Niall J. Finnerty

Prefrontal GABAB Receptor Activation Attenuates Phencyclidine-Induced Impairments of Prepulse Inhibition: Involvement of Nitric Oxide

Recent theories propose that both GABA and glutamate signaling are compromised in patients with schizophrenia. These deficits can be observed in several brain regions including the prefrontal cortex (PFC), an area extensively linked to the cognitive dysfunction in this disease and notably affected by NMDA receptor antagonists such as phencyclidine (PCP). We have previously demonstrated that inhibition of the nitric oxide (NO) pathways in the brain, particularly in the PFC, prevents a wide range of PCP-induced behavioral deficits including disruption of prepulse inhibition (PPI). This study investigated the role of GABAB receptor signaling and NO in the effects of PCP on PPI. Mice received systemic or prefrontal injections of the GABAB receptor agonist baclofen (2.5–5 mg/kg and 1 mM) before PCP treatment (5 mg/kg) and were thereafter tested for PPI. GABA/NO interactions were studied by combining baclofen and the NO synthase inhibitor L-NAME (20 mg/kg) in subthreshold doses. The role of GABAB receptors for NO production in vivo was assessed using NO-sensors implanted into the rat PFC. PCP-induced PPI deficits were attenuated in an additive manner by systemic baclofen treatment, whereas prefrontal microinjections of baclofen completely blocked the effects of PCP, without affecting PPI per se. The combination of baclofen and L-NAME was more effective in preventing the effects of PCP than any compound by itself. Additionally, baclofen decreased NO release in the PFC in a dose-related manner. This study proposes a role for GABAB receptor signaling in the effects of PCP, with altered NO levels as a downstream consequence. Thus, prefrontal NO signaling mirrors an altered level of cortical inhibition that may be of importance for information processing deficits in schizophrenia.

Brain nitric oxide: regional characterisation of a real-time microelectrochemical sensor.

A reliable method of directly measuring endogenously generated nitric oxide (NO) in real-time and in various brain regions is presented. An extensive characterisation of a previously described amperometric sensor has been carried out in the prefrontal cortex and nucleus accumbens of freely moving rats. Systemic administration of saline caused a transient increase in signal from baseline levels in both the prefrontal cortex (13 ± 3pA, n=17) and nucleus accumbens (12 ± 3pA, n=8). NO levels in the prefrontal cortex were significantly increased by 43 ± 9pA (n=9) following administration of l-arginine. A similar trend was observed in the nucleus accumbens, where an increase of 44 ± 9pA (n=8) was observed when compared against baseline levels. Systemic injections of the non-selective NOS inhibitor l-NAME produced a significant decrease in current recorded in the prefrontal cortex (24 ± 6pA, n=5) and nucleus accumbens (17 ± 3pA, n=6). Finally it was necessary to validate the sensors functionality in vivo by investigating the effect of the interferent ascorbate on the oxidation current. The current showed no variation in both regions over the selected time interval of 60 min, indicating no deterioration of the polymer membrane. A detailed comparison identified significantly greater affects of administrations on NO sensors implanted in the striatum than those inserted in the prefrontal cortex and the nucleus accumbens.

Increased cortical nitric oxide release after phencyclidine administration

Phencyclidine exerts psychotomimetic effects in humans and is used as a pharmacological animal model for schizophrenia. We, and others, have demonstrated that phencyclidine induces cognitive deficits in rats that are associated with schizophrenia. These cognitive deficits can be normalized by inhibition of nitric oxide synthase. The development of selective microelectrochemical nitric oxide sensors may provide direct evidence for the involvement of nitric oxide in these effects. The aim of the present study was to use LIVE (long term in vivo electrochemistry) to investigate the effect of phencyclidine, alone or in combination with the nitric oxide synthase inhibitor L‐NAME, on nitric oxide levels in the medial prefrontal cortex of freely moving rats. Phencyclidine (2 mg kg−1) produced an increase in cortical nitric oxide levels and this increase was ameliorated by L‐NAME (10 mg kg−1). Tentatively, the results from the present study provide a biochemical rationale for the involvement of nitric oxide in the phencyclidine model of schizophrenia. Synapse 63:1083–1088, 2009.

An investigation of hypofrontality in an animal model of schizophrenia using real-time microelectrochemical sensors for glucose, oxygen, and nitric oxide.

Glucose, O2, and nitric oxide (NO) were monitored in real time in the prefrontal cortex of freely moving animals using microelectrochemical sensors following phencyclidine (PCP) administration. Injection of saline controls produced a decrease in glucose and increases in both O2 and NO. These changes were short-lived and typical of injection stress, lasting ca. 30 s for glucose and between 2 and 6 min for O2 and NO, respectively. Subchronic PCP (10 mg/kg) resulted in increased motor activity and increases in all three analytes lasting several hours: O2 and glucose were uncoupled with O2 increasing rapidly following injection reaching a maximum of 70% (ca. 62 μM) after ca. 15 min and then slowly returning to baseline over a period of ca. 3 h. The time course of changes in glucose and NO were similar; both signals increased gradually over the first hour post injection reaching maxima of 55% (ca. 982 μM) and 8% (ca. 31 nM), respectively, and remaining elevated to within 1 h of returning to baseline levels (after ca. 5 and 7 h, respectively). While supporting increased utilization of glucose and O2 and suggesting overcompensating supply mechanisms, this neurochemical data indicates a hyperfrontal effect following acute PCP administration which is potentially mediated by NO. It also confirms that long-term in vivo electrochemical sensors and data offer a real-time biochemical perspective of the underlying mechanisms.

Increased brain nitric oxide levels following ethanol administration.

Nitric oxide is a ubiquitous messenger molecule, which at elevated concentrations has been implicated in the pathogenesis of several neurological disorders. Its role in oxidative stress, attributed in particular to the formation of peroxynitrite, proceeds through its high affinity for the superoxide radical. Alcoholism has recently been associated with the induction of oxidative stress, which is generally defined as a shift in equilibrium between pro-oxidant and anti-oxidant species in the direction of the former. Furthermore, its primary metabolite acetaldehyde, has been extensively associated with oxidative damage related toxic effects following alcohol ingestion. The principal objective of this study was the application of long term in vivo electrochemistry (LIVE) to investigate the effect of ethanol (0.125, 0.5 and 2.0 g kg(-1)) and acetaldehyde (12.5, 50 and 200 mg kg(-1)) on NO levels in the nucleus accumbens of freely moving rats. Systemic administrations of ethanol and acetaldehyde resulted in a dose-dependent increases in NO levels, albeit with very differing time courses. Subsequent to this the effect on accumbal NO levels, of subjecting the animal to different drug combinations, was also elucidated. The nitric oxide synthase inhibitor L-NAME (20 mg kg(-1)) and acetaldehyde sequestering agent D-penicillamine (50 mg kg(-1)) both attenuated the increase in NO levels following ethanol (1 g kg(-1)) administration. Conversely, the alcohol dehydrogenase inhibitor 4-methylpyrazole (25 mg kg(-1)) and catalase inhibitor sodium azide (10 mg kg(-1)) potentiated the increase in NO levels following ethanol administration. Finally, dual inhibition of aldehyde dehydrogenase and catalase by cyanamide (25 mg kg(-1)) caused an attenuation of ethanol effects on NO levels. Taken together these data highlight a robust increase in brain NO levels following systemic alcohol administration which is dependent on NO synthase activity and may involve both alcohol- and acetaldehyde-dependent mechanisms.

In vivo characterisation of a Nafion ® -modified Pt electrode for real-time nitric oxide monitoring in brain extracellular fluid

A Nafion®-modified Pt sensor developed for real-time neurochemical monitoring in freely-moving animals has now been characterised in vivo for the detection of nitric oxide (NO). Experiments were undertaken to test sensitivity, selectivity and stability. In control experiments, local administration of aCSF resulted in a decrease in signal (42 ± 12 pA, n = 9), while systemic administration of saline caused a transient increase in signal (22 ± 3 pA, n = 9) from baseline levels. Local MD perfusions of known concentrations of NO (1 mM) and L-arginine (720 μM) caused significant increases in current (4 ± 1 nA (n = 7) and 271 ± 37 pA (n = 5) respectively) while systemic administration of the non-selective NOS inhibitor L-NAME produced a significant decrease in current (91 ± 19 pA, n = 4) compared to baseline levels. Confirmation that the sensor remained selective was achieved by injection of ascorbic acid (2 g kg−1, i.p). The oxidation current showed no variation over the selected time interval of 60 min, indicating no deterioration of the polymer membrane. Finally, after an initial decrease in sensitivity (ca. 50%) within the first 24 h of implantation the Nafion®-modified Pt sensor showed excellent stability under physiological conditions over extended periods (8 days) validating its use for continuous chronic in vivo recordings.

Long Term Amperometric Recordings in the Brain Extracellular Fluid of Freely Moving Immunocompromised NOD SCID Mice

We describe the in vivo characterization of microamperometric sensors for the real-time monitoring of nitric oxide (NO) and oxygen (O2) in the striatum of immunocompromised NOD SCID mice. The latter strain has been utilized routinely in the establishment of humanized models of disease e.g., Parkinson’s disease. NOD SCID mice were implanted with highly sensitive and selective NO and O2 sensors that have been previously characterized both in vitro and in freely moving rats. Animals were systemically administered compounds that perturbed the amperometric current and confirmed sensor performance. Furthermore, the stability of the amperometric current was investigated and 24 h recordings examined. Saline injections caused transient changes in both currents that were not significant from baseline. l-NAME caused significant decreases in NO (p < 0.05) and O2 (p < 0.001) currents compared to saline. l-Arginine produced a significant increase (p < 0.001) in NO current, and chloral hydrate and Diamox (acetazolamide) caused significant increases in O2 signal (p < 0.01) compared against saline. The stability of both currents were confirmed over an eight-day period and analysis of 24-h recordings identified diurnal variations in both signals. These findings confirm the efficacy of the amperometric sensors to perform continuous and reliable recordings in immunocompromised mice.

Continuous Real-Time in vivo Measurement of Cerebral Nitric Oxide Supports Theoretical Predictions of an Irreversible Switching in Cerebral ROS after Sufficient Exposure to External Toxins

BACKGROUND Mathematical models of the interactions between alphasynuclein (αS) and reactive oxygen species (ROS) predict a systematic and irreversible switching to damagingly high levels of ROS after sufficient exposure to risk factors associated with Parkinsons disease (PD). OBJECTIVES We tested this prediction by continuously monitoring real-time changes in neurochemical levels over periods of several days in animals exposed to a toxin known to cause Parkinsonian symptoms. METHODS Nitric oxide (NO) sensors were implanted in the brains of freely moving rats and the NO levels continuously recorded while the animals were exposed to paraquat (PQ) injections of various amounts and frequencies. RESULTS Long-term, real-time measurement of NO in a cohort of animals showed systematic switching in levels when PQ injections of sufficient size and frequency were administered. The experimental observations of changes in NO imply a corresponding switching in endogenous ROS levels and support theoretical predictions of an irreversible change to damagingly high levels of endogenous ROS when PD risks are sufficiently large. CONCLUSIONS Our current results only consider one form of PD risk, however, we are sufficiently confident in them to conclude that: (i) continuous long-term measurement of neurochemical dynamics provide a novel way to measure the temporal change and system dynamics which determine Parkinsonian damage, and (ii) the bistable feedback switching predicted by mathematical modelling seems to exist and that a deeper analysis of its characteristics would provide a way of understanding the pathogenic mechanisms that initiate Parkinsonian cell damage.

In vitro development and in vivo application of a platinum-based electrochemical device for continuous measurements of peripheral tissue oxygen

Acute limb ischaemia is caused by compromised tissue perfusion and requires immediate attention to reduce the occurrence of secondary complications that could lead to amputation or death. To address this, we have developed a novel platinum (Pt)-based electrochemical oxygen (O2) device for future applications in clinical monitoring of peripheral tissue ischaemia. The effect of integrating a Pt pseudo-reference electrode into the O2 device was investigated in vitro with an optimum reduction potential of -0.80V. A non-significant (p=0.11) decrease in sensitivity was recorded when compared against an established Pt-based O2 sensor operating at -0.65V. Furthermore, a biocompatible clinical sensor (ClinOX) was designed, demonstrating excellent linearity (R2=0.99) and sensitivity (1.41±0.02nAμM-1) for O2 detection. Significant rapid decreases in the O2 current during in vivo ischaemic insults in rodent limbs were reported for Pt-Pt (p<0.001) and ClinOX (p<0.01) and for ClinOX (p<0.001) in porcine limbs. Ex vivo sensocompatibility investigations identified no significant difference (p=0.08) in sensitivity values over 14days of exposure to tissue homogenate. The Pt-Pt based O2 design demonstrated high sensitivity for tissue ischaemia detection and thus warrants future clinical investigation.

Real-Time Amperometric Recording of Extracellular H2O2 in the Brain of Immunocompromised Mice: An In Vitro, Ex Vivo and In Vivo Characterisation Study

We detail an extensive characterisation study on a previously described dual amperometric H2O2 biosensor consisting of H2O2 detection (blank) and degradation (catalase) electrodes. In vitro investigations demonstrated excellent H2O2 sensitivity and selectivity against the interferent, ascorbic acid. Ex vivo studies were performed to mimic physiological conditions prior to in vivo deployment. Exposure to brain tissue homogenate identified reliable sensitivity and selectivity recordings up to seven days for both blank and catalase electrodes. Furthermore, there was no compromise in pre- and post-implanted catalase electrode sensitivity in ex vivo mouse brain. In vivo investigations performed in anaesthetised mice confirmed the ability of the H2O2 biosensor to detect increases in amperometric current following locally perfused/infused H2O2 and antioxidant inhibitors mercaptosuccinic acid and sodium azide. Subsequent recordings in freely moving mice identified negligible effects of control saline and sodium ascorbate interference injections on amperometric H2O2 current. Furthermore, the stability of the amperometric current was confirmed over a five-day period and analysis of 24-h signal recordings identified the absence of diurnal variations in amperometric current. Collectively, these findings confirm the biosensor current responds in vivo to increasing exogenous and endogenous H2O2 and tentatively supports measurement of H2O2 dynamics in freely moving NOD SCID mice.