5-[125I]iodo-2'-deoxyuridine in the radiotherapy of brain tumors in rats

Glial neoplasms of the human central nervous system have defied treatment, in part because of the limited selectivity of available cytotoxic agents. The thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter 125I (125IUdR) is highly toxic to dividing cells when it is deoxyribonucleic acid incorporated, but it is relatively innocuous when located outside the nucleus. Previous studies have shown that 125IUdR has significant antineoplastic potential against mammalian cells in vitro and direct administration of 125IUdR is effective therapy for ovarian ascites tumors in mice and neoplastic meningitis in rats. Studies using external gamma imaging and autoradiography have also shown that direct intratumoral administration of 123IUdR/125IUdR into intracerebral 9L gliosarcomas in rats results in selective uptake of the radionuclide into tumor cells. Based on these encouraging results, we have evaluated the therapeutic potential of 125IUdR in rats bearing intracerebral 9L gliosarcomas. METHODS: Iodine-125-IUdR was infused intracerebrally over a 2-day period into rats bearing 1-day-old 9L tumors and over a 6-day period into animals with 9-day-old 9L tumors; equimolar concentrations of 127IUdR were infused into control animals. Tumor growth was monitored by contrast-enhanced 1H MRI and animal survival was followed over time. RESULTS: Intracerebral tumors (3-7 mm) were readily detected by MRI. Tumor-bearing rats treated with 127IUdR succumbed within 17-24 days, whereas tumor-bearing animals treated with 125IUdR survived significantly longer, and 10%-20% of the animals were cured of tumors. CONCLUSION: These data substantiate the antineoplastic potential of 5-[125I]iodo-2'-deoxyuridine and indicate that it may be a useful agent for the therapy of solid tumors that are accessible to direct radiopharmaceutical administration.     

Encapsulation of plasmid DNA in biodegradable poly(D, L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery

Transferrin receptor is a key protein for the cellular uptake of transferrin iron. The highest number of transferrin receptors is on the surface of erythroblasts. The released iron is used for hemoglobinosynthesis. Regulation occurs at mRNA level depending on the intracellular iron concentration. The synthesis of ferritin and transferrin receptor are regulated in an opposite manner. Serum transferrin receptor is a truncated monomeric form of the cellular receptor. Most of the circulating receptors come from erythroid marrow precursors. Its level mirrors the total tissue receptor mass, it depends on the rate of erythropoiesis and on the iron status. Serum transferrin receptor is easily measured by Elisa methods but the lack of standardization triggers large differences in the results. Unlike ferritin, the concentration of serum transferrin receptors is unaffected in inflammatory diseases, infections, malignancies or cytolysis. In these conditions its measurement is particularly valuable for assessing an associated iron deficiency. It is a very useful tool for the diagnosis of different causes of anemia. In chronic renal failure serum transferrin receptor can predict whether patients will respond to rHu EPO therapy.     

Distribution of drugs following controlled delivery to the brain interstitium

Intracranial controlled release polymers have been used for drug delivery to the brain, bypassing the blood brain barrier (BBB). By understanding the rates and patterns of transport in the local tissues, it is possible to design delivery systems that provide the optimal spatial and temporal pattern of chemotherapy within the intracranial space. This paper reviews the kinetics of drug release from polymeric controlled release implants, and describes the fate of drug molecules following release into the brain interstitium. Potential improvements in drug delivery based on the understanding of the mechanisms of drug release, transport and elimination are discussed.     

In vivo characteristics of injectable poly(DL-lactic acid) microspheres for long-acting drug delivery

Poly(DL-lactic acid) (PLA) microspheres containing testosterone (T) were prepared by the solvent evaporation process to evaluate their physical properties such as size distribution, shape, drug content, in vivo controlled drug release, pharmacological influences on the prostate gland in castrated rats, and histopathological findings of tissues surrounding the implants. The in vivo release of T from PLA microspheres containing 30 mg of drug obtained with chloroform was continued over a 6-week period. This effect is attributed to high dispersibility of T in the device when obtained with chloroform. Both serum drug levels and prostate gland weight recovery suggested the effects of a long-acting drug delivery system. The histopathological findings showed that the devices used were completely degraded 10 weeks after injection.     

Controlled drug delivery by biodegradable poly(ester) devices: different preparative approaches

There has been extensive research on drug delivery by biodegradable polymeric devices since bioresorbable surgical sutures entered the market two decades ago. Among the different classes of biodegradable polymers, the thermoplastic aliphatic poly(esters) such as poly(lactide) (PLA), poly(glycolide) (PGA), and especially the copolymer of lactide and glycolide referred to as poly(lactide-co-glycolide) (PLGA) have generated tremendous interest because of their excellent biocompatibility, biodegradability, and mechanical strength. They are easy to formulate into various devices for carrying a variety of drug classes such as vaccines, peptides, proteins, and micromolecules. Most importantly, they have been approved by the United States Food and Drug Administration (FDA) for drug delivery. This review presents different preparation techniques of various drug-loaded PLGA devices, with special emphasis on preparing microparticles. Certain issues about other related biodegradable polyesters are discussed.     

Optimizing interstitial delivery of BCNU from controlled release polymers for the treatment of brain tumors

OBJECTIVE: To determine quality of hip fracture services provided by "generalist" general surgeons (generalists) in Nova Scotia. DESIGN: Chart review and postoperative, blinded, random-ordered radiologic analysis. SETTING: Three community hospitals and 1 tertiary care hospital in Nova Scotia. PARTICIPANTS: Seven generalists who performed 120 hip fracture repairs and 7 orthopedic surgeons (specialists) who performed 135 hip fracture repairs. OUTCOME MEASURES: Patient demographics, preoperative, perioperative, postoperative and discharge information, technical quality of reduction as determined through postoperative radiologic assessment. RESULTS: There were no differences between patients treated by generalists and those treated by specialists with respect to age, sex, American Society of Anesthesiologists' class, level of function and fracture type. Intraoperatively, the patient groups were similar with respect to type of anesthesia, use of antibiotics, number of transfusions and surgical complications. Significant differences were noted in length of operation (54.4 v. 41.1 minutes), use of C-arm imaging (6.7% v. 85.9%) and management of Garden classes 1 and 2 subcapital fractures. Postoperatively, the 2 groups had similar numbers of medical complications, wound complications, reoperations, readmissions and deaths, and a similar level of function on discharge. Significant differences included the number of intensive care unit admissions (5.8% v. 15.6%) and length of stay there (5.7 v. 2.8 days) and of postoperative stay (14.5 v. 10.7 days). The assessment of radiographs did not demonstrate any significant difference in the quality of reduction. CONCLUSION: In Nova Scotia the outcomes of hip fracture surgery performed by generalists are comparable to those performed by specialists.     

Synthesis and biological activity of bis(pivaloyloxymethyl) ester of 2'-azido-2'-deoxyuridine 5'-monophosphate

Bis(pivaloyloxymethyl) ester of 2'-azido-2'-deoxyuridine 5'-monophosphate was prepared as a prodrug to generate 2'-azido-2'-deoxyuridine 5'-diphosphate inside the cell. A synthetic route utilizing stannyl phosphate was adopted in the preparation. The prodrug was evaluated for cell growth inhibition against a variety of tumor cell lines along with 2'-azido-2'-deoxyuridine and 2'-azido-2'-deoxycytidine.     

The radiotoxicity of iodine-125 in mammalian cells II. A comparative study on cell survival and cytogenetic responses to 125IUdR, 131TUdR, and 3HTdR

1. The effect of iron binding on the conformation of transferrins has been studied using analytical gel filtration and differential velocity sedimentation. 2. In all cases where bicarbonate is the anion the transferrin molecules undergo changes in Stokes' radius and sedimentation coefficient of the same order of magnitude, but of opposite sign. 3. Replacement of the bicarbonate ion with oxalate results in smaller changes in these two parameters. 4. In all cases except ovotranferrin, sheep serum transferrin, and human transferrin with oxalate as the anion, changes in Stokes' radius for the addition of the first and second iron atoms are similar. 5. For ovotransferrin and sheep transferrin it is necessary to postulate interaction between the iron-binding sites to account for the change in Stokes' radius caused by the second iron atom binding being greater than for the first. 6. Human transferrin with oxalate as the anion shows a greater change for the first atom bound than for the second which is consistent with the increased size of the oxalate ion compared with bicarbonate.     

Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) microspheres for controlled release of GM-CSF

PURPOSE: This study describes the preparation and characterization of a controlled release formulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) encapsulated in poly(glycolide-co-D,L-lactide) (PLGA) and poly(D,L-lactide) (PLA) microspheres. METHODS: GM-CSF was encapsulated in PLGA/PLA microspheres by a novel silicone oil based phase separation process. Several different blends of PLGA and low molecular weight PLA were used to prepare the microspheres. The microspheres and the encapsulated GM-CSF were extensively characterized both in vitro and in vivo. RESULTS: Steady release of GM-CSF was achieved over a period of about one week without significant "burst" of protein from the microspheres. Analysis of microsphere degradation kinetics by gel permeation chromatography (GPC) indicated that low molecular weight PLA enhanced the degradation of the PLGA and thereby affected release kinetics. GM-CSF released from the microspheres was found to be biologically active and physically intact by bioassay and chromatographic analysis. Analysis of serum from mice receiving huGM-CSF indicated that the GM-CSF was biologically active and that a concentration of greater than 10 ng/mL was maintained for a period lasting at least nine days. MuGM-CSF was not detected following in vivo administration of muGM-CSF microspheres. The tissues of mice receiving muGM-CSF microspheres were characterized by infiltration of neutrophils, and macrophages which were in significant excess of those found in mice administered with placebo controls (i.e. microspheres without GM-CSF). CONCLUSIONS: This study demonstrates the influence of formulation parameters on the encapsulation of GM-CSF in PLGA/PLA microspheres and its controlled release in biologically active form. The intense local tissue reaction in mice to muGM-CSF microspheres demonstrates the importance of the mode of delivery on the pharmacologic activity of GM-CSF.     

Daily and circadian variations in 2-[125I]-iodomelatonin binding sites in the pike brain (Esox lucius)

The fish pineal organ, through its 24 h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g. reproduction, growth). We have investigated the binding of 2-[125I]iodomelatonin to whole brain membrane preparations from pikes (Esox lucius L., teleost) maintained for 24-48 h under different photoperiodic conditions. Specific binding was stable, reversible, saturable and sensitive to the presence of a GTP analogue. Scatchard analysis revealed one class of binding sites. Displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations, respectively. The Bmax exhibited monophasic nycthemeral variations, with higher values at the light-to-dark transition (34.0 +/- 4.5 fmol/mg protein) and low values during the second half of night (10.0 +/- 1.0 fmol/mg protein). Under the same conditions, the KD exhibited biphasic variations: values were low during daytime and at the middle of the dark phase (approximately 100 pM); they were high at the beginning (approximately 225 pM) and at the end (approximately 330 pM) of the night. These variations were maintained under constant light (LL) and constant darkness (DD). Thus, the variations in the number and affinity of the melatonin binding sites were controlled by circadian oscillators, synchronized by the photoperiod. The nature of these oscillators is not known. Therefore, in fish, we suggest that the photodependent effects of melatonin result from the circadian variations of both its production by the pineal and its binding sites in the brain.     

Preparation and characterization of pH-sensitive proteinoid microspheres for the oral delivery of methotrexate

Prot A7, a polypeptidic proteinoid composed of seven different amino acids, was synthesized and microspheres of 1-5 microm size were prepared by the self-assembly process. The morphological characterization of the microspheres was carried out using optical microscopy and SEM (scanning electron microscopy). Emphasis also has been made on studying the mechanism behind the microsphere formation and to relate it with the conformation of the polypeptide. These self-assembled microspheres were found to be pH-sensitive in aqueous medium. The suitability of the Prot A7 microspheres as a carrier for gastric irritant drugs was verified by choosing methotrexate (MTX) as a model drug. MTX was entrapped in proteinoid microspheres and its utility for the oral delivery system was verified by carrying out the drug dissolution studies in simulated gastric medium (pH 1.2) and neutral medium of the blood (pH 7) under physiological conditions. The pH responsive dissolution behaviour of the microspheres was clarified.     

Synthesis and cellular uptake of 2'-substituted analogues of (E)-5-(2-[125I]iodovinyl)-2'-deoxyuridine in tumor cells transduced with the herpes simplex type-1 thymidine kinase gene. Evaluation as probes for monitoring gene therapy

A useful synthetic methodology was developed to synthesize and radiolabel a series of (E)-5-(2-[125I]iodovinyl)uracil nucleoside substrates for herpes simplex virus type-1 thymidine kinase (HSV-1 TK). (E)-5-(2-[125I]Iodovinyl)-2'-deoxyuridine ([125I]IVDU, 10), (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyuridine ([125I]IVFRU, 11), (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyarabinouridine ([125I]IVFAU, 12), and (E)-5-(2-[125I]iodovinyl)arabinouridine ([125I]IVAU, 13) were synthesized in 63-83% radiochemical yield by reaction of the unprotected (E)-5-(2-(trimethylsilyl)vinyl) precursors (6-9) with [125I]ICl. Cellular uptake of these labeled compounds (10-13) was evaluated in vitro. All compounds showed minimal uptake in the KBALB cell line. However, increased uptake was observed for all compounds in KBALB-STK cells which are transduced with a replication incompetent Moloney murine leukemia virus vector encoding the HSV-1 TK gene. The results indicate that uptake of these compounds in KBALB-STK cells is variable and highly dependent on the nature of the sugar 2'-substituent. When a fluoro (12) or a hydroxy (13) substituent is present in the arabinofuranosyl (up) configuration at the 2'-position, there is diminished cellular uptake in KBALB-STK cells relative to hydrogen (10) or fluorine (11) in the ribofuranosyl (down) configuration at the 2'-position. Our results indicate that radiolabeled IVFRU (11) is most promising for further in vivo studies.     

Intrathecal 5-fluoro-2'-deoxyuridine (FdUrd) for the treatment of solid tumor neoplastic meningitis: an in vivo study

To evaluate the possible intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) for neoplastic meningitis, its antitumor activity and neurotoxicity in vivo were assessed. FdUrd at doses in the range 5-100 microg/animal was effective against meningeal carcinomatosis using Walker 256 carcinoma cells in rats and MM46 mammary cancer cells in mice and against meningeal gliomatosis using 203 glioma cells in mice. After four intrathecal injections, FdUrd at these doses also showed minimal neurotoxicity in the C57BL/6 mouse brain. To estimate the mechanism of FdUrd efficacy, thymidine phosphorylase (TPase) and thymidine kinase (TK), key enzymes in the metabolism of FdUrd, were measured in rat, mouse and normal human brain tissue, and in human brain tumor tissues and cerebrospinal fluid (CSF) from patients with malignant brain tumors including meningeal carcinomatosis. TPase levels were lower in brain and malignant brain tumors than in other organs and their tumors. Moreover, the activity of TPase in the gray matter of human brain, which faces the cerebrospinal fluid across the cortical surface and into which malignant cells invade in meningeal carcinomatosis, was lower than that in the white matter. TK was undetectable, and TPase was detected (at very low concentrations) in only 4 of 56 patients with brain tumors or meningeal carcinomatosis. These findings indicate that brain tissue and CSF are favorable sites for FdUrd chemotherapy because the rate of conversion of FdUrd to 5-FU would be minimal. In conclusion, FdUrd is potentially useful for intrathecal treatment of neoplastic meningitis from primary brain tumors and systemic cancer.     

Poly (DL-lactide-co-glycolide) microspheres as carriers for peptide vaccines

Peptides carrying an immunodominant T-helper cell epitope delineated from the rabies virus nucleoprotein either alone or in combination with a linear B-cell epitope from the same protein were incorporated into three different formulations of poly(DL-lactide-co-glycolide) (PLG) which were distinct in their composition, and consequently in their peptide release rates. In vitro peptides incorporated into any of the PLG formulations stimulated a peptide-specific T-cell line. Upon subcutaneous immunization of mice, the PLG formulation that showed the fastest peptide release rate induced the best immune response. This immune response was in magnitude comparable or even superior to that induced by peptide emulsified in complete Freund's adjuvant.     

125I] beta-CIT-FE and [125I] beta-CIT-FP are superior to [125I] beta-CIT for dopamine transporter visualization: autoradiographic evaluation in the human brain

The binding of the three dopamine transporter radioligands ([125I] beta-CIT, [125I] beta-CIT-FE, and [125I] beta-CIT-FP) was studied using whole-hemisphere autoradiography on postmortem human brains. The autoradiograms revealed an intense and homogeneous labeling of the nucleus caudatus and putamen but also to varying extent to serotonergic and noradrenergic transporters of neocortex and thalamus. The order of specificity estimated (striatum over neocortex ratios) was beta-CIT-FP > beta-CIT-FE > > beta-CIT, suggesting that beta-CIT-FE and beta-CIT-FP should be preferred for in vivo studies of the dopamine transporter in the human brain.     

pH-sensitive liposomes for receptor-mediated delivery to chicken hepatoma (LMH) cells

The purpose of this study was to evaluate pediatric patients with systemic lupus erythematosus (SLE) to determine 1) the incidence of thrombosis, 2) the incidence of antiphospholipid antibodies, and 3) whether there is an association between the presence of antiphospholipid antibodies and thrombosis. We performed a cross-sectional cohort study in 59 consecutive SLE patients who had been managed at rheumatology clinics in two pediatric hospitals. A history, questionnaire, and chart review were completed by the study nurse blinded to laboratory results. Only the thrombotic events that could be substantiated by review of radiographic tests were accepted. The presence of antiphospholipid antibodies was determined by prospective analysis for a lupus anticoagulant and anticardiolipin antibodies on two separate occasions at least 3 mo apart. Patients were considered to be positive if one or more tests were positive on both occasions. Thirteen thrombotic events occurred in 10 of the 59 patients (17%). Fourteen patients (24%) were classified as positive for lupus anticoagulant, and 19 patients (27%) were classified as positive for anticardiolipin antibodies. A significant relationship between the presence of a lupus anticoagulant and a thrombotic event was shown: odds ratio 28.7 (95% confidence interval 4.03-138.2, p < 0.001). A nonsignificant trend was seen for the presence of an anticardiolipin antibody and a thrombotic event: odds ratio 2.12 (95% confidence interval 0.71-22.8, p=0.08). We conclude that in pediatric patients with SLE: 1) a significant proportion of patients have thrombotic events, 2) a significant proportion of patients have antiphospholipid antibodies, and 3) there is a significant relationship between the presence of a lupus anticoagulant and thrombotic events.     

Drug delivery of antisense molecules to the brain for treatment of Alzheimer's disease and cerebral AIDS

Antisense oligonucleotides (ODNs) and peptide nucleic acids (PNAs) are potential therapeutics for eradication of malignancies, viral infections, and other pathologies. However, ODNs and PNAs in general are unable to cross cellular membranes and blood-tissue barriers, such as the blood-brain barrier (BBB), which is only permeable to lipophilic molecules of molecular weight <600 Da. Cellular delivery systems based on conjugates of streptavidin (SA) and the OX26 monoclonal antibody directed to the transferrin receptor may be employed as a universal carrier for the transport of mono-biotinylated peptides, ODNs, or PNAs. 3'-Biotinylation of phosphodiester (PO)-ODN produces complete protection of ODN against serum and cellular 3'-exonucleases, facilitating the conjugation to avidin-based delivery systems and maintaining the activation of RNase H. These delivery systems markedly increased the cellular uptake and antisense efficacy of 3'-biotinylated ODNs in models of Alzheimer's disease and HIV-AIDS. In vivo brain delivery studies demonstrated that 3'-protected PO-ODNs and PO-phosphorothioate(PS)-ODN hybrids containing a single PO linkage are subjected to endonuclease degradation in vivo. On the contrary PS-ODNs, which were also protected at 3'-terminus by biotinylation, are metabolically stable in vivo and resistant to exo/endonuclease degradation. However, because of the strong binding of these oligomers to plasma protein, PS-ODNs are poorly transported into the brain through the BBB by the OX26-SA delivery vector following intravenous administration. PNAs are also resistant to exo/endonuclease and protease degradation, and these molecules biotinylated at the amino terminal group were transported into the brain by the OX26-SA delivery system with brain uptake levels comparable to that of morphine. Using the rev gene of HIV as a model target, RNase protection assays and cell-free translation arrest showed that the PNA-OX26-SA conjugate maintained active recognition and inactivation of target mRNA, respectively. The overall experimental evidence suggests that PNA-OX26-SA conjugates represent optimal antisense molecules for drug delivery to the brain.     

Assessment of correlation between skin target site free drug concentration and the in vivo topical antiviral efficacy in hairless mice for (E)-5-(2-bromovinyl)-2'-deoxyuridine and acyclovir formulations

Recently, we reported that the in vivo efficacy of acyclovir (ACV) formulations was a single valued function of skin target site free drug concentration (C) irrespective of the formulation compositions. A long-term objective of this research has been to generalize the C concept using model drugs which are similar to as well as different from ACV in their mechanism of actions. (Bromovinyl)deoxyuridine (BVDU) was selected as a model drug based on the reported similarity in its mechanism of action with ACV. The relationship between the C predictions and the in vivo efficacies for some topical formulations containing different concentrations (0.05-10%) of either ACV or BVDU in 95% DMSO as a vehicle with or without 5% Azone as skin permeation enhancer was examined. Hairless mice infected cutaneously with HSV-1 were used to quantitatively estimate the in vivo topical antiviral efficacy. A finite dose of the test antiviral formulation was applied twice a day for 4 days, starting the day after virus inoculation. On the fifth day, the lesions were scored and the efficacy values were calculated. For each formulation, in vitro flux experiments were performed in an in vivo-in vitro experimental design that closely approximated the in vivo study protocol. As was previously shown, with all ACV formulations, a good correlation was found between the C predictions and the in vivo topical efficacy. With the BVDU formulations, on the other hand, this was found not to be the case. BVDU formulations with 5% Azone were generally much more effective than those without Azone at comparable C values. This finding is believed to be the first of its kind showing that skin "permeation enhancers" may enhance efficacy by more than simply increasing skin permeation rates.     

Binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane to serotonin and dopamine transporters: different ionic requirements for substrate and 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binding

The iodinated cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane (beta-[125I]CIT) binds with high affinity to the platelet plasma membrane serotonin transporter, as previously reported for dopamine transporters from rat brain [Eur. J. Pharmacol. 194:133-134 (1991)]. Unlabeled beta-CIT also inhibits serotonin transport by platelet membrane vesicles. In both rat striatal membranes and platelet plasma membranes, beta-[125I]CIT binding was found to be pH dependent, with a pKa of 6.4-6.9, and did not require the presence of Cl-. Na+ dramatically stimulated beta-[125I]CIT binding to both serotonin and dopamine transporters, although a small fraction of beta-[125I]CIT binding to the serotonin transporter was observed in the absence of Na+. The substrates serotonin and dopamine competed with beta-[125I]CIT for binding to their respective transporters. However, substrate affinity was enhanced by Cl-, whereas beta-[125I]CIT binding affinity was not. [3H]Imipramine binding to the platelet serotonin transporter and [3H]GBR-12935 binding to the dopamine transporter were not inhibited by decreasing the pH from 8 to 6.5. Likewise, the ability of serotonin to compete with [3H]imipramine binding and that of dopamine to inhibit [3H]GBR-12935 binding were equal at pH 6.5 or 8. Thus, beta-[125I]CIT binding to biogenic amine transporters is distinct from serotonin or dopamine binding by virtue of its inhibition by H+ and its insensitivity to Cl-.     

Molecular and functional adaptation of the GABA(A) receptor complex during pregnancy and after delivery in the rat brain

The abundance of gamma-aminobutyric acid receptor type A (GABAA receptor) subunit mRNAs and polypeptides as well as muscimol-stimulated 36Cl- uptake were measured in rat cerebral cortex or hippocampus at various times during pregnancy and after delivery. RNase protection assays revealed that the amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, and then returned to control values around the time of delivery. A similar pattern was observed for the alpha5 subunit mRNA in the cerebral cortex, whereas no significant changes were apparent for alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3 and gamma2S subunit mRNAs. The amounts of gamma2 and alpha1 proteins in the cerebral cortex were measured by immunoblot analysis; whereas the abundance of gamma2 protein decreased during pregnancy, no change was detected in the amount of alpha1 protein. Evaluation for functional significance of the down-regulated gamma2 and alpha5 subunit was made by determining the GABAA receptor function assessed by measurement of muscimol-stimulated 36Cl- uptake in cerebral cortical membrane vesicles. Muscimol-induced 36Cl- uptake was markedly reduced during of pregnancy compared with rats in oestrus. At this same time, the potentiating effects of diazepam and allopregnanolone on muscimol stimulation of 36Cl- uptake also were reduced. In contrast, the effects of muscimol, allopregnanolone and diazepam were significantly increased, relative to animals in oestrus, after delivery.