Curved Beam Computed Tomography based Structural Rigidity Analysis of Bones with Simulated Lytic Defect: A Comparative Study with Finite Element Analysis.

In this paper, a CT based structural rigidity analysis (CTRA) method that incorporates bone intrinsic local curvature is introduced to assess the compressive failure load of human femur with simulated lytic defects. The proposed CTRA is based on a three dimensional curved beam theory to obtain critical stresses within the human femur model. To test the proposed method, ten human cadaveric femurs with and without simulated defects were mechanically tested under axial compression to failure. Quantitative computed tomography images were acquired from the samples, and CTRA and finite element analysis were performed to obtain the failure load as well as rigidities in both straight and curved cross sections. Experimental results were compared to the results obtained from FEA and CTRA. The failure loads predicated by curved beam CTRA and FEA are in agreement with experimental results. The results also show that the proposed method is an efficient and reliable method to find both the location and magnitude of failure load. Moreover, the results show that the proposed curved CTRA outperforms the regular straight beam CTRA, which ignores the bone intrinsic curvature and can be used as a useful tool in clinical practices.

Sex differences in formalin-evoked primary afferent release of substance P.

BACKGROUND: Sex differences in pain have been well documented; however, the mechanisms involved remain to be elucidated. The present study examined whether sex differences exist in the functioning of primary afferent fibres by assessing formalin-evoked release of substance P by way of neurokinin 1 receptor (NK1r) internalization. The study also investigated whether the observed effects would be oestradiol-sensitive. METHODS: Intact and gonadectomized male and female rats were given intraplantar formalin and then euthanized either 5 or 30 min later, representing phase 1 or 2 of the formalin response, respectively. In a subsequent experiment, ovariectomized females received oestradiol prior to formalin administration. Lastly, formalin-evoked NK1r internalization was assessed across the female oestrous cycle. RESULTS: Intraplantar formalin evoked significant NK1r internalization, during phase 1 and 2, in both males and females. During phase 1, no differences in NK1r internalization were detected between males or females, regardless of the gonadal status. In contrast, during phase 2, intact females exhibited greater NK1r internalization than intact males. Moreover, ovariectomy reduced NK1r internalization as compared to intact females, whereas castration had no effect as compared to intact males. Oestradiol supplementation in ovariectomized females increased NK1r internalization to levels observed in intact females. Formalin-evoked NK1r internalization did not differ across the oestrous cycle. CONCLUSIONS: These findings suggest that oestradiol mediates sex differences in formalin-evoked substance P release, which may contribute to a differential development of central sensitization and pain behaviours in males and females.

Assessment of axial bone rigidity in rats with metabolic diseases using CT-based structural rigidity analysis.

OBJECTIVES: This study aims to assess the correlation of CT-based structural rigidity analysis with mechanically determined axial rigidity in normal and metabolically diseased rat bone. METHODS: A total of 30 rats were divided equally into normal, ovariectomized, and partially nephrectomized groups. Cortical and trabecular bone segments from each animal underwent micro-CT to assess their average and minimum axial rigidities using structural rigidity analysis. Following imaging, all specimens were subjected to uniaxial compression and assessment of mechanically-derived axial rigidity. RESULTS: The average structural rigidity-based axial rigidity was well correlated with the average mechanically-derived axial rigidity results (R(2) = 0.74). This correlation improved significantly (p < 0.0001) when the CT-based Structural Rigidity Analysis (CTRA) minimum axial rigidity was correlated to the mechanically-derived minimum axial rigidity results (R(2) = 0.84). Tests of slopes in the mixed model regression analysis indicated a significantly steeper slope for the average axial rigidity compared with the minimum axial rigidity (p = 0.028) and a significant difference in the intercepts (p = 0.022). The CTRA average and minimum axial rigidities were correlated with the mechanically-derived average and minimum axial rigidities using paired t-test analysis (p = 0.37 and p = 0.18, respectively). CONCLUSIONS: In summary, the results of this study suggest that structural rigidity analysis of micro-CT data can be used to accurately and quantitatively measure the axial rigidity of bones with metabolic pathologies in an experimental rat model. It appears that minimum axial rigidity is a better model for measuring bone rigidity than average axial rigidity.

Design and manufacture of a novel system to simulate the biomechanics of basic and pitching shoulder motion.

OBJECTIVES: Cadaveric models of the shoulder evaluate discrete motion segments using the glenohumeral joint in isolation over a defined trajectory. The aim of this study was to design, manufacture and validate a robotic system to accurately create three-dimensional movement of the upper body and capture it using high-speed motion cameras. METHODS: In particular, we intended to use the robotic system to simulate the normal throwing motion in an intact cadaver. The robotic system consists of a lower frame (to move the torso) and an upper frame (to move an arm) using seven actuators. The actuators accurately reproduced planned trajectories. The marker setup used for motion capture was able to determine the six degrees of freedom of all involved joints during the planned motion of the end effector. RESULTS: The testing system demonstrated high precision and accuracy based on the expected versus observed displacements of individual axes. The maximum coefficient of variation for displacement of unloaded axes was less than 0.5% for all axes. The expected and observed actual displacements had a high level of correlation with coefficients of determination of 1.0 for all axes. CONCLUSIONS: Given that this system can accurately simulate and track simple and complex motion, there is a new opportunity to study kinematics of the shoulder under normal and pathological conditions in a cadaveric shoulder model.

Radiographic trabecular 2D and 3D parameters of proximal femoral bone cores correlate with each other and with yield stress.

UNLABELLED: Radiographic images of bone cores taken from cadaver proximal femora provided two-dimensional parameters of projected trabecular patterns that correlated highly with conceptually equivalent three-dimensional parameters in the same cores. Measurements also highly correlated with yield stress, suggesting that both parameters have similar biomechanical qualities. INTRODUCTION: We compared morphometric measurements of trabecular patterns in two-dimensional (2D) projection radiographic images of cores from cadaver proximal femoral bones with conceptually equivalent measurements from three-dimensional microcomputed tomography (3D microCT) images. METHODS: Seven cadaver proximal femora provided 47 excised cores from seven regions. Digitized radiographs of those cores were processed with software that extracts trabecular patterns. Measurements of their distribution, geometry, and connectivity were compared with 3D parameters of similar definition derived from microCT of those cores. The relationship between 2D and 3D measurements and yield stress was also examined. RESULTS: 2D measurements strongly correlated with conceptually equivalent measurements obtained using 3D microCT. In all cases, the correlation coefficients were high, ranging from r = 0.84 (p < 0.001) to r = 0.93 (p < 0.001). The correlation coefficients between 2D and 3D measurements and yield stress of the cores were also high (r = 0.60 and 0.82, p < 0.001, respectively). CONCLUSIONS: These findings provide correlative and biomechanical evidence supporting the qualitative similarity of 2D microstructural parameters extracted from plain proximal femoral core X-ray images to conceptually equivalent 3D microstructural measurements of those same cores.

Shortcomings of DXA to assess changes in bone tissue density and microstructure induced by metabolic bone diseases in rat models.

UNLABELLED: The aim of this study is to demonstrate the deficiencies of dual-energy X-ray absorptiometry (DXA), compared with quantitative computed tomography, to reflect and differentiate between changes in bone mineral density and microstructure that contribute to a well-defined finding of altered skeletal state for both osteoporosis and renal osteodystrophy induced by chronic renal insufficiency. INTRODUCTION: The aim of this study is to demonstrate the deficiencies of dual-energy X-ray absorptiometry (DXA), compared with quantitative CT, to reflect and differentiate between changes in bone mineral density and microstructure that contribute to a well-defined finding of altered skeletal state for both osteoporosis and renal osteodystrophy induced by chronic renal insufficiency. METHODS: Forty-five female Sprague-Dawley rats were divided into three equal groups: control, ovariectomy, and nephrectomy. Following euthanasia, femurs were excised, divided into diaphyseal and distal metaphyseal sections, and subjected to DXA and micro-CT imaging and mechanical testing. RESULTS: Ovariectomy does not affect the structural and mechanical properties of cortical bone material, but partial nephrectomy does adversely affect these properties. Both are verified by DXA and micro-CT imaging and mechanical testing. Meanwhile, nephrectomy does not affect trabecular bone microstructure or equivalent density, yet ovariectomy affects the trabecular microstructure. DXA is unable to detect changes in trabecular bone microstructure in relation to changes in their mechanical properties. DISCUSSION: Dual energy X-ray absorptiometry measures the average bone mineral content in a 2D projected area and cannot differentiate whether the changes occur in the bone microstructure or equivalent bone tissue density. In contrast, micro-CT provides an accurate measurement of the changes in both equivalent bone tissue mineral density and microstructure for cancellous and cortical bone.

Dexmedetomidine and ST-91 analgesia in the formalin model is mediated by alpha2A-adrenoceptors: a mechanism of action distinct from morphine.

BACKGROUND AND PURPOSE: Intrathecal administration of alpha(2)-adrenoceptor agonists produces potent analgesia. This study addressed the subtype of spinal alpha(2)-adrenoceptor responsible for the analgesic effects of i.t. dexmedetomidine and ST-91 in the formalin behavioural model and their effects on primary afferent substance P (SP) release and spinal Fos activation. EXPERIMENTAL APPROACH: The analgesic effects of i.t. dexmedetomidine and ST-91 (alpha(2) agonists) were tested on the formalin behavioural model. To determine the subtype of alpha(2)-adrenoceptor involved in the analgesia, i.t. BRL44408 (alpha(2A) antagonist) or ARC239 (alpha(2B/C) antagonist) were given before dexmedetomidine or ST-91. Moreover, the ability of dexmedetomidine and ST-91 to inhibit formalin-induced release of SP from primary afferent terminals was measured by the internalization of neurokinin(1) (NK(1)) receptors. Finally, the effects of dexmedetomidine on formalin-induced Fos expression were assessed in the dorsal horn. KEY RESULTS: Intrathecal administration of dexmedetomidine or ST-91 dose-dependently reduced the formalin-induced paw-flinching behaviour in rats. BRL44408 dose-dependently blocked, whereas ARC239 had no effect on the analgesic actions of dexmedetomidine and ST-91. Dexmedetomidine and ST-91 had no effect on the formalin-induced NK(1) receptor internalization, while morphine significantly reduced the NK(1) receptor internalization. On the other hand, both dexmedetomidine and morphine diminished the formalin-induced Fos activation. The effect of dexmedetomidine on formalin-induced Fos activation was reversed by BRL44408, but not ARC239. CONCLUSION AND IMPLICATIONS: These findings suggest that alpha(2A)-adrenoceptors mediate dexmedetomidine and ST-91 analgesia. This effect could be through a mechanism postsynaptic to primary afferent terminals, distinct from that of morphine.

Association of TGIFLX/Y mRNA expression with azoospermia in infertile men.

HOX genes are well-known to encode transcriptional regulatory proteins that play essential roles in directing embryonic development. TGIFLX/Y contains two genes, TGIFLX (X-linked) and TGIFLY (Y-linked), which are specifically expressed in human adult testes. The function(s) of these genes in normal and abnormal development are unknown. To investigate the potential role(s) of the TGIFLX/Y gene in infertile males, a nested reverse transcriptase polymerase chain reaction (RT-PCR) was performed on testicular samples from 110 patients with nonobstructive azoospermia. Although the only 51 (46.4%) of the 110 patients had detectable levels of TGIFLY expression, none of the patients with various spermatogenesis defects showed any of the TGIFLX gene expression found in normal testes. These results suggest that the function of TGIFLX may be required for the regulation of spermatogonial stem cell specification and proliferation. While functional similarity has been demonstrated among some homeobox genes, these results may refute the suggestion of redundancy between TGIFLX and TGIFLY. Furthermore, TGIFLX might be a potential biomarker candidate for male infertility assessment.

Spinal N-methyl-D-aspartate receptors and nociception-evoked release of primary afferent substance P.

Dorsal horn N-methyl-D-aspartate (NMDA) receptors contribute significantly to spinal nociceptive processing through an effect postsynaptic to non-primary glutamatergic axons, and perhaps presynaptic to the primary afferent terminals. The present study sought to examine the regulatory effects of NMDA receptors on primary afferent release of substance P (SP), as measured by neurokinin 1 receptor (NK1r) internalization in the spinal dorsal horn of rats. The effects of intrathecal NMDA alone or in combination with D-serine (a glycine site agonist) were initially examined on basal levels of NK1r internalization. NMDA alone or when co-administered with D-serine failed to induce NK1r internalization, whereas activation of spinal TRPV1 receptors by capsaicin resulted in a notable NK1r internalization. To determine whether NMDA receptor activation could potentiate NK1r internalization or pain behavior induced by a peripheral noxious stimulus, intrathecal NMDA was given prior to an intraplantar injection of formalin. NMDA did not alter the formalin-induced NK1r internalization nor did it enhance the formalin paw flinching behavior. To further characterize the effects of presynaptic NMDA receptors, the NMDA antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 were intrathecally administered to assess their regulatory effects on formalin-induced NK1r internalization and pain behavior. AP-5 had no effect on formalin-induced NK1r internalization, whereas MK-801 produced only a modest reduction. Both antagonists, however, reduced the formalin paw flinching behavior. In subsequent in vitro experiments, perfusion of NMDA in spinal cord slice preparations did not evoke basal release of SP or calcitonin gene-related peptide (CGRP). Likewise, perfusion of NMDA did not enhance capsaicin-evoked release of the two peptides. These results suggest that presynaptic NMDA receptors in the spinal cord play little if any role on the primary afferent release of SP.

BMP-silk composite matrices heal critically sized femoral defects.

Clinical drawbacks of bone grafting prompt the search for alternative bone augmentation technologies such as use of growth and differentiation factors, gene therapy, and cell therapy. Osteopromotive matrices are frequently employed for the local delivery and controlled release of these augmentation agents. Some matrices also provide an osteoconductive scaffold to support new bone growth. In this study, silkworm-derived silk fibroin was evaluated as an osteoconductive matrix for healing critical sized mid-femoral segmental defects in nude rats. Four treatment groups were assessed over eight weeks: silk scaffolds (SS) with recombinant human BMP-2 (rhBMP-2) and human mesenchymal stem cells (HMSC) that had been pre-differentiated along an osteoblastic lineage ex vivo (Group I; pdHMSC/rhBMP-2/SS); SS with rhBMP-2 and undifferentiated HMSCs (Group II; udHMSC/rhBMP-2/SS); SS and rhBMP-2 alone (Group III; rhBMP-2/SS); and empty defects (Group IV). Bi-weekly radiographs revealed a progressive and similar increase in Group I-III mean defect mineralization through post-operative week (POW) 8. Radiographs, dual energy x-ray absorptiometry, and micro-computed tomography confirmed that Groups I-III exhibited similar substantial and significantly (p<0.05) greater defect mineralization at POW 8 than the unfilled Group IV defects which remained void of bone. No significant differences in Groups I-III defect healing at POW 8 were apparent using these same assays or mechanical testing. Histology at POW 8 revealed moderately good bridging of the parent diaphyseal cortices with woven and lamellar bone bridging islands of silk matrix in Groups I and III. Group II defects possessed comparatively less new bone which was most abundant adjacent to the parent bone margins. Elsewhere the silk matrix was more often enveloped by poorly differentiated loose fibrous connective tissue. Group IV defects showed minimal new bone formation. None of the treatment groups attained the mean mineralization or the mean biomechanical strength of identical defects implanted with SS and pdHMSCs alone in a previous study. However, addition of rhBMP-2 to SS prompted more bone than was previously generated using udHMSC/SS or SS alone. These data imply the clinical potential of silk scaffolds and rhBMP-2 as composite osteopromotive implants when used alone or with select stem cell populations. Additional studies in larger species are now warranted.

Delayed administration of adenoviral BMP-2 vector improves the formation of bone in osseous defects.

The direct, local, administration of adenovirus carrying human BMP-2 cDNA (Ad.BMP-2) heals critical-sized femoral bone defects in rabbit and rat models. However, the outcome is suboptimal and the technology needs to provide a more reliable and uniform outcome. To this end, we investigated whether the timing of Ad.BMP-2 administration influenced the formation of mineralized tissue within the defect. Critical-sized defects were created in the femora of 28 Sprague-Dawley rats. Animals were injected intralesionally with a single, percutaneous injection of Ad.BMP-2 (4 x 10(8) plaque-forming units) either intraoperatively (day 0) or 24 h (day 1), 5 days or 10 days after surgery. The femora were evaluated 8 weeks after surgery by X-ray, microcomputed tomography, dual-energy X-ray absorptiometry and biomechanical testing. The incidence of radiological union was markedly increased when administration of Ad.BMP-2 was delayed until days 5 and 10, at which point 86% of the defects healed. These time points also provided greater bone mineral content within the defect site and improved the average mechanical strength of the healed bone. Thus, delaying the injection of Ad.BMP-2 until 5 or 10 days after surgery enables a greater percentage of critical-sized, segmental defects to achieve radiological union, producing a repair tissue with enhanced mineralization and greater mechanical strength.

Silk based biomaterials to heal critical sized femur defects.

Bone auto- and allografts have inherent drawbacks, therefore the treatment of non-unions and critical size defects in load bearing long bones would benefit from the use of osteopromotive biodegradable, biocompatible and mechanically durable matrices to enhance migration or delivery of cell populations and/or morphogens/cytokines. Silk fibroin biomaterial scaffolds were evaluated as osteopromotive matrices in critical sized mid-femoral segmental defects in nude rats. Four treatment groups were assessed over 8 weeks in vivo: silk scaffolds (SS) with human mesenchymal stem cells (hMSCs) that had previously been differentiated along an osteoblastic lineage in vitro (group I; pdHMSC/SS); SS with undifferentiated hMSCs (group II; udHMSC/SS); SS alone (group III; SS); and empty defects (group IV). When hMSCs were cultured in vitro in osteogenic medium for 5 weeks, bone formation was characterized with bimodal peak activities for alkaline phosphatase at 2 and 4 weeks. Calcium deposition started after 1 week and progressively increased to peak at 4 weeks, reaching cumulative levels of deposited calcium at 16 mug per mg scaffold wet weight. In vivo osteogenesis was characterized by almost bridged defects with newly formed bone after 8 weeks in group I. Significantly (P < 0.01) greater bone volumes were observed with the pdHMSC/SS (group I) implants than with groups II, III or IV. These three groups failed to induce substantial new bone formation and resulted in the ingrowth of cells with fibroblast-like morphology into the defect zone. The implantation of pdHMSC/SS resulted in significantly (P < 0.05) greater maximal load and torque when compared to the other treatment regimens. The pdHMSC/SS implants demonstrated osteogenic ability in vitro and capacity to thrive towards the healing of critical size femoral segmental defects in vivo. Thus, these new constructs provide an alternative protein-based biomaterial for load bearing applications.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments.

FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line

Effects of acute and repeated methamphetamine treatment on the ultrasonic vocalizations of postnatal rats.

Repeated exposure to cocaine (COC) has been reported to both increase ultrasonic vocalizations (USVs) of postnatal rats and enhance the crying of human infants. The purpose of the present study was to determine whether acute or repeated treatment with another commonly abused psychostimulant, methamphetamine (MAP), would affect the USV production of postnatal rats. In the first experiment, USVs were measured 30 min after rats were given an acute injection of saline or MAP (1, 2, 4, or 8 mg/kg ip) on postnatal day (PD) 10. In the second experiment, rats were exposed to MAP (0, 1, or 4 mg/kg/day ip) on PD 2-8 or PD 2-9. On PD 10, rats were given an acute injection of saline or MAP (1 or 4 mg/kg ip) 30 min prior to behavioral assessment. Results showed that acute treatment with MAP (4 or 8 mg/kg) decreased the USVs of rats on PD 10, while repeated exposure to MAP did not affect the USV emissions of rats subsequently treated with saline or MAP. The reason why acute MAP treatment decreased USV production is uncertain, but it is possible that MAP alleviates isolation distress by stimulating reward processes. Alternatively, MAP increases heart rate and blood pressure, so acute treatment with this drug may decrease USV emissions through peripheral physiological mechanisms (i.e., by reducing abdominal compression reactions).

Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction.

Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.

Ultrasonic vocalizations of preweanling rats: involvement of both alpha(2)-adrenoceptor and kappa-opioid receptor systems.

Stimulation of alpha(2)-adrenoceptors and kappa-opioid receptors increases the ultrasonic vocalizations of preweanling rats. The purpose of the present study was to determine whether alpha(2)-adrenoceptors and kappa-opioid receptors modulate ultrasonic vocalization production via a common mechanism. To that end, 11-day-old rats were injected with the alpha(2)-adrenoceptor antagonist yohimbine (0, 0.5, or 1.0 mg/kg, i.p.) or the kappa-opioid receptor antagonist nor-binaltorphimine (0, 5, or 10 mg/kg, i.p.). After 15 min, the same rats were injected with saline, the alpha(2)-adrenoceptor agonist clonidine (0.25 mg/kg, i.p.), or the kappa-opioid receptor agonist trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate (U-50,488; 2.5 mg/kg, i.p.). Results showed that both clonidine and U-50,488 increased the ultrasonic vocalizations of preweanling rats. Not surprisingly, clonidine-induced ultrasonic vocalizations were blocked by yohimbine, while U-50,488-induced vocalizations were blocked by nor-binaltorphimine. Importantly, yohimbine also attenuated the vocalizations produced by U-50,488, whereas nor-binaltorphimine did not alter clonidine-induced ultrasonic vocalizations. Thus, it appears that alpha(2)-adrenoceptor and kappa-opioid receptor stimulation increases ultrasonic vocalization production via a common mechanism. It is likely that the kappa-opioid receptors responsible for modulating ultrasonic vocalizations are located "upstream" from the alpha(2)-adrenoceptors.

Cocaine-induced behavioral sensitization in the young rat.

RATIONALE: Repeated psychostimulant treatment has been shown to sensitize the locomotor activity of young rats, but there is conflicting evidence suggesting that this sensitized response will persist across only a few drug abstinence days. OBJECTIVE: The purpose of the present study was to determine whether: (a) young rats are capable of expressing a sensitized locomotor response after an extended drug abstinence period, and (b) the longevity of the sensitized response is critically affected by either the number of drug pretreatment days or environmental conditioning factors. METHODS: Young rats were pretreated with saline or cocaine (15 mg/kg or 30 mg/kg, i.p.) for either five or ten consecutive days [i.e., on postnatal days (PD) 16-20 or PD 11-20]. After each daily injection, rats were placed in activity chambers, and locomotion was measured for 30 min. To assess environmental conditioning factors, some rats were injected with saline prior to being placed in the activity chambers and then injected with cocaine prior to being returned to the home cage. After one or seven abstinence days (i.e., on PD 22 or PD 28), rats received a challenge injection of saline or cocaine (15 mg/kg) in the activity chamber and locomotion was assessed. RESULTS: Young rats exhibit cocaine-induced locomotor sensitization after either a short (1-day) or long (7-day) drug abstinence period. When a long abstinence period was used, locomotor sensitization was only apparent when cocaine pretreatment lasted for 10 days. Conditioning factors were also important for determining whether locomotor sensitization was expressed, because young rats pretreated with cocaine in the home cage did not show a sensitized locomotor response after seven abstinence days. CONCLUSIONS: Young rats are capable of showing cocaine-induced locomotor sensitization after an extended abstinence period. Both the number of drug pretreatment days and the environmental context in which cocaine was given (i.e., the activity chamber or home cage) influenced the longevity of cocaine-induced locomotor sensitization.

Role of dopamine D(1) receptors for kappa-opioid-mediated locomotor activity and antinociception during the preweanling period: a study using D(1) receptor knockout mice.

kappa-Opioid receptor agonists both increase the locomotor activity of preweanling rats and induce antinociception. To determine whether dopamine (DA) D(1) receptors are necessary for either of these kappa-opioid-mediated effects we used D(1) (D(1A)) receptor knockout mice (i.e., D(1)-deficient mice). Heterozygous, wild-type, and D(1)-deficient mice (13 days old at testing) were injected with the kappa-opioid receptor agonist U-50,488 methanesulfonate (0.0, 0.2, 1. 0, 2.5, or 5.0 mg/kg, s.c.) and locomotor activity was measured for 60 min. In a separate experiment, tail-flick latencies of heterozygous, wild-type, and D(1)-deficient 13-day-old mice were assessed both before and after treatment with U-50,488 (0.0, 1.0, 2. 5, 5.0, or 10.0 mg/kg, s.c.). Results showed that lower doses of U-50,488 (0.2 and 1.0 mg/kg) increased the locomotor activity of 13-day-old mice regardless of genotype. Besides affecting locomotion, kappa-opioid receptor stimulation induced antinociception in preweanling mice, as U-50,488 caused a dose-dependent increase in the tail-flick latencies of heterozygous, wild-type, and D(1)-deficient mice. U-50,488's locomotor activating and analgesic effects did not differ according to genotype, thus suggesting that D(1) receptors are not necessary for kappa-opioid-mediated locomotor activity and antinociception during the preweanling period.

kappa-Opioid receptors in the substantia nigra pars reticulata mediate the U-50,488-induced locomotor activity of preweanling rats.

The purpose of the present study was to determine the neuroanatomical location where kappa-opioid receptor stimulation induces locomotor activity in the preweanling rat. To confirm that the U-50,488-induced locomotor activity of preweanling rats is mediated by kappa-opioid receptors, 18-day-old rats were initially injected with vehicle or the kappa-opioid receptor agonist U-50,488 (5 mg/kg, s.c.) followed, 15 min later, by an injection of the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI; 0, 2, 4, 8, or 12 mg/kg, s.c.). In subsequent experiments, 18-day-old rats were injected with vehicle or U-50,488 (5 mg/kg, s.c.) 15 min prior to bilateral administration (0.25 or 0.5 microl per side) of nor-BNI (0, 5, 10, or 20 microg) into the substantia nigra pars reticulata (SNR) or medial dorsal striatum (MDS). In the final experiment, 18-day-old rats received bilateral administration (0.25 microl per side) of vehicle or U-50,488 (0.0, 0.8, 1.6, or 3.2 microg) into the SNR. Results showed that systemically administered nor-BNI (0-12 mg/kg, s.c.) produced a dose-dependent reduction in the U-50, 488-induced locomotor activity of preweanling rats. The site of action for U-50,488's locomotor-activating effects appeared to be the SNR, because (a) bilateral administration of nor-BNI (5, 10, or 20 microg) into the SNR caused a complete attenuation of U-50, 488-induced locomotion, and (b) bilateral administration of U-50,488 into the SNR caused a dose-dependent increase in the locomotor activity of preweanling rats. Striatal injections of nor-BNI did not affect U-50,488-induced locomotor activity. When these findings are considered together it is apparent that stimulation of kappa-opioid receptors in the SNR is both necessary and sufficient for the occurrence of U-50,488-induced locomotor activity in the preweanling rat.