Optical analysis of glutamate spread in the neuropil.

Fast synaptic communication uses diffusible transmitters whose spread is limited by uptake mechanisms. However, on the submicron-scale, the distance between two synapses, the extent of glutamate spread has so far remained difficult to measure. Here, we show that quantal glutamate release from individual hippocampal synapses activates extracellular iGluSnFr molecules at a distance of >1.5 µm. 2P-glutamate uncaging near spines further showed that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-Rs and N-methyl-D-aspartate (NMDA)-Rs respond to distant uncaging spots at approximately 800 and 2000 nm, respectively, when releasing the amount of glutamate contained in approximately five synaptic vesicles. The uncaging-induced remote activation of AMPA-Rs was facilitated by blocking glutamate transporters but only modestly decreased by elevating the recording temperature. When mimicking release from neighboring synapses by three simultaneous uncaging spots in the microenvironment of a spine, AMPA-R-mediated responses increased supra-additively. Interfering with extracellular glutamate diffusion through a glutamate scavenger system weakly reduced field synaptic responses but not the quantal amplitude. Together, our data suggest that the neuropil is more permissive to short-range spread of transmitter than suggested by theory, that multivesicular release could regularly coactivate nearest neighbor synapses and that on this scale glutamate buffering by transporters primarily limits the spread of transmitter and allows for cooperative glutamate signaling in extracellular microdomains.

Categorization of survival and death after cardiac arrest.

BACKGROUND: Most cardiac arrest (CA) patients remain comatose post-resuscitation, prompting goals-of-care (GOC) conversations. The impact of these conversations on patient outcomes has not been well described. METHODS: Patients (n=385) treated for CA in Columbia University ICUs between 2008-2015 were retrospectively categorized into various modes of survival and death based on documented GOC discussions. Patients were deemed "medically unstable" if there was evidence of hemodynamic instability at the time of discussion. Cerebral performance category (CPC) greater than 2 was defined as poor outcome at discharge and one-year post-arrest. RESULTS: The survival rate was 31% (n=118); most commonly after early recovery without any discussions (57%, n=67), followed by survival due to family wishes despite physicians predicting poor neurological prognosis (20%, n=24), and then survival after physician/family agreement of favorable prognosis (17%, n=20). The survivors due to family wishes had significantly worse outcomes compared to the early recovery group (discharge: p=0.01; one-year: p=0.06) and agreement group (p<0.001; p<0.001), though 2 patients did achieve favorable recovery. Among nonsurvivors (n=267), withdrawal of life-sustaining therapy (WLST) while medically unstable was most common (31%; n=83), followed by death after care was capped (24%, n=65), then WLST while medically stable (17%, n=45). Death despite full support, brain death and WLST due to advanced directives were less common causes. CONCLUSIONS: Most survivors due to family wishes despite poor neurological prognosis die or have poor outcomes at one-year. However, a small number achieve favorable recovery, demonstrating limitations with current prognostication methods. Among nonsurvivors, most WLST occurs while medically unstable, suggesting an overestimation of WLST due to unfavorable neurological prognosis.

Effects of spinally delivered N- and P-type voltage-dependent calcium channel antagonists on dorsal horn neuronal responses in a rat model of neuropathy.

Neuropathic pain, due to peripheral nerve damage, can include allodynia (perception of innocuous stimuli as being painful), hyperalgesia (increased sensitivity to noxious stimuli) and spontaneous pain, often accompanied by sensory deficits. Plasticity in transmission and modulatory systems are implicated in the underlying mechanisms. The Kim and Chung rodent model of neuropathy (Kim and Chung, Pain 50 (1992) 355) employed here involves unilateral tight ligation of two (L5 and L6) of the three (L4, L5, and L6) spinal nerves of the sciatic nerve and reproducibly induced mechanical and cold allodynia in the ipsilateral hindpaw over the 14 day post-operative period. In vivo electrophysiological techniques have then been used to record the response of dorsal horn neurones to innocuous and noxious electrical and natural (mechanical and thermal) stimuli after spinal nerve ligation (SNL). Activation of voltage-dependent calcium channels (VDCCs) is critical for neurotransmitter release and neuronal excitability, and antagonists can be antinociceptive. Here, for the first time, the effect of N- and P-type VDCC antagonists (omega-conotoxin-GVIA and omega-agatoxin-IVA, respectively) on the evoked dorsal horn neuronal responses after neuropathy have been investigated. Spinal omega-conotoxin-GVIA (0.1-3.2 microg) produced prolonged inhibitions of both the electrically- and low- and high-intensity naturally-evoked neuronal responses in SNL and control rats. Spinal omega-agatoxin-IVA (0.1-3.2 microg) also had an inhibitory effect but to a lesser extent. After neuropathy the potency of omega-conotoxin-GVIA was increased at lower doses in comparison to control. This indicates an altered role for N-type but not P-type VDCCs in sensory transmission after neuropathy and selective plasticity in these channels after nerve injury. Both pre- and post-synaptic VDCCs appear to be important.

Comparison of the effects of MK-801, ketamine and memantine on responses of spinal dorsal horn neurones in a rat model of mononeuropathy.

Selective ligation of the L5/L6 spinal nerves produces a partial denervation of the hindpaw and has proved to be a useful model for studying the mechanisms underlying neuropathic pain. Two weeks after surgery, in vivo electrophysiological studies were performed in sham operated and nerve injured rats and the responses of spinal dorsal horn neurones to controlled electrical and natural (mechanical and heat) stimuli were recorded. The systemic effects of three N-methyl-D-aspartate receptor (NMDA) antagonists, ketamine (1-10 mg/kg), memantine (1-20 mg/kg) and MK-801 (0.1-5 mg/kg) were compared. Ketamine a clinically available NMDA receptor antagonist, produced greater reductions of the postdischarge, thermal (10 mg/kg, P=0.02), and mechanical evoked responses in spinal nerve ligated (SNL) rats (von Frey 9 g, 1 mg/kg, P=0.04; 5 mg/kg, P=0.01; 10 mg/kg, P=0.05; von Frey 50 g, 5 mg/kg, P=0.02; 10 mg/kg, P=0.003). The inhibition of wind-up was comparable in both animal groups. Memantine produced powerful inhibitions of wind-up after nerve injury with little effect in sham controls (5 mg/kg, P=0.02). The postdischarge, mechanical and thermal evoked responses were reduced to similar extents by memantine in both experimental groups. The effects of MK-801 were comparable between SNL and sham operated rats for all neuronal measures (wind-up, postdischarge, thermal and noxious mechanical evoked responses). The differential blocking abilities of these antagonists on the various neuronal responses may relate to the characteristics of their voltage-dependent blockage of the channel associated with the receptor. The favourable side effect profile of memantine supports its potential use for the treatment of neuropathic pain.

Effects of ethosuximide, a T-type Ca(2+) channel blocker, on dorsal horn neuronal responses in rats.

Plasticity in transmission and modulatory systems are implicated in mechanisms of neuropathic pain. Studies demonstrate the importance of high voltage-activated Ca(2+) channels in pain transmission, but the role of low voltage-activated, T-type Ca(2+) channels in nociception has not been investigated. The Kim and Chung rodent model of neuropathy [Pain 50 (1992) 355] was used to induce mechanical and cold allodynia in the ipsilateral hindpaw. In vivo electrophysiological techniques were used to record the response of dorsal horn neurones to innocuous and noxious electrical and natural (mechanical and thermal) stimuli after spinal nerve ligation. Spinal ethosuximide (5-1055 microg) exerted dose-related inhibitions of both the electrically and low- and high-intensity mechanical and thermal evoked neuronal responses and its profile remained unaltered after neuropathy. Measures of spinal cord hyperexcitability were most susceptible to ethosuximide. This study, for the first time, indicates a possible role for low voltage-activated Ca(2+) channels in sensory transmission.

Effects of an environmental manipulation emphasizing client-centred care on agitation and sleep in dementia sufferers in a nursing home.

This study was designed to determine whether a change from a task-oriented care approach to a client-oriented care approach affects (a) the level of agitation and (b) 24-hour sleep in residents suffering from dementia in a nursing home. The levels of dementia and sleep of 33 nursing home residents were measured four times over 12 weeks (twice before and twice after the change in care approach) using the Cohen-Mansfield agitation inventory and the dementia mood assessment scale. Verbal agitation levels significantly decreased 6 to 8 weeks following the change, whereas more infrequent agitated behaviours, which were classified as 'other', significantly increased. Daytime sleep increased initially after the change but then returned to baseline levels after 6 weeks. While the main focus of the study was on residents' behaviour following an environmental manipulation, anecdotal observations of staff members interactions with residents indicated that they felt less rushed and were more tolerant of residents' behaviour following the intervention.

Where have all the student nurses gone?

Since the mid-eighties the elective allocation of student nurses to the operating department during their nurse education programme, has steadily diminished. Nursing programmes prior to 1988 usually incorporated a period of supervised learning of four to six weeks in theatres. During this time the student was expected to observe and participate in the care of the peri-operative surgical patient. The student was generally on rostered service and would be supervised by an experience Operating Department nurse.

Distinct chromosomal abnormalities in murine leukemia virus-induced T- and B-cell lymphomas.

We performed a cytogenetic study on 16 murine mature B-cell lymphomas and 10 T-cell lymphomas, using G-banding techniques. All tumors, with the exception of 3 spontaneous B-cell tumors, were induced by various slowly transforming murine leukemia viruses (MuLV). Metaphases were obtained from primary (10 B-cell tumors) and first or second transplant generation lymphomas (6 B-cell and 10 T-cell tumors), all of which were well characterized with respect to phenotypic, histologic and genotypic features. In the T-cell tumors we found relatively simple karyotypic abnormalities, including various numerical aberrations, such as trisomy 15, in line with many earlier reports. However, the majority of B-cell tumors showed a great variety of both structural and numerical chromosomal anomalies. Three B-cell lymphomas had an apparently normal karyotype. No single cytogenetic abnormality occurred commonly in the B-cell lymphomas, but some structural abnormalities were found in more than one stemline, in particular, ins (II) (A1; A2) in 3 tumors, and deletions involving the D-region of chromosome 14 in 3 other lymphomas. These cytogenetic results clearly indicate that the pathogenic mechanisms involved in MuLV-induced (long latency) B-cell lymphomagenesis and (short latency) T-cell lymphomagenesis differ considerably.

Retrovirally induced murine B-cell tumors rarely show proviral integration in sites common in T-cell tumors.

The molecular etiology of retrovirally induced T-cell tumors has been shown in many cases to involve proviral integration near a cellular oncogene, c-myc, N-myc, Pim-1 and pvt-1 being frequent targets for insertional activation. Murine B-cell tumors induced by infection with murine leukemia virus have been studied for rearrangements in these and other loci. In contrast to the T-cell lymphomas, tumors of the B-cell lineage, either early B-cell tumors induced in nude mice or late B-cell tumors in immunocompetent mice, did not show disruption of N-myc or Pim-1 in any of the tumors studied, although those lymphomas had acquired many new proviruses. The loci c-abl, bcl-2, fis-1, c-erbB, c-myb, and neu were likewise not involved. Rearrangement of c-myc was seen in 1 out of 71 and rearrangement of the pvt-1 locus in 4 out of 73 (5%) of the B-cell tumors. Thus it appears that mechanistic differences exist in the development of T-cell tumors and B-cell tumors caused by the same etiological agent.

Primary virus-induced lymphomas evade T cell immunity by failure to express viral antigens.

T lymphoma induction by the mink cell focus-inducing murine leukemia virus MCF 1233 in C57BL/10 and C57BL/6 mice is influenced by a strongly Th-dependent, H-2I-A-restricted antiviral immune response (25). We compared the MHC class I as well as viral env and gag antigenic cell surface profiles of frequent T lymphomas of H-2I-A nonresponder-type mice to that of rare T lymphomas of H-2I-A responder-type mice. Membrane immunofluorescence studies, with a panel of anti-env mAbs (reactive with the highly conserved gp70f epitope, the p15Ec epitope, and the gp70-p15E complex), a polyclonal anti-p30 serum, and anti-H-2 class I mAbs, showed that all 17 nonresponder tumors tested expressed high levels of both env and gag viral proteins, and 15 of these 17 nonresponder tumors expressed high levels of H-2 class I K and D antigens. In contrast, 10 of 11 responder lymphomas lacked env and/or gag determinants. The only responder lymphoma with both strong env and gag expression failed to express H-2K and -D antigens. Preferential loss of env or gag expression did not correlate with H-2 class I allelic specificities. Both responder and nonresponder T lymphoma DNA contained multiple, predominantly MCF-like, newly acquired proviral integrations. Differences in viral antigen cell surface expression were confirmed at cytoplasmic and RNA levels. The amounts of 8.2- and 3.2-kb viral RNA were greatly reduced in two responder lymphomas when compared with four nonresponder lymphomas. In both responder lymphomas, aberrantly sized viral RNA species were found. Upon in vivo passage of these responder lymphomas in either immunocompetent or T cell-deficient nu/nu mice, it was found that various molecular mechanisms may underlie the lack of viral antigen expression at the cell surface of these lymphomas. One lymphoma re-expressed viral antigens when transplanted with nu/nu mice, whereas the other remained stably gag negative. The combined findings indicate that an H-2I-A-regulated antiviral immune response not only strongly reduces T lymphoma incidence, but also forces T lymphomas that still arise to poorly express viral antigens, thus explaining their escape from immunosurveillance.

Complexity of T cell receptor recognition sites for defined alloantigens.

Three monoclonal antibodies react with the T cell receptor on the tumor line HPB-ALL and in addition with 3 to 13% of human peripheral blood T cells of normal donors. These antibodies are shown to react with an epitope encoded by the V beta 5 family of T cell receptor beta-chain variable region gene segments. Cells expressing V beta 5 gene segments can have cytotoxic or helper function, be of the T4+ or T8+ phenotype, and have specificity for either class I or class II major histocompatibility complex alloantigens. Seven T cell clones were generated, which express V beta 5 and are specific for the HLA-A2 molecule. With the use of these clones, we illustrate how isoelectric focusing can be used to analyze T cell receptor alpha- and beta-chain structure. The seven clones recognize five distinct conformational determinants on HLA-A2. They procure different binding sites by the use of different alpha-chains, J beta sequences, or both.

Conserved 5S rRNA complement to tRNA is not required for protein synthesis.

The notion that tRNA and 5S rRNA interact through evolutionarily conserved complementary sequences has been tested by nucleolytic modification of the 5S rRNA, using the modified rRNA to reconstitute the large ribosomal subunit, and assaying for poly(uridylic acid)-directed polyphenylalanine synthesis. The 5S rRNA sequence C-G-A-A (residues 43-46) and several residues surrounding it are not essential for protein synthesis.

An efficient in vitro total reconstitution of the Escherichia coli 50S ribosomal subunit.

A new, relatively simple technique for the total invitro reconstitution of E. coli 50S ribosomes has been developed. It is a two-step procedure like that previously reported by Nierhaus and Dohme [Proc. Natl. Acad. Sci. 71, 4713 (1974)], but it differs in a number of important aspects. Ribosomal RNA is prepared by direct phenol extraction of 70S particles to minimize nuclease fragmentation. A mixture of 50S proteins is prepared by acetic acid extraction and immediate removal of the acetic acid by thin film dialysis. The resulting protein mixture is soluble and stable. Separate RNA and protein fractions are mixed, incubated first at 44 degrees C in 7.5 mM Mg(2+), and then at 50 degrees C in 20 mM Mg(2+). The resulting 50S particles comigrate with native 50S particles in analytical gradients. They range from 50 to 100% active in five different functional assays. This is a fairly stringent test of the effectiveness of reconstitution since 50S particles derived from highly active vacant couples were used as a control.Images