Comprehensive characterization of commercially available canine training aids.

Effective and reliable training aids for victim recovery canine teams is essential for law enforcement and investigative purposes. Without adequate training aids, the rate of recovery for sub surface or surface human remains deposition using canine teams may be adversely affected and result in confusing information. The composition of three commercially available canine training aids that purportedly generate volatile components responsible for the odor of human decomposition is relatively simple and not closely related to those compounds experimentally determined to be present at the site of surface or sub-surface human remains. In this study, these different commercial formulations were chemically characterized using six different sampling approaches, including two applications of direct liquid injection, solid-phase microextraction (SPME), purge and trap, ambient preconcentration/thermal desorption, and cryogenic preconcentration/thermal desorption. Direct liquid injections resulted in the fewest number of detected compounds, while a cryogen based thermal desorption method detected the greatest number of compounds in each formulation. Based solely upon the direct liquid injection analysis, Pseudo™ Scent I was composed of approximately 29±4% and 71±5% of 2-pyrrolidinone and 4-aminobutanoic acid, respectively. This same analysis showed that Pseudo™ Scent II was composed of approximately 11±1, 11±1, 24±5, and 54±7% of putrescine, cadaverine, 2-pyrrolidinone, and 4-aminobutanoic acid, respectively. Headspace analysis was conducted to more closely simulate the process whereby a canine's nose would capture a volatiles profile. More compounds were detected using the headspace sampling method; however, the vast majority was not consistent with current data on human decomposition. Additionally, the three formulations were tested in outdoor and indoor scenarios by a double-blinded canine team, using a certified and specifically trained victim recovery canine with multiple confirmed recoveries, to determine if the formulations would be recognized by that canine as being related to human decomposition. The canine used in this study did not provide a positive response to any of the formulations tested in either test scenario. The implications for locating residual human decomposition odor in the absence of recoverable material are discussed in light of these data.

Optimizing the Temporal Resolution of Fast-Scan Cyclic Voltammetry.

Electrochemical detection with carbon-fiber microelectrodes has become an established method to monitor directly the release of dopamine from neurons and its uptake by the dopamine transporter. With constant potential amperometry (CPA) the measured current provides a real time view of the rapid concentration changes, but the method lacks chemical identification of the monitored species and markedly increases the difficulty of signal calibration. Monitoring with fast-scan cyclic voltammetry (FSCV) allows species identification and concentration measurements, but often exhibits a delayed response time due to the time-dependent adsorption/desorption of electroactive species at the electrode. We sought to improve the temporal resolution of FSCV to make it more comparable to CPA by increasing the waveform repetition rate from 10 to 60 Hz with uncoated carbon-fiber electrodes. The faster acquisition led to diminished time delays of the recordings that tracked more closely with CPA measurements. The measurements reveal that FSCV at 10 Hz underestimates the normal rate of dopamine uptake by about 18%. However, FSCV collection at 10 Hz and 60 Hz provide identical results when a dopamine transporter (DAT) blocker such as cocaine is bath applied. To verify further the utility of this method, we used transgenic mice that over-express DAT. After accounting for the slight adsorption delay time, FSCV at 60 Hz adequately monitored the increased uptake rate that arose from overexpression of DAT and, again, was similar to CPA results. Furthermore, the utility of collecting data at 60 Hz was verified in an anesthetized rat by using a higher scan rate (2400 V/s) to increase sensitivity and the overall signal.

Synapsins differentially control dopamine and serotonin release.

Synapsins are a family of synaptic vesicle proteins that are important for neurotransmitter release. Here we have used triple knock-out (TKO) mice lacking all three synapsin genes to determine the roles of synapsins in the release of two monoamine neurotransmitters, dopamine and serotonin. Serotonin release evoked by electrical stimulation was identical in substantia nigra pars reticulata slices prepared from TKO and wild-type mice. In contrast, release of dopamine in response to electrical stimulation was approximately doubled in striatum of TKO mice, both in vivo and in striatal slices, in comparison to wild-type controls. This was due to loss of synapsin III, because deletion of synapsin III alone was sufficient to increase dopamine release. Deletion of synapsins also increased the sensitivity of dopamine release to extracellular calcium ions. Although cocaine did not affect the release of serotonin from nigral tissue, this drug did enhance dopamine release. Cocaine-induced facilitation of dopamine release was a function of external calcium, an effect that was reduced in TKO mice. We conclude that synapsins play different roles in the control of release of dopamine and serotonin, with release of dopamine being negatively regulated by synapsins, specifically synapsin III, while serotonin release appears to be relatively independent of synapsins. These results provide further support for the concept that synapsin function in presynaptic terminals varies according to the neurotransmitter being released.

Neuropeptide Release is Impaired in a Mouse Model of Fragile X Mental Retardation Syndrome.

Fragile X syndrome (FXS), an inherited disorder characterized by mental retardation and autismlike behaviors, is caused by the failure to transcribe the gene for fragile X mental retardation protein (FMRP), a translational regulator and transporter of select mRNAs. FXS model mice (Fmr1 KO mice) exhibit impaired neuropeptide release. Release of biogenic amines does not differ between wild-type (WT) and Fmr1 KO mice. Rab3A, an mRNA cargo of FMRP involved in the recruitment of vesicles, is decreased by ∼50% in synaptoneurosomes of Fmr1 KO mice; however, the number of dense-core vesicles (DCVs) does not differ between WT and Fmr1 KO mice. Therefore, deficits associated with FXS may reflect this aberrant vesicle release, specifically involving docking and fusion of peptidergic DCVs, and may lead to defective maturation/maintenance of synaptic connections.

In vivo measurement of somatodendritic release of dopamine in the ventral tegmental area.

The ventral tegmental area (VTA), the locus of mesolimbic dopamine cell bodies, contains dopamine. Experiments in brain slices have demonstrated that VTA dopamine can be released by local electrical stimulation. Measurements with both push-pull cannula and microdialysis in intact animals have also obtained evidence for releasable dopamine. Here we demonstrate that dopamine release in the VTA can be evoked by remote stimulations of the medial forebrain bundle (MFB) in the anesthetized rat. In initial experiments, the MFB was electrically stimulated while a carbon-fiber electrode was lowered to the VTA, with recording by fast-scan cyclic voltammetry. While release was not observed with the carbon fiber 4-6 mm below dura, a voltammetric response was observed at 6-8 mm below dura, but the voltammogram was poorly defined. At lower depths, in the VTA, dopamine release was evoked. Immunohistochemistry experiments with antibodies for tyrosine hydroxylase (TH) confirmed that dopamine processes were primarily found below 8 mm. Similarly, tissue content determined by liquid chromatography revealed serotonin but not dopamine dorsal to 8 mm with both dopamine and serotonin at lower depths. Evaluation of the VTA signal by pharmacological means showed that it increased with inhibitors of dopamine uptake, but release was not altered by D2 agents. Dopamine release in the VTA was frequency dependent and could be exhausted by stimulations longer than 5 s. Thus, VTA dopamine release can be evoked in vivo by remote stimulations and it resembles release in terminal regions, possessing a similar uptake mechanism and a finite releasable storage pool.

In vivo voltammetric monitoring of norepinephrine release in the rat ventral bed nucleus of the stria terminalis and anteroventral thalamic nucleus.

The role and contribution of the dense noradrenergic innervation in the ventral bed nucleus of the stria terminalis (vBNST) and anteroventral thalamic nucleus (AV) to biological function and animal behaviors is poorly understood due to the small size of these nuclei. The aim of this study was to compare norepinephrine release and uptake in the vBNST with that in the AV of anesthetized rats. Measurements were made in vivo with fast-scan cyclic voltammetry following electrical stimulation of noradrenergic projection pathways, either the dorsal noradrenergic bundle (DNB) or the ventral noradrenergic bundle (VNB). The substance detected was identified as norepinephrine based upon voltammetric, anatomical, neurochemical and pharmacological evidence. Fast-scan cyclic voltammetry enables the selective monitoring of local norepinephrine overflow in the vBNST evoked by the stimulation of either the DNB or the VNB while norepinephrine in the AV was only evoked by DNB stimulation. The alpha2-adrenoceptor antagonist yohimbine and the norepinephrine uptake inhibitor desipramine increased norepinephrine overflow and slowed its disappearance in both regions. However, control of extracellular norepinephrine by both autoreceptors and uptake was greater in the AV. The greater control exerted by autoreceptors and uptake in the AV resulted in reduced extracellular concentration compared with the v BNST when large numbers of stimulation pulses were employed. The differences in noradrenergic transmission observed in the terminal fields of the v BNST and the AV may differentially regulate activity in these two regions that both contain high densities of norepinephrine terminals.

Electroosmotic flow and its contribution to iontophoretic delivery.

Iontophoresis is the movement of charged molecules in solution under applied current using pulled multibarrel glass capillaries drawn to a sharp tip. The technique is generally nonquantitative, and to address this, we have characterized the ejection of charged and neutral species using carbon-fiber electrodes attached to iontophoretic barrels. Our results show that observed ejections are due to the sum of iontophoretic and electroosmotic forces. With the use of the neutral, electroactive molecule 2-(4-nitrophenoxy) ethanol (NPE), which is only transported by electroosmotic flow (EOF), a positive correlation between the amount ejected and the diameter of each barrel's tip was found. In addition, using various charged and neutral electroactive compounds we found that, when each compound is paired with the EOF marker, the percentage of the ejection due to EOF remains constant. This percentage varies for each pair of compounds, and the differences in mobility are positively correlated to differences in electrophoretic mobility. Overall, the results show that capillary electrophoresis (CE) can be used to predict the percentage of ejection that will be due to EOF. With this information, quantitative iontophoresis is possible for electrochemically inactive drugs by using NPE as a marker for EOF.