Contribution of NMDA and non-NMDA receptors to synaptic transmission from the brachium of the inferior colliculus to the medial subdivision of the medial geniculate nucleus in the rabbit

Previous work from this laboratory has demonstrated that monosynaptic inputs from the brachium of the inferior colliculus (BIC) to the medial subdivision of the medial geniculate nucleus (mMG) strengthen as a result of associative conditioning with an acoustic conditioned stimulus (i.e., fear conditioning). One model that has been proposed to underlie certain types of neuronal plasticity involves the recruitment of N-methyl-D-aspartic acid (NMDA)-type glutamate receptors. The purpose of the present study was to examine the relative contributions of glutamatergic NMDA and non-NMDA receptors to synaptic transmission within this pathway. Individual contributions of the specific receptor types were assessed through the use of 2-amino-5-phosphonovaleric acid (AP5), a selective NMDA receptor antagonist, and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist. Bipolar stimulating electrodes were stereotaxically implanted in BIC and recording electrodes (attached to dual 32-gauge cannulae for delivery of drug) were positioned in mMG of New Zealand albino rabbits. Single pulses (150 micros, 100-350 microA) delivered to BIC resulted in short-latency (<4 ms) responses in mMG. BIC-evoked single-unit activity was recorded from mMG before, during, and at several intervals after injection of AP5, CNQX, and/or artificial cerebrospinal fluid (ACSF). Injection of either AP5 or CNQX, but not ACSF, significantly attenuated the short-latency BIC-evoked responses in the vast majority of cells tested. These findings suggest that the monosynaptic pathway from BIC to mMG is glutamatergic and that this pathway frequently employs NMDA-type receptors during electrically stimulated synaptic transmission. Due to the NMDA receptors' proposed role in plasticity (e.g., long-term potentiation), these results may have implications for understanding the mechanisms of synaptic plasticity observed at this synapse during associative learning.     

Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.     

The surgical risk of pancreas transplantation in the cyclosporine era: an overview

BACKGROUND: Pancreas transplants are still associated with the highest surgical complication rate of all routinely performed solid organ transplants. To date, the impact of serious surgical complications in the cyclosporine era on perioperative patient morbidity, graft and patient survival, and hospital costs has not been analyzed in detail. STUDY DESIGN: We retrospectively studied surgical complications after 445 consecutive pancreas transplants (45% simultaneous pancreas-kidney [SPK], 24% pancreas after kidney [PAK], and 31% pancreas transplant alone [PTA]). Of these, 80% were primary transplants, 20% were retransplants. Cadaver donors were used in 92%, living related donors in 8%. To develop guidelines for their prevention and management, we studied the impact of significant surgical complications (intra-abdominal infections, vascular graft thrombosis, and anastomotic leak) requiring relaparotomy on graft and patient survival. RESULTS: Relaparotomy was required after 32% of all pancreas transplants (SPK: 36%, PAK: 25%, PTA: 16% [p = 0.04]). Perioperative mortality was 9%. Graft and patient survival rates were significantly lower for recipients with (versus without) relaparotomy. The most common procedures were drainage of intra-abdominal abscess with graft necrosectomy (50% of all relaparotomies) and transplant pancreatectomy (34%). The most common causes of relaparotomy were intra-abdominal infection, vascular graft thrombosis, and anastomotic leak. Intra-abdominal infection occurred in 20% (SPK: 18%, PAK: 24%, PTA: 20% [p = NS]). The rate was significantly higher for living related donor (42%) versus cadaver donor (18%) recipients and for those with enteric-drained (39%) versus bladder-drained (18%) transplants. Graft and patient survival rates were significantly lower for recipients with (versus without) intra-abdominal infection. Outcome was better after bacterial (versus fungal) infections. For SPK recipients, those not on dialysis before the transplant had significantly higher graft survival than those on dialysis. Vascular graft thrombosis occurred in 12% of all recipients. The rate was significantly higher for PAK (21%) than for PTA (10%) and SPK (9%) recipients. It was significantly lower for recipients of grafts with donor iliac Y-graft reconstruction (versus all other types of arterial reconstruction) and with right-sided (versus left-sided) graft placement. Of note, patient survival was not different for recipients with versus without vascular graft thrombosis. The incidence of anastomotic or duodenal stump leaks was 10%; of these recipients, 70% required relaparotomy. Patient and graft survival rates were no different for recipients with versus without leaks. CONCLUSIONS: Serious surgical complications occurred in 35% of pancreas recipients and had a significant impact on patient and graft survival. Based on multivariate risk factor analyses, we recommend the following: donors over 45 years and those dying of cerebrocardiovascular disease should not be used; recipients over 45 years and those with a history of cardiac disease should be considered for a kidney transplant alone (KTA); surgical technique for graft procurement, preparation, and implantation should be meticulous; right-sided implantation and arterial Y-graft reconstruction should be performed when possible, since they had the highest success rates; when complications require relaparotomy, the focus must switch from graft salvage to life preservation; and the threshold for pancreatectomy should be low. Diagnosis should be timely, and treatment and relaparotomy expeditious. These cornerstones of success should help decrease the risk of surgical complications and mortality after pancreas transplants.     

Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels

In contrast to other kinds of voltage-gated Ca2+ channels, the underlying molecular basis of T-type and R-type channels is not well-understood. To facilitate comparisons with cloned Ca2+ channel subunits, we have carried out a systematic analysis of the properties of T-type currents in undifferentiated NG108-15 cells and R-type currents in cerebellar granule neurons. Marked differences were found in their biophysical and pharmacological features under identical recording conditions. T-type channels became activated at potentials approximately 25 mV more negative than R-type channels; however, T-type channels required potentials approximately 15 mV less negative than R-type channels to be available. Accordingly, T-type channels display a much larger overlap between the curves describing inactivation and activation, making them more suitable for generating sustained Ca2+ entry in support of secretion or pacemaker activity. In contrast, R-type channels are not equipped to provide a steady current, but are very capable of supplying transient surges of Ca2+ influx. In response to a series of increasingly strong depolarizations T-type and R-type Ca2+ channels gave rise to very different kinetic patterns. T-type current records crossed each other in a characteristic pattern not found for R-type currents. These biophysical distinctions were independent of absolute membrane potential and were, therefore, complementary to the conventional categorization of T- and R-type Ca2+ channels as low- and high-voltage activated. R-type channels deactivated approximately eight-fold more quickly than T-type channels, with clear consequences for the generation of divalent cation influx during simulated action potentials. Pharmacological comparisons revealed additional contrasts. R-type current was responsive to block by omega-Aga IIIA but not nimodipine, while the opposite was true for T-type current. Both channel types were potently inhibited by the non-dihydropyridine compound mibefradil. In all respects examined, R-type currents were similar to currents derived from expression of the alpha1E subunit whereas T-type currents were not.     

Allosteric gating of a large conductance Ca-activated K+ channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.     

Fluoxetine increases norepinephrine release in rat hypothalamus as measured by tissue levels of MHPG-SO4 and microdialysis in conscious rats

The selective serotonin uptake inhibitor fluoxetine (10 mg/kg i.p.) increased tissue levels of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethylene glycol sulfate (MHPG-SO4) in rat hypothalamus, indicating an increased release of norepinephrine. Microdialysis studies in conscious rats showed that fluoxetine (10 mg/kg i.p.) increased extracellular concentrations of norepinephrine as well as serotonin in the hypothalamus. In contrast, desipramine (10 mg/kg i.p.) increased extracellular concentration of norepinephrine but not serotonin in the hypothalamus. Consistent with its mechanism of being a selective serotonin uptake inhibitor, local perfusion of fluoxetine (10 microM) caused a 7-fold increase in hypothalamic extracellular serotonin and a small non-significant increase in extracellular norepinephrine. The subsequent systemic injection of fluoxetine (10 mg/kg s.c.) after local perfusion caused a 3-fold increase in extracellular norepinephrine, indicating that fluoxetine's action leading to an increase in extracellular norepinephrine was not occurring in the terminal areas of the hypothalamus but elsewhere in the brain, possibly cell bodies in the locus coeruleus.