Datura quids at Pinwheel Cave, California, provide unambiguous confirmation of the ingestion of hallucinogens at a rock art site.

While debates have raged over the relationship between trance and rock art, unambiguous evidence of the consumption of hallucinogens has not been reported from any rock art site in the world. A painting possibly representing the flowers of Datura on the ceiling of a Californian rock art site called Pinwheel Cave was discovered alongside fibrous quids in the same ceiling. Even though Native Californians are historically documented to have used Datura to enter trance states, little evidence exists to associate it with rock art. A multianalytical approach to the rock art, the quids, and the archaeological context of this site was undertaken. Liquid chromatography-mass spectrometry (LC-MS) results found hallucinogenic alkaloids scopolamine and atropine in the quids, while scanning electron microscope analysis confirms most to be Datura wrightii Three-dimensional (3D) analyses of the quids indicate the quids were likely masticated and thus consumed in the cave under the paintings. Archaeological evidence and chronological dating shows the site was well utilized as a temporary residence for a range of activities from Late Prehistory through Colonial Periods. This indicates that Datura was ingested in the cave and that the rock painting represents the plant itself, serving to codify communal rituals involving this powerful entheogen. These results confirm the use of hallucinogens at a rock art site while calling into question previous assumptions concerning trance and rock art imagery.

Utilization of an Electrochemiluminescence Sensor for Atropine Determination in Complex Matrices.

A major challenge within forensic science is the development of accurate and robust methodologies that can be utilized on-site for detection at crime scenes and can be used for analyzing multiple sample types. The recent expansion of electrochemical sensors to tackle this hurdle requires sensors that can undergo analysis without any pretreatment. Given the vast array of samples that are submitted for forensic analysis, this can pose a major challenge for all electrochemical sensors, including electrochemiluminescent (ECL)-based sensors. Within this contribution, we demonstrate the capacity for an ECL-based sensor to address this challenge and it is potential to detect and quantify atropine from a wide range of samples directly from herbal material to spiked solutions. This portable platform demonstrates satisfactory analytical parameters with linearity across a concentration range of 0.75 to 100 µM, reproducibility of 3.0%, repeatability of 9.2%, and a detection limit of ∼0.75 µM. The sensor displays good selectivity toward alkaloid species and, in particular, the hallucinogenic tropane alkaloid functionality within complex matrices. This portable sensor provides rapid detection alongside low cost and operational simplicity, thus, providing a basis for the exploitation of ECL-based sensors within the forensic arena.

Mice with early retinal degeneration show differences in neuropeptide expression in the suprachiasmatic nucleus.

BACKGROUND: In mammals, the brain clock responsible for generating circadian rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light entrainment of the clock occurs through intrinsically photosensitive retinal ganglion cells (ipRGCs) whose axons project to the SCN via the retinohypothalamic tract. Although ipRGCs are sufficient for photoentrainment, rod and cone photoreceptors also contribute. Adult CBA/J mice, which exhibit loss of rod and cone photoreceptors during early postnatal development, have greater numbers of ipRGCs compared to CBA/N control mice. A greater number of photosensitive cells might argue for enhanced light responses, however, these mice exhibit attenuated phase shifting behaviors. To reconcile these findings, we looked for potential differences in SCN neurons of CBA/J mice that might underly the altered circadian behaviors. We hypothesized that CBA/J mice have differences in the expression of neuropeptides in the SCN, where ipRGCs synapse. The neuropeptides vasoactive intestinal peptide (VIP) and vasopressin (VP) are expressed by many SCN neurons and play an important role in the generation of circadian rhythms and photic entrainment. METHODS: Using immunohistochemistry, we looked for differences in the expression of VIP and VP in the SCN of CBA/J mice, and using a light-induced FOS assay, we also examined the degree of retinal innervation of the SCN by ipRGCs. RESULTS: Our data demonstrate greater numbers of VIP-and VP-positive cells in the SCN of CBA/J mice and a greater degree of light-induced FOS expression. CONCLUSIONS: These results implicate changes in neuropeptide expression in the SCN which may underlie the altered circadian responses to light in these animals.

The development of melanopsin-containing retinal ganglion cells in mice with early retinal degeneration.

In mammals, the neuronal pathways by which rod and cone photoreceptors mediate vision have been well documented. The roles that classical photoreceptors play in photoentrainment, however, have been less clear. In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the photopigment melanopsin project directly to the suprachiasmatic nucleus of the hypothalamus, the site of the circadian clock, and thereby contribute to non-image-forming responses to light. Classical photoreceptors are not necessary for photoentrainment as loss of rods and cones does not eliminate light entrainment. Conflicting evidence arose, however, when attenuated phase-shifting responses were observed in the retinal-degenerate CBA/J mouse. In this study, we examined the time course of retinal degeneration in CBA/J mice and used these animals to determine if maturation of the outer retina regulates the morphology, number and distribution of ipRGCs. We also examined whether degeneration during the early development of the outer retina can alter the function of the adult circadian system. We report that dendritic stratification and distribution of ipRGCs was unaltered in mice with early retinal degeneration, suggesting that normal development of the outer retina was not necessary for these processes. We found, however, that adult CBA/J mice have greater numbers of ipRGCs than controls, implicating a role for the outer retinal photoreceptors in regulating developmental cell death of ipRGCs.

Synaptic inputs to retinal ganglion cells that set the circadian clock.

Melanopsin-containing retinal ganglion cells (RGCs) project to the suprachiasmatic nuclei (SCN) and mediate photoentrainment of the circadian system. Melanopsin is a novel retinal-based photopigment that renders these cells intrinsically photosensitive (ip). Although genetic ablation of melanopsin abolishes the intrinsic light response, it has a surprisingly minor effect on circadian photoentrainment. This and other non-visual responses to light are lost only when the melanopsin deficiency is coupled with mutations that disable classical rod and cone photoreceptors, suggesting that melanopsin-containing RGCs also receive rod- and cone-driven synaptic inputs. Using whole-cell patch-clamp recording, we demonstrate that light triggers synaptic currents in ipRGCs via activation of ionotropic glutamate and gamma-aminobutyric acid (GABA) receptors. Miniature postsynaptic currents (mPSCs) were clearly observed in ipRGCs, although they were less robust and were seen less frequently than those seen in non-ip cells. Pharmacological treatments revealed that the majority of ipRGCs receive excitatory glutamatergic inputs that were blocked by DNQX and/or kynurenic acid, as well as inhibitory GABAergic inputs that were blocked by bicuculline. Other ipRGCs received either glutamatergic or GABAergic inputs nearly exclusively. Although strychnine (Strych)-sensitive mPSCs were evident on many non-ipRGCs, indicating the presence of glycinergic inputs, we saw no evidence of Strych-sensitive events in ipRGCs. Based on these results, it is clear that SCN-projecting RGCs can respond to light both via an intrinsic melanopsin-based signaling cascade and via a synaptic pathway driven by classical rod and/or cone photoreceptors. It remains to be determined how the ipRGCs integrate these temporally distinct inputs to generate the signals that mediate circadian photoentrainment and other non-visual responses to light.

The light-activated signaling pathway in SCN-projecting rat retinal ganglion cells.

In mammals, the master circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus. The period and phase of the circadian pacemaker are calibrated by direct photic input from retinal ganglion cells (RGCs). SCN-projecting RGCs respond to light in the absence of rod- and cone-driven synaptic input, a property for which they are termed intrinsically photosensitive. In SCN-projecting RGCs, light activates a nonselective cationic current that displays inward and outward rectification. The goal of the present study was to investigate the identity of the light-activated ion channel and the intracellular signaling pathway leading to its activation. We considered two candidate channels, cyclic nucleotide-gated (CNG) channels and transient receptor potential (TRP) channels, which mediate vertebrate and invertebrate phototransduction, respectively. We report that the intrinsic light response relies upon a G-protein-dependent process. Although our data indicate that cyclic nucleotides modulate the signaling pathway, CNG channels do not appear to conduct the light-activated current because (i) cyclic nucleotides in the pipette solution do not activate a conductance or completely block the light response, (ii) CNG channel blockers fail to inhibit the light response, (iii) the effects of internal and external divalent cations are inconsistent with their effects on CNG channels, and (iv) immunohistochemistry reveals no CNG channels in SCN-projecting RGCs. Finally, we show that the pharmacology of the light-activated channel resembles that of some TRPC channel family members; the response is blocked by lanthanides and ruthenium red and SK&F 96365, and is enhanced by flufenamic acid and 1-oleoyl-2-acetyl-sn-glycerol. Furthermore, immunohistochemical experiments reveal that TRPC6 is expressed in many RGCs, including those that express melanopsin.

Physiological response properties of displaced amacrine cells of the adult ferret retina.

The ganglion cell layer (GCL) of the mammalian retina contains a large number of neurons called displaced amacrine cells (DACs) that do not project to the optic nerve. However, with the exception of the rabbit starburst amacrine cell little is known regarding the function of this large population due to the difficulty experienced in making physiological recordings from these neurons. We have overcome these difficulties and have used whole-cell patch-clamp techniques to examine the intrinsic membrane properties of DACs in the ferret retina. Our results indicate a large degree of diversity in their intrinsic membrane properties. In response to maintained depolarizing current injection, DACs responded with graded depolarization or by eliciting either transient or sustained bursts of spiking activity. At the resting membrane potential, 10% of the DACs generated spontaneous spikes in either an apparently random manner or at the peak of intrinsic waves of depolarization. The resting membrane activity of the remaining DACs recorded could be classified into three groups that were quiescent (28%), had robust uncorrelated synaptic activity (30%), or underwent slow waves of depolarization (42%). Diversity was also revealed in the membrane currents recorded in voltage-clamp where some DACs were quiescent (19%), or exhibited robust nonrhythmic synaptic events (42%). The remaining DACs exhibited waves of oscillatory activity (39%), characterized by either rhythmic bursts of synaptic events (17%) or slow inward currents (22%). Bath application of 50 microM biccuculine or 150 microM picrotoxin had no effect on the waves of activity, however, the gap junction blocker, carbenoxolone (100 microm), blocked both oscillatory patterns. By including Lucifer yellow and biocytin in the recording pipette, it was possible to determine the morphology of recorded neurons and group them based on dendritic extent as small-, medium-, or large-field DACs. There were few relationships between these morphologically defined groups and their intrinsic membrane properties. The present study provides the first in-depth examination of the intrinsic membrane properties of DACs in the ferret retina and provides new insights into the potential roles these neurons play in the processing of visual information in the mammalian retina.

Intrinsic light responses of retinal ganglion cells projecting to the circadian system.

In mammals, light entrainment of the circadian clock, located in the suprachiasmatic nuclei (SCN), requires retinal input. Traditional rod and cone photoreceptors, however, are not required. Instead, the SCN-projecting retinal ganglion cells (RGCs) function as autonomous photoreceptors and exhibit light responses independent of rod- and cone-driven input. Using whole-cell patch-clamp recording techniques, we have investigated the morphological and electrophysiological properties of this unique class of RGCs. Although SCN-projecting RGCs resemble Type III cells in form, they display strikingly different physiological properties from these neurons. First, in response to the injection of a sustained depolarizing current, SCN-projecting cells fired in a transient fashion, in contrast to most RGCs which fired robust trains of action potentials. Second, in response to light, SCN-projecting RGCs exhibited an intensity-dependent transient depolarization in the absence of rod and cone input. This depolarization reached a peak within 5 s and generated increased spiking activity before decaying to a plateau. Voltage-clamp recordings were used to characterize the light-activated conductance which generated this depolarization. In response to varying light intensities, SCN-projecting RGCs exhibited a graded transient inward current which peaked within 5 s and decayed to a plateau. The voltage dependence of the light-activated current was obtained by subtracting currents elicited by a voltage ramp before and during illumination. The light-activated current displayed both inward and outward rectification and was largely unaffected by substitution of extracellular Na+ with choline. In both respects, the intrinsic light-activated current observed in SCN-projecting RGCs resembles currents carried by ion channels of the transient receptor potential (trp) family, which are known to mediate the light response of invertebrate photoreceptors.

Evaluation of the Roche Amplicor polymerase chain reaction assay for detection of enteroviruses in cerebrospinal fluid and its potential impact on patient management.

OBJECTIVE: To evaluate the Roche Amplicor polymerase chain reaction assay (APCR) by comparing the detection of enteroviruses from cerebrospinal fluid (CSF) by the Roche assay with detection by viral culture and to determine whether routine use of enteroviral PCR will affect patient management. METHODS: One hundred and sixty-three CSF specimens were tested by APCR and viral culture. Some of the discrepant specimens were resolved by retesting with an in-house PCR assay. Other discrepant results were resolved by testing the patients' serum by APCR or by viral culture of throat and stool specimens. RESULTS: Thirty CSF specimens were positive by APCR, and 18 of these were positive by viral culture. There were no APCR-negative, viral-culture-positive CSF specimens. Six of the 12 discrepant specimens were resolved as true positives. CONCLUSIONS: The APCR assay was more rapid and sensitive than viral culture for detection of enteroviruses from CSF. Routine use of this assay has the potential to reduce the amount of antibiotics used and the number of patient days spent in hospital.