Evidence on extracellular dopamine level in rat striatum: implications for the validity of quantitative microdialysis

Microdialysis zero-net-flux (ZNF) method is commonly used to monitor drug-induced changes in neurotransmitter baseline and release/uptake processes. Recent studies in this field suggest that microdialysis ZNF method seriously underestimates the resting concentration of extracellular dopamine in the rat neostriatum because probe implantation preferentially damages nearby dopamine release sites and that dopamine uptake inhibition increases the relative recovery of dopamine by microdialysis. This study assessed the validity of these claims by examining current data on extracellular dopamine levels at rest and after drug application obtained by voltammetry, a technique thought to induce less tissue disruption than microdialysis. To obtain the extracellular baseline value for dopamine from the evoked overflow data, we modified the existing dopamine kinetic model to suit both the resting and stimulated circumstances. It was found that dopamine uptake inhibition did in fact decrease the microdialysis relative recovery of dopamine, implying that the average basal extracellular dopamine level is within the range of 7-20 nm in rat striatum. This study concludes that the microdialysis ZNF method indeed underestimates the extracellular dopamine concentration, although not by as much as had been thought. Chronic microdialysis damages both neurotransmitter release and uptake, but it does so in a somewhat relative and proportional way for both processes. Thus the validity of the microdialysis ZNF method is not seriously undermined.     

Effects of tissue trauma on the characteristics of microdialysis zero-net-flux method sampling neurotransmitters

Microdialysis has been used for studying neurochemistry in brain regions that respond to afferent inputs or administered drugs. As the knowledge derived from and concerning microdialysis grows, so do the concerns over its invasiveness and, hence, the credibility of resulting data. Recent experimental and theoretical studies impugned the validity of the microdialysis zero-net-flux (ZNF) method in measuring brain extracellular neurotransmitters, suggesting that the tissue trauma resulting from probe implantation seriously compromises its worth. This paper developed a theoretical model to study the influences of two categories of tissue trauma on microdialysis ZNF operation: (1) morphological alterations in tissue extracellular structure and (2) physiological impairment of neurotransmitter release and uptake processes. Model results show that alterations of tissue extracellular structure negligibly affect the accuracy of the ZNF method in determining the basal level of extracellular neurotransmitter but do affect the fundamental characteristics of microdialysis: the extraction efficiency and relative recovery. An inhibited or damaged neurotransmitter uptake process always decreases the efficiency of microdialysis extraction, but rise of the relative recovery of neurotransmitters with the same uptake inhibition/damage occurs only when there is far more damage to the neurotransmitter release than to the uptake process in the tissue. A criterion for this rising trend of microdialysis relative recovery is discussed in terms of trauma parameters and neurotransmitter uptake inhibition.     

Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.     

Coupled Effects of Mass Transfer and Uptake Kinetics on In Vivo Microdialysis of Dopamine

Abstract: Voltammetric microelectrodes and microdialysis probes were used simultaneously to monitor extracellular dopamine in rat striatum during electrical stimulation of the medial forebrain bundle. Microelectrodes were placed far away (1 mm) from, immediately adjacent to, and at the outlet of microdialysis probes. In drug-naive rats, electrical stimulation (45 Hz, 25 s) evoked a robust response at microelectrodes far away from the probes, but there was no response at microelectrodes adjacent to and at the outlet of the probes. After nomifensine administration (20 mg/kg i.p.), stimulation evoked robust responses at all three microelectrode placements. These results demonstrate first that evoked release in tissue adjacent to microdialysis probes is suppressed in comparison with evoked release in tissue far away from the probes and second that equilibration of the dopamine concentration in the extracellular fluid adjacent to and far away from the probes is prevented by the high-affinity dopamine transporter. Hence, models of microdialysis, which assume the properties of tissue to be spatially uniform, require modification to account for the distance that separates viable sites of evoked dopamine release from the probe. We introduce new mass transfer resistance parameters that qualitatively explain the observed effects of uptake inhibition on stimulation responses recorded with microdialysis and voltammetry.     

In vivo monitoring of brain neurotransmitter release for the assessment of neuroendocrine interactions

Summary 1. The neurotransmitter mechanisms regulating neuroendocrine processes have been traditionally inferred from the effects of drugs purportedly acting through specific transmitter systems. The direct appraisal of changes in endogenous neuromediators had to rely initially on analyses of brain samples obtained post-morten.2. Currently, a more physiological assessment is available through the monitoring ot the extracellular levels of neurotransmitters and their metabolites in discrete brain areas of living animals. Two methodologies, namely in vivo voltammetry and microdialysis, are being increasingly used for this purpose. This article summarizes their principles, relative merits, and limitations and presents some relevant applications.3. Thus, microdialysis data show a differential response in the amphetamine-induced dopamine release in the nucleus accumbens in adult male and female rats castrated prepuberally. Given their high time-resolution, in vivo electrochemistry techniques seem especially suited for studying the fast, non-genomic effects of steroid hormones. This is illustrated by the voltammetric detection of a rapid release of dopamine in the corpus striatum induced by progesterone in males.4. These methodologies should be regarded as complementary tools for the assessment of the neurochemical correlates of neuroendocrine interactions.     

NMDA-mediated release of glutamate and GABA in the subthalamic nucleus is mediated by dopamine: an in vivo microdialysis study in rats

The present study investigated the effects of N-methyl-D-aspartic acid.H2O (NMDA) on the dopamine, glutamate and GABA release in the subthalamic nucleus (STN) by using in vivo microdialysis in rats. NMDA (100 micromol/L) perfused through the microdialysis probe evoked an increase in extracellular dopamine in the STN of the intact rat of about 170%. This coincided with significant increases in both extracellular glutamate (350%) and GABA (250%). The effect of NMDA perfusion on neurotransmitter release at the level of the STN was completely abolished by co-perfusion of the selective NMDA-receptor antagonist MK-801 (10 micromol/L), whereas subthalamic perfusion of MK-801 alone had no effect on extracellular neurotransmitter concentrations. Furthermore, NMDA induced increases in glutamate were abolished by both SCH23390 (8 micromol/L), a selective D1 antagonist, and remoxipride (4 micromol/L), a selective D2 antagonist. The NMDA induced increase in GABA was abolished by remoxipride but not by SCH23390. Perfusion of the STN with SCH23390 or remoxipride alone had no effect on extracellular neurotransmitter concentrations. The observed effects in intact animals depend on the nigral dopaminergic innervation, as dopamine denervation, by means of 6-hydroxydopamine lesioning of the substantia nigra, clearly abolished the effects of NMDA on neurotransmitter release at the level of the STN. Our work points to a complex interaction between dopamine, glutamate and GABA with a crucial role for dopamine at the level of the STN.     

Effect of resuscitative mild hypothermia on glutamate and dopamine release,apoptosis and ischaemic brain damage in the endothelin-1 rat model for focal cerebral ischaemia

The relationship between glutamate and dopamine release, apoptosis and ischaemic damage was studied following induction of transient focal cerebral ischaemia under normothermic (37 degrees C) and postischaemic (resuscitative) mild hypothermic (34 degrees C for 2 h) conditions in sevoflurane anaesthetized male Wistar rats. Focal ischaemia was induced by infusing endothelin-1 adjacent to the middle cerebral artery. In vivo microdialysis was used to sample glutamate and dopamine from striatum and parietal cortex of the ipsilateral hemisphere. The volume of ischaemic damage and the degree of apoptosis were determined 24 h after the insult. In both striatum and cortex of the normothermic group an initial increase in extracellular glutamate and dopamine levels following endothelin-1 infusion was observed. Striatal glutamate levels remained enhanced (250% of baseline) throughout the experiment, while the other neurotransmitter levels returned to baseline values. Hypothermia significantly attenuated the endothelin-1 induced glutamate release in the striatum. It also reduced apoptosis and infarct volume in the cortex. These results indicate that: (i) postischaemic mild hypothermia exerts its neuroprotective effect by inhibiting apoptosis in the ischaemic penumbral region; and (ii) this effect is not associated with an attenuation of glutamate or dopamine release in the cortex.     

In vivo Effects of the Anatoxin-a on Striatal Dopamine Release

Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of anatoxin-a on striatal in vivo dopamine release by microdialysis.The results of this work show that anatoxin-a evoked dopamine release in a concentration-dependent way. Atropine had not any effect on dopamine release evoked by 3.5 mM anatoxin-a. However, perfusion of nicotinic antagonists mecamylamine and α-bungarotoxin induced a total inhibition of the striatal dopamine release. Perfusion of α7*-receptors antagonists, metillycaconitine or α-bungarotoxin, partially inhibits the release of dopamine stimulated by anatoxin-a. These results show that anatoxin-a can be used as an important nicotinic agonist in the study of nicotinic receptor by in vivo microdialysis technique and also support further in vivo evidences that α7*nicotinic AChRs are implicated in the regulation of striatal dopamine release.     

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson''s and Huntington''s disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 μm) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.     

Use of Calcium Antagonism for the Characterization of Drug-Evoked Dopamine Release from the Brain of Conscious Rats Determined by Microdialysis

Dopamine was determined by microdialysis of the striatum of conscious rats. We investigated whether the release of dopamine, induced by nine different pharmacological treatments, was sensitive to calcium antagonism. Calcium antagonism was determined by Mg2+ or Cd2+ infusion. The following conditions were investigated: haloperidol, haloperidol plus GBR 12909, nomifensine, (+)-amphetamine (all administered intraperitoneally), KCl, 1-methyl-4-phenyl-pyridinium ion (MPP+), glutamate, ouabain, and 120 mmol/L magnesium (all applied by infusion through the dialysis membrane). The results on calcium antagonism were combined with data on tetrodotoxin (TTX) sensitivity. With the combined data, three different types of dopamine release were characterized. First, action potential-dependent dopamine release was observed in animals treated with saline, haloperidol, haloperidol plus GBR 12909, nomifensine, and ouabain. Second, action potential-independent release was established in the case of (+)-amphetamine, glutamate, MPP+, and 120 mmol/L Mg2+. Finally, K+-induced dopamine release was classified as TTX independent and calcium dependent. It is concluded that brain dialysis is a powerful method for differentiating between different types of neurotransmitter release.     

Dysfunction of Cholinergic and Dopaminergic Neuronal Systems in β-Amyloid Protein-Infused Rats

Abstract: Accumulations of β-amyloid protein are characteristic and diagnostic features of the brain of Alzheimer's disease patients; however, the physiological role of this protein in CNS is unknown. We have previously reported that continuous infusion of β-amyloid protein into rat cerebral ventricle impairs learning ability and decreases choline acetyltransferase activity, a marker enzyme of cholinergic neuron. In this study, the effects of β-amyloid protein infusion on the release of neurotransmitters in cholinergic and dopaminergic neuronal systems were investigated by using an in vivo brain microdialysis method. Nicotine-stimulated release of acetylcholine and dopamine in these animals was significantly lower than that in vehicle-infused rats. Further, dopamine release induced by high-K stimulation was decreased in β-amyloid protein-infused rats compared with vehicle-infused rats. These results suggest that the release of the two transmitters, acetylcholine and dopamine, was decreased by β-amyloid protein and that learning deficits observed in the β-amyloid protein-infused rats are partly due to the impairment of neurotransmitter release. Furthermore, continuous infusion of β-amyloid protein may be a useful method to produce the animal model of Alzheimer's disease.     

Effect of Precursor Loading on the Synthesis Rate and Release of Dopamine and Serotonin in the Striatum: A Microdialysis Study in Conscious Rats

The effects of systemic administration of tyrosine and phenylalanine on the extracellular levels of tyrosine and dopamine were determined by microdialysis in the striatum of awake rats. In addition, the effects of these precursors on in vivo 3,4-dihydroxyphenylalanine (DOPA) formation were determined during continuous infusion of a decarboxylase inhibitor. Both precursors increased the dialysate levels of tyrosine sixfold, but only phenylalanine administration stimulated DOPA formation. However, neither precursor affected the release of dopamine. When the precursor administration was repeated in rats in which the release of dopamine was stimulated by haloperidol pretreatment, again no effect was seen on the release of dopamine. Systemic administration of tryptophan (100 mg/kg, i.p.) during continuous infusion of a decarboxylase inhibitor induced a threefold increase in the formation of 5-hydroxytryptophan and caused an increase in the release of serotonin during infusion of an uptake inhibitor to about 150% of controls. Finally, we investigated whether dietary precursors were able to influence neurotransmitter formation and release. Rats trained to consume their daily food in a period of 2 h were implanted with microdialysis probes. Scheduled eating induced a small increase in the extracellular levels of tyrosine (135% of controls), but the release of dopamine and the formation of 5-hydroxytryptophan during continuous infusion of a decarboxylase inhibitor were not affected.     

First messengers. Methods and approaches for assaying their release

The review is focused on methods and approaches, which are widely used to monitor neurotransmitter release from diverse cells and to explore underlying mechanisms. A variety of methods and technical approaches for ex vivo and in vivo experiments are discussed, including microdialysis, microelectrode voltammetry, and enzymatic biosensors. The method of the cellular biosensor is specifically discussed as an effective approach for assaying neurotransmitter release from single cells.     

Effect of Ischemia on the In Vivo Release of Striatal Dopamine, Glutamate, and γ-Aminobutyric Acid Studied by Intracerebral Microdialysis

We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesioning. The present study was carried out to evaluate whether the protective effect of the lesion involves changes in the degree of local cerebral blood flow (ICBF) reduction, energy metabolite depletion, or alterations in the extracellular release of striatal dopamine (DA), glutamate (Glu), or gamma-aminobutyric acid (GABA). Control and SN-lesioned rats were subjected to 20 min of forebrain ischemia by four-vessel occlusion combined with systemic hypotension. Levels of ICBF, as measured by the autoradiographic method, and energy metabolites were uniformly reduced in both the ipsi- and contralateral striata at the end of the ischemic period, a finding implying that the lesion did not affect the severity of the ischemic insult itself. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. An approximately 500-fold increase in DA content, a 7-fold increase in Glu content, and a 5-fold increase in GABA content were observed during ischemia in nonlesioned animals. These levels gradually returned to baseline by 30 min of reperfusion. In SN-lesioned rats, the release of DA was completely prevented, the release of GABA was not affected, and the release of Glu was partially attenuated. However, excessive extracellular Glu concentrations were still attained, which are potentially toxic. This, taken together with the previous neuropathological findings, suggests that excessive release of DA is important for the development of ischemic cell damage in the striatum.     

Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+.

Ringer's solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.     

Neurotransmitter release statistics: Moment estimates for inhomogeneous Bernoulli trials

Summary Evoked release of quanta of neurotransmitter is generally treated as a set of homogeneous, stationary Bernoulli trials, hence governed by the binomial distribution. Relaxing the assumptions of uniformity and stationarity leads to a more realistic physiological model of transmitter release but also introduces systematic biases in the moment estimates of the binomial parameters. We derive probability generating functions for quantal release and expressions for the moment estimates of ¯n and ¯p for a generalized model that incorporates temporal variation and nonuniformity in individual release probabilities and in numbers of release sites.     

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.     

Neurocircuitry of the basal ganglia studied by monitoring neurotransmitter release

The neurocircuitries of the basal ganglia are studied with in vivo microdialysis, with special consideration to dopamine transmission and its interaction with other neurotransmitter systems. The aim is to develop experimental models to study the pathophysiology and therapy of neurodegenerative disorders of the basal ganglia, as well as to develop models to study the short- and long-term consequences of perinatal asphyctic lesions. A main goal of these studies is to find and to characterize new treatments for these disorders.     

Differential regulation of somatodendritic and nerve terminal dopamine release by serotonergic innervation of substantia nigra

Nigrostriatal dopaminergic neurons release dopamine from dendrites in substantia nigra and axon terminals in striatum. The cellular mechanisms for somatodendritic and axonal dopamine release are similar, but somatodendritic and nerve terminal dopamine release may not always occur in parallel. The current studies used in vivo microdialysis to simultaneously measure changes in dendritic and nerve terminal dopamine efflux in substantia nigra and ipsilateral striatum respectively, following intranigral application of various drugs by reverse dialysis through the nigral probe. The serotonin releasers (+/-)-fenfluramine (100 micro m) and (+)-fenfluramine (100 micro m) significantly increased dendritic dopamine efflux without affecting extracellular dopamine in striatum. The non-selective serotonin receptor agonist 1-(m-chlorophenyl)-piperazine (100 micro m) elicited a similar pattern of dopamine release in substantia nigra and striatum. NMDA (33 micro m) produced an increase in nigral dopamine of a similar magnitude to mCPP or either fenfluramine drug. However, NMDA also induced a concurrent increase in striatal dopamine. The D2 agonist quinpirole (100 micro m) had a parallel inhibitory effect on dopamine release from dendritic and terminal sites as well. Taken together, these data suggest that serotonergic afferents to substantia nigra may evoke dendritic dopamine release through a mechanism that is uncoupled from the impulse-dependent control of nerve terminal dopamine release.     

Effect of co-administration of varenicline and antidepressants on extracellular monoamine concentrations in rat prefrontal cortex

Since a substantial proportion of smokers have comorbid mood disorders, the smoking cessation aid varenicline might occasionally be prescribed to patients who are simultaneously treated with antidepressants. Given that varenicline is a selective nicotinic acetylcholine receptor partial agonist and not a substrate or inhibitor of drug metabolizing enzymes, pharmacokinetic interactions with various classes of antidepressants are highly unlikely. It is, however, conceivable that varenicline may have a pharmacodynamic effect on antidepressant-evoked increases in central monoamine release. Interactions resulting in excessive transmitter release could cause adverse events such as serotonin syndrome, while attenuation of monoamine release could impact the clinical efficacy of antidepressants. To investigate this we examined whether varenicline administration modulates the effects of the selective serotonin reuptake inhibitor sertraline and the monoamine oxidase inhibitor clorgyline, given alone and combined, on extracellular concentrations of the monoamines serotonin, dopamine, and norepinephrine in rat brain by microdialysis. Given the important role attributed to cortical monoamine release in serotonin syndrome as well as antidepressant activity, the effects on extracellular monoamine concentrations were measured in the medial prefrontal cortex. Responses to maximally effective doses of sertraline or clorgyline and of sertraline plus clorgyline were the same in the absence as in the presence of a relatively high dose of varenicline, which by itself had no significant effect on cortical monoamine release. This is consistent with the binding profile of varenicline that has insufficient affinity for receptors, enzymes, or transporters to inhibit or potentiate the pharmacologic effects of antidepressants. Since varenicline neither diminished nor potentiated sertraline- or clorgyline-induced increases in neurotransmitter levels, combining varenicline with serotonergic antidepressants is unlikely to cause excessive serotonin release or to attenuate antidepressant efficacy via effects on cortical serotonin, dopamine or norepinephrine release.