Binge-like ethanol exposure during the early postnatal period impairs eyeblink conditioning at short and long CS-US intervals in rats

Binge-like ethanol exposure on postnatal days (PD) 4-9 in rodents causes cerebellar cell loss and impaired acquisition of conditioned responses (CRs) during "short-delay" eyeblink classical conditioning (ECC), using optimal (280-350 ms) interstimulus intervals (ISIs). We extended those earlier findings by comparing acquisition of delay ECC under two different ISIs. From PD 4 to 9, rats were intubated with either 5.25 g/kg of ethanol (2/day), sham intubated, or were not intubated. They were then trained either as periadolescents (about PD 35) or as adults (>PD 90) with either the optimal short-delay (280-ms) ISI, a long-delay (880-ms) ISI, or explicitly unpaired CS and US presentations. Neonatal binge ethanol treatment significantly impaired acquisition of conditioning at both ages regardless of ISI, and deficits in the acquisition and expression of CRs were comparable across ISIs. These deficits are consistent with the previously documented ethanol-induced damage to the cerebellar-brainstem circuit essential for Pavlovian ECC.     

Synapses, circuits, and the ontogeny of learning

This article summarizes the proceedings of a symposium organized by Mark Stanton and Pamela Hunt and presented at the annual meeting of the International Society for Developmental Psychobiology. The purpose of the symposium was to review recent advances in neurobiological and developmental studies of fear and eyeblink conditioning with the hope of discovering how neural circuitry might inform the ontogenetic analyses of learning and memory, and vice versa. The presentations were: (1) Multiple Brain Regions Contribute to the Acquisition of Pavlovian Fear by Michael S. Fanselow; (2) Expression of Learned Fear: Appropriate to Age of Training or Age of Testing by Rick Richardson; (3) Trying to Understand the Cerebellum Well Enough to Build One by Michael D. Mauk; and (4) The Ontogeny of Eyeblink Conditioning: Neural Mechanisms by John H. Freeman. Taken together, these presentations converge on the conclusions that (1) seemingly simple forms of associative learning are governed by multiple "engrams" and by temporally dynamic interactions among these engrams and other circuit elements and (2) developmental changes in these interactions determine when and how learning emerges during ontogeny.     

The segway personal transporter as an alternative mobility device for people with disabilities: a pilot study

Sawatzky B, Denison I, Langrish S, Richardson S, Hiller K, Slobogean B. The Segway Personal Transporter as an alternative mobility device for people with disabilities: a pilot study.

Objectives

To determine the functional measures that best correlate with the skill levels of people with disabilities who operate a Segway Personal Transporter, and—using a qualitative analysis—to explore subjects’ experience with the Segway.

Design

A prospective study encompassing 3 training sessions with the Segway to correlate subjects’ functional ability (eg, cognition, balance, mobility, muscle strength) with their skill level on the device.

Setting

A provincial adult rehabilitation center.

Participants

Twenty-three subjects (age range, 19−65y) with a wide range of disabilities (eg, multiple sclerosis, spinal cord injury, amputation) who could walk at least 6m with or without assistance.

Interventions

Not applicable.

Main Outcome Measures

Segway Task Assessment, Berg Balance Scale, and Timed Up & Go test.

Results

No correlation was found because all participants successfully completed the final Segway Task Assessment, regardless of scores on functional assessments.

Conclusions

The Segway is a useful device for a broad range of populations with functional disabilities. Subjects found the Segway easy to use and were excited about its potential as an assistive device for use in their communities.     

Project Kealahou: Improving Hawai‘i's System of Care for At-Risk Girls and Young Women through Gender-Responsive,Trauma-Informed Care

Project Kealahou (PK) is a six-year, federally-funded program aimed at improving services and outcomes for Hawai‘i''s female youth who are at risk for running away, truancy, abuse, suicide, arrest and incarceration. PK builds upon two decades of sustained cross-agency efforts among the state''s mental health, juvenile justice, education, and child welfare systems to promote system-of-care (SOC) principles of community-based, individualized, culturally and linguistically competent, family driven, youth-guided, and evidence-based services. In addition, PK emphasizes trauma-informed and gender-responsive care in serving its target population of females ages 11–18 years who have experienced psychological trauma.Results from the first four years of the implementation of PK in the Department of Health''s (DOH) Child and Adolescent Mental Health Division (CAMHD) highlight the serious familial, socioeconomic, functional, and interpersonal challenges faced by the young women who receive services in Hawai‘i''s SOC. Despite the challenges faced by PK youth and their families, preliminary results of the evaluation of PK show significant improvements across multiple clinical and functional domains of service recipients. A financial analysis indicates that these outcomes were obtained with a minimal overall increase in costs when compared to standard care alone. Overall, these results suggest that PK may offer a cost effective way to improve access, care, and outcomes for at-risk youth and their families in Hawai‘i.     

Modulation of α1β3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a β-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABA_A α1β3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the β⁺α⁻ interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in β3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [β3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1β3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the β⁺α⁻ interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to β3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the β⁺α⁻ interface for the anesthetic propofol.

While photoswitches have been a popular topic in numerous reviews (1), their application in research is still very rare. Photoswitches are freely diffusible molecules containing a photosensitive moiety which can alternate between two isomeric forms depending on light irradiation at specific wavelengths. These isomeric forms of the photoswitch possess different affinities or efficacies toward their biological target, such that their pharmacological activity can be turned on or off depending on the light wavelength used, thus providing temporal and spatial control. When photoswitches covalently tether to a native or engineered residue at the specific biological target, the resulting conformation would determine the corresponding pharmacological activity. One way the tethered photoswitch can be activated is by positioning the tether in such a way that the ligand moiety can reach its binding site in only one of the photoswitch conformations. Additional major advantages of the tethered photoswitches are high local concentration (the residue cannot diffuse away) and spatial restriction within the biological target. The ligand groups of photoswitches can possess diverse pharmacological activities, acting as agonists, inverse agonists, or antagonists at specific binding sites.The GABA_A receptor is formed by five subunits (usually two α, two β, and one γ or δ) arranged in pseudosymmetry around a central channel, in the following order (counterclockwise, viewed from the extracellular side): γ-β-α-β-α (2). Each interface is named after the subunits that form it, with “+” and “−” designated following the counterclockwise order (a model of the α1β3γ2 GABA_A receptor can be found in SI Appendix, Fig. S1). Each subunit consists of an extracellular domain, attached to a sequence of four transmembrane helices (TMs), with a large intracellular loop inserted between TM3 and TM4. Multiple anesthetic drugs that act through this receptor possess relatively low binding affinities; therefore, precise identification and characterization of their binding sites require the use of techniques like mutagenesis, substituted cysteine modification protection (SCAMP), and photolabeling with photoreactive anesthetic analogs (3). Structures of the GABA_A receptor with bound anesthetic drugs have recently been made available (4), but one unexplored way of obtaining valuable, functional information would be by using appropriately designed tethered photoswitches. In previous studies of GABA_A receptors, the photoswitches used have included diffusible propofol photoswitches (5, 6) and a tethered propofol photoswitch with a very long spacer between the tethering cysteine (located in the extracellular domain) and the propofol moiety (6). Though all three positively modulated GABA_A receptors, none could be used to define the propofol binding site.Multiple studies point to propofol binding cavities being located in the TM interfaces between GABA_A receptor subunits. The observation that specific mutations in TM2 or TM3 of GABA_A β subunits could dramatically decrease propofol potentiation of GABA responses, and even direct activation of the receptor (7–9) led to the development of the β3(N265M) knockin mouse, which showed either a greatly decreased or absent hypnotic effect of propofol, depending on the test used (10). More recent studies have focused on a more complete characterization of binding sites for propofol as well as other intravenous anesthetics. A photoreactive propofol analog labeled three amino acids in the β⁺α⁻ interface (β3M286, α1M236, and α1I239) and one in the α⁺β⁻ interface (β3M227) in α1β3 receptors. Using etomidate and R-mTFD-MPAB [R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid] to inhibit photolabeling established that propofol also binds to the β⁺β⁻ interface, suggesting that propofol shows little selectivity for either interface (11). Another study mutated photolabeled residues (α1M236, β3M227, and their homologs, all located in TM1) to tryptophan (causing steric occupancy of the pocket) and cysteine (to test propofol protection against cysteine-specific labeling) (12). SCAMP studies confirmed that propofol binds to the β⁺α⁻ and α⁺β⁻ interfaces, and also to the γ⁺β⁻ interface; there was no evidence of binding to the α⁺γ⁻ interface. A more recent study has expanded the analysis of residues at the β⁺α⁻ interface that line a putative propofol binding site (13) (SI Appendix, Fig. S1). And cryoelectron microscopy (cryo-EM) structures of α1β2γ2 GABA_A receptors bound to intravenous anesthetics and benzodiazepines have recently been published (4), consolidating the evidence toward a propofol binding site at the β⁺α⁻ interfaces.Our aim was to develop a β-subunit-tethered propofol photoswitch, as a tool to better study the mechanism of anesthesia through the GABA_A α1β3ɣ2 receptor, merging both structural and functional approaches. This tethered photoswitch (Fig. 1A) consists of propofol as the ligand group, linked to an azobenzene group (which is photosensitive and can change between cis and trans isomeric forms, Fig. 1B), and finally a tethering group (methanethiosulfonate, that spontaneously forms a covalent bond with the thiolate group of cysteines located in water-filled cavities). This photoswitch was abbreviated as MAP20 (methanethiosulfonate azobenzene propofol 2020). These three basic components of this tethered photoswitch are connected by short spacers, decreasing the range between tethering and target residues. We used β3 subunits in our study because the immobilizing and hypnotic effects of propofol are mostly mediated by β3-containing receptors (10).Open in a separate windowFig. 1.Tethered photoswitch. (A) Methanethiosulfonate azobenzene propofol 2020 (MAP20) consists of propofol (ligand group), an azobenzene moiety (photosensitive), and a tether (methanethiosulfonate). (B) The azobenzene moiety can change between cis and trans isomeric forms depending on the wavelength of the light irradiated.     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.

We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.     

Transient protein kinase C activation primes long-term depression and suppresses long-term potentiation of synaptic transmission in hippocampus.

Activity-dependent long-lasting plasticity in hippocampus and neocortex includes long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength. Recent studies have confirmed theoretical predictions that the sensitivity of LTP- and LTD-inducing mechanisms is dynamically regulated by previous synaptic history. In particular, prior induction of either repeated short-term potentiations or LTP lowers the threshold for induction of LTD and raises the threshold for LTP. In the current study, transient activation of protein kinase C with phorbol 12,13-diacetate was able to substitute for synaptic activity in priming synapses to exhibit enhanced homosynaptic LTD and to suppress the induction of LTP at Schaffer collateral synapses in area CA1 of hippocampal slices. This priming lasted 30 min, but not 3 hr, following phorbol 12,13-diacetate bath application. These data suggest that a protein kinase C-sensitive phosphorylation site may be an activity-sensitive target mediating the rapid expression of LTP and LTD.