently related to apparent ZDV clearance. CONCLUSIONS: The pharmacokinetic profile of ZDV in several subpopulations has been evaluated, as well as the observation of possible drug-drug interactions between ZDV and 14 different drugs or groups of drugs. These data suggest that patient-individualized antiretroviral therapy may be appropriate once pharmacokinetic-pharmacodynamic relationships have been established.

STEP: a family of brain-enriched PTPs. Alternative splicing produces transmembrane, cytosolic and truncated isoforms.

The family of striatal enriched phosphatases (STEPs) consists of protein tyrosine phosphatases (PTPs) that are enriched within the central nervous system. Previous biochemical studies have shown that the STEP family includes transmembrane, as well as soluble, cytosolic proteins. We now extend these findings with the isolation and characterization of a new, truncated member of this family, termed STEP38. The cDNA of STEP38 encodes a protein of 346 amino acids with a predicted mobility of 38 kDa. In contrast to the cytosolic variants, it contains two hydrophobic amino acid sequences at its N-terminus, two sequences enriched in Pro, Glu, Asp, Ser and Thr residues (PEST sequences), and two polyproline domains. We have used differential centrifugation, continuous sucrose gradients, and transfection experiments to clarify the subcellular localization of STEP38 within membrane compartments. These experiments suggest that a pool of STEP38 is targeted to membrane compartments of the endoplasmic reticulum. The STEP38 cDNA contains a stop codon upstream of the catalytic phosphatase domain that is normally present in other STEP variants, and enzymatic assays conform that STEP38 is inactive. Thus, the STEP family consists of cytosolic, transmembrane, and truncated isoforms. These findings are similar to what has been found for some members of the protein tyrosine kinase (PTK) family that uses alternative splicing mechanisms to produce active and inactive variants. By analogy with suggested mechanisms of action for the truncated PTKs, inactive STEP isoforms may participate in signaling events by protecting potential substrates from dephosphorylation by other members of this family.

Liposomes and immunoassays.

Various aspects of the application of liposomes as a label in immunoassays are reviewed. Methods for the preparation of liposomes, from the basic film method to the more advanced dehydration-rehydration method, are discussed. Furthermore, the markers used in liposome labels, as well as the methods to conjugate liposomes to antigens or antibodies, are summarized. Liposome immunoassays are applied as homogeneous or heterogeneous assays. Homogeneous assays often rely on the lytic activity of complement on antibody-associated liposomes. Another group of homogeneous assays utilizes the inhibitory action of antibodies on the activity of conjugates of mellitin (a bee venom protein) with a hapten. Free mellitin conjugates are able to lyse liposomes effectively. Heterogeneous liposome immunoassays, performed either competitively or non-competitively, resemble more closely standard enzyme linked immunosorbent assays, with the enzyme being replaced by a liposome label. Washing steps are used to separate antigen-specifically bound liposomes from unbound liposomes. All bound liposomes are lysed with a detergent, giving an instantaneous amplification. Flow-injection liposome immunoassays and liposome immunosensors are also described as examples of other possible immunoassay formats.

Application of xanthine oxidase-catalyzed luminol chemiluminescence in a mouse interleukin-5 immunoassay.

A chemiluminescent substrate reagent for use in a sandwich immunoassay for the model antigen mouse interleukin-5 (IL-5) was developed using xanthine oxidase and luminol. Various parameters involved in this chemiluminescent reaction have been studied, including the substrate hypoxanthine, luminol and the Fe(II)-EDTA complex. Addition of the Fe(II)-EDTA complex enhances the chemiluminescence signal considerably. The xanthine oxidase-catalyzed chemiluminescent immunoassay was compared to horseradish peroxidase-linked immunoassays with luminol as chemiluminescent, and tetramethyl benzidine as colorimetric substrate. The detection limit of the xanthine oxidase-luminol assay was found to be about 0.6 pg/ml IL-5, whereas the peroxidase-catalyzed immunoassays have detection limits of about 1.3 (HRP-TMB) and 2.9 pg/ml (HRP-luminol) IL-5.

Identification of two alternatively spliced transcripts of STEP: a subfamily of brain-enriched protein tyrosine phosphatases.

A brain-enriched protein tyrosine phosphatase termed STEP46 (striatal enriched phosphatase) was previously isolated and characterized. Immunological studies with a STEP monoclonal antibody recognized several STEP-immunoreactive proteins, and suggested that additional STEP-related polypeptides existed. This study reports the isolation of two alternatively spliced transcripts of the STEP gene. One of these, STEP20 (with a predicted molecular mass of 20 kDa) was further characterized and found to lack the conserved tyrosine phosphatase domain. Northern analysis detected a 2.8 kb STEP20 message in mouse brain. The second alternatively spliced transcript, STEP61, has a 5'-extended open reading frame that encodes a protein with a predicted molecular mass of 61 kDa and contains a single tyrosine phosphatase domain. The exon-intron organization responsible for the novel STEP20 and STEP61 sequences was determined in the mouse STEP genomic DNA. We propose that the original STEP46, along with STEP20 and STEP61, are members of a brain-enriched subfamily of protein tyrosine phosphatases, and that STEP isoforms may have distinct functions within the central nervous system.

Quantitative determination of Ecteinascidin 743 in human plasma by miniaturized high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A method was developed for the bio-analysis of Ecteinascidin 743 (ET-743) using miniaturized liquid chromatography (LC) coupled to an electrospray ionization sample inlet (TurbolonSpray) and two quadrupole mass analyzers (LC/ESI-MS/MS). Solid-phase extraction was used as a sample pretreatment procedure. Ecteinascidin 743 is a very potent anticancer compound and is administered in microgram m-2 dosages, which demands special requirements in terms of sensitivity for the analytical method supporting clinical pharmacokinetic studies. Using conventional LC/UV, a lower limit of quantitation (LLQ) of 1 ng ml-1 plasma was reached using a 500 microliters sample volume, but LC/ESI-MS/MS permitted an LLQ of 10 pg ml-1. The latter method was found to be accurate and precise, and provided a broad linear concentration range of 0.010-2.50 ng ml-1.

A putative poststreptococcal case of OCD with chronic tic disorder, not otherwise specified.

A 12-year-old girl presented with an atypical, recurrent, increasingly treatment-resistant case of obsessive-compulsive disorder and chronic tic disorder associated with profound separation anxiety, learning difficulty, and intermittent upper respiratory symptoms. In addition to detailed reviews of history and findings from many clinical caretakers from the prior 7 years, current pediatric, psychiatric, neuropsychological, neuroimaging, and clinical laboratory data were also available. Treatment options were considered from multiple perspectives: psychoanalytically oriented psychotherapy, conventional pharmacotherapy, family interventions, cognitive-behavioral therapy, and learning-supportive strategies. Psychological, neuropsychiatric, and neuroimmunological formulations of etiology were considered. Subsequent treatments included supportive psychotherapy, neuroleptic augmentation of selective serotonin reuptake inhibitors, prophylactic penicillin, and a course of six sessions of plasmapheresis over a 2-week period. The case raises questions for ongoing consideration that juxtapose dynamic, neuropsychiatric, and neuroimmunological perspectives.

Tissue disposition, excretion and metabolism of vinblastine in mice as determined by high-performance liquid chromatography.

We have developed and validated a selective analytical procedure, based on ion-exchange normal-phase liquid chromatography with fluorescence detection and liquid-liquid extraction, for the analysis of vinblastine (VBL) in biological matrices. The assay is suitable for the determination of the parent compound and its metabolites in plasma, tissue, faeces and urine specimens. Pharmacokinetics studies were performed in male FVB mice receiving VBL by intravenous (i.v.) bolus injection at a dose of 6 mg/kg. Plasma concentrations were monitored until 48 h after drug administration. Urine and faeces samples were collected in 24-h portions for up to 72 h and tissue samples were obtained at 4, 24, 72 and 168 h after drug administration. To facilitate a comparison between the findings we obtained by high-performance liquid chromatography (HPLC) and the results of previous studies using radiolabeled drug monitoring, some of the animals were also given radiolabeled drug. Large discrepancies were observed between the results obtained by the two methods. Excretion of the radiolabel in faeces and urine was 85% of the dose within 72 h. HPLC revealed that only 18% of the dose was excreted as unchanged drug and 19%, as measurable metabolites [O4-deacetylvinblastine (DVBL) and two unknown compounds]. In most of the tissues taken at 4 h after drug administration, virtually all of the radioactivity represented VBL or DVBL. In all tissues taken at 72 h after drug administration, however, only very little of the radioactivity remained in the form of these compounds. Following the administration, VBL and DVBL were distributed extensively to most tissues. Many tissues appeared to possess effective means of extruding the cytotoxic drug with decreasing plasma levels. However, in some organs, including those from the genital tract and lymphatic tissues, VBL and DVBL were retained for prolonged periods. Our studies confirm previous indications that selective retention may be the basis of the activity of VBL against malignant transformations derived from these tissues.

Pharmaceutical development of a parenteral lyophilized formulation of the novel indoloquinone antitumor agent EO9.

The aim of this study was to design a stable parenteral dosing form of the investigational cytotoxic drug, encoded EO9. EO9 exhibits poor aqueous solubility and stability characteristics. Freeze-drying was selected as the manufacturing process. Differential scanning calorimetry studies were conducted to determine the freeze-drying cycle parameters. A stable lyophilized formulation of EO9 was developed. The prototype, containing 8.0 mg EO9 and 200 mg lactose/vial, was found to be the optimal formulation in terms of solubility, length of the freeze-drying cycle, stability, and dosing requirements for phase I clinical trials. Quality control of the freeze-dried formulation showed that the manufacturing process does not change the integrity of EO9. Shelf-life studies demonstrated that the formulation remains stable for at least 1 year when stored at +4 degrees C in a dark environment.

Plasma pharmacokinetics, tissue disposition, excretion and metabolism of vinleucinol in mice as determined by high-performance liquid chromatography.

We investigated the pharmacokinetics of the experimental semisynthetic vinca alkaloid vinleucinol (VileE; O4-deacetyl-3-de(methoxycarbonyl)-3-[[[1-ethoxycarbonyl-2- methylbutyl]amino]carbonyl]-vincaleukoblastine). The study was performed in male FVB mice receiving 10.5 mg/kg VileE i.v. or p.o. Plasma, urine, faeces and tissue samples were analysed by a selective method based on ion-exchange normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection and liquid-liquid extraction for sample clean-up. Apart from the parent drug, two other metabolic compounds were detected. One of these metabolites is vinleucinol acid (VileA; O4-deacetyl-3-de(methoxycarbonyl)-3-[[[1-carboxyl-2- methylbutyl]amino]carbonyl]-vincaleukoblastine), which possesses no cytotoxic activity. The structure proposed for the second metabolite (VileX) was based on tandem mass spectrometry (MS-MS) and infrared (IR) spectroscopy data. Metabolization of VileE to VileX must occur in the amino acid moiety of the molecule, with a (beta- or gamma-) lactone ring being formed after oxidation of the (beta- or gamma) carbon of the amino acid. VileX is a major metabolite, which is excreted in faeces and urine after i.v. administration and accounting for up to 23% of the administered dose. The activity of VileX against cultured L1210 cells is four times that of the parent drug VileE and comparable with that of vinblastine (VBL). At 48 h after administration of VileE, the concentration of VileX exceeds that of the parent drug in many tissues. These findings indicate that the metabolite VileX may be at least largely responsible for the activity observed against xenografts in mice after administration of the parent drug, VileE.

Pharmacokinetics and metabolism of docetaxel administered as a 1-h intravenous infusion.

Docetaxel, a taxane antitumor agent, was administered to 24 patients by a 1-h intravenous infusion at a dose level of 100 mg/m(2) with pharmacokinetic monitoring. The plasma concentration-versus-time data were fitted with a three-compartment model. The mean area under the curve (AUC) for docetaxel was 3.1 +/- 0.9 h. mg/l and the clearance was 34.8 +/- 9.3 l/h per m(2). There was considerable interpatient pharmacokinetic variability. In 33% of the patient population, metabolites were detected in plasma samples collected 5-30 min after the end of the infusion. The cyclized oxazolidinedione metabolite M4 was most frequently present and was detected in 8 out of 24 patients with maximal concentrations between 0.022 and 0.23 mg/l. Logistic regression analysis was performed to predict M4 docetaxel metabolism. In the final model, alanine aminotransferase and alkaline phosphatase levels were the strongest predictors. No relationship was found between M4 metabolism and percentage decrease in neutrophil count in this study. Three patients with high M4 concentrations in plasma during course 1 suffered from most pronounced fluid retention (grade 2-3) after two to five courses.

Distribution of Ca2+-dependent protein kinase C isoforms in the suprachiasmatic nucleus of the diurnal murid rodent, Arvicanthis niloticus.

The suprachiasmatic nuclei (SCN) contain the major 'biological clock' in mammals that controls most circadian rhythms expressed by these animals. The functional importance of protein phosphorylation and intracellular Ca2+ in the mammalian circadian pacemaker is becoming increasingly apparent. Here we report the immunocytochemical localization of the four Ca2+-dependent protein kinase C (PKC) isoforms (alpha, betaI, betaII, gamma) within the SCN of the diurnal murid rodent, Arvicanthis niloticus, and the nocturnal golden hamster. In the SCN of A. niloticus, PKCalpha was the most abundant of the four isoforms. Cells containing PKCalpha were homogeneously distributed throughout the SCN. PKCbetaI cells were sparsely distributed in the perimeter of the SCN and were absent in its central area. PKCbetaII and -gamma were not found in the SCN of A. niloticus. In the SCN of the golden hamster, PKCalpha cells were most heavily concentrated in the dorsomedial region, though some were also present laterally and ventrally. The distribution of arginine-vasopressin (AVP) cells in the SCN overlapped with that of PKC in both species. Species differences in the location of the Ca2+-dependent PKC isoforms suggest differences in function such as the relaying of photic or non-photic information to the clock mechanism, or the synchronization of AVP neurons and their subsequent output signals.

Distribution of AVP and Ca(2+)-dependent PKC-isozymes in the suprachiasmatic nucleus of the mouse and rabbit.

The suprachiasmatic nucleus (SCN) is the circadian pacemaker in mammals and contains a network of arginine-vasopressin-immunoreactive (AVP-ir) neurons. AVP-recipient cells contain the V1a class of receptors linked to phosphoinositol turnover and protein kinase C (PKC). The present study describes the localization of AVP and the four Ca(2+)-dependent PKC-isoforms in the mouse and rabbit SCN. An estimate of the numerical density of AVP-ir neurons at the rostral, medial, and caudal level of the SCN revealed that the mouse SCN contains more than twice the number of AVP-ir neurons than the rabbit SCN. Neurons immunostained for AVP or PKC dominated in the dorsomedial and ventrolateral aspects of the mouse SCN, while the central area of the SCN revealed only weakly stained neurons. The rabbit SCN was characterized by a more homogeneous distribution of AVP-ir and PKC-ir neurons. PKC alpha was the most abundantly expressed isozyme in both species, whereas the presence of the other isoforms differed (mouse: PKC alpha > PKC beta I >> PKC beta II > PKC gamma; rabbit: PKC alpha > PKC beta II > or = PKC gamma > PKC beta I). Clear PKC gamma-positive neurons were only observed in the rabbit SCN, while the mouse SCN predominantly contained immunolabeled fiber tracts for this PKC isozyme. Astrocytes immunoreactive for each PKC isoform were frequently encountered in the rabbit SCN, but were absent in mice. Immunofluorescence double labeling showed that numerous AVP-recipient cells in the mouse SCN were immunopositive for PKC alpha, and that nearly all AVP-ir neurons express PKC alpha abundantly. These results substantiate the putative role for PKC alpha in vasopressinergic signal transduction in the SCN. The differential expression in degree and cell type of the Ca(2+)-dependent PKC-isoforms in the mouse and rabbit SCN may be related to the differences observed in circadian timekeeping between the two species.

Calretinin is not a marker for subdivisions within the suprachiasmatic nucleus.

In this study, we report the immunocytochemical localization of the calcium-binding protein calretinin (CAL) in the suprachiasmatic nuclei (SCN) of male and female rodents including rats, mice, golden hamsters, and Arvicanthis niloticus. The results revealed that CAL is present in different subdivisions of the SCN in the different species studied and CAL can, therefore, not be considered a marker for particular subdivisions within the SCN. No differences were found between males and females.

Differential expression of protein kinase C betaI (PKCbetaI) but not PKCalpha and PKCbetaII in the suprachiasmatic nucleus of selected house mouse lines, and the relationship to arginine-vasopressin.

The functional significance of the suprachiasmatic nucleus (SCN) in circadian rhythm control of mammals has been well documented. The role of protein phosphorylation mediated by protein kinase C (PKC), however, is not well known. We report the immunocytochemical localization of three Ca(2+)-dependent PKC isoforms (alpha, betaI, betaII) within the SCN of selected house mouse lines that differ in behavioral circadian rhythm parameters. Optical density measurements revealed that the adult mice selected for low levels of nest-building behavior (small nest-builders) had more than threefold higher PKCbetaI immunostaining in the SCN than the mice selected for high levels of nest-building behavior (big nest-builders). A similar twofold difference between the adult small and big nest-builders was observed for the number of PKCbetaI-containing cells in the SCN. The non-selected control lines were intermediate. Ten-day-old pups revealed similar differences in PKCbetaI immunostaining in the SCN between the small and big nest-builders. PKCalpha and PKCbetaII immunostaining in the SCN was not different among the lines. PKCbetaI immunostaining was not different among the selected lines in the lateroanterior hypothalamic nucleus (LA) and the cornu ammonis field 1 (CA1) of the dorsal hippocampus and confirms the specificity of the difference in PKCbetaI immunostaining in the SCN among the selected lines. The significance of these findings is discussed in the context of differences among the lines in arginine-vasopressin (AVP) and light-induced Fos expression in the SCN, behavioral phase-delay responses to 15-min light pulses in constant darkness, and measures of the strength of the circadian activity rhythm expressed.