W Timmerman

Brain microdialysis of GABA and glutamate : What does it signify?

Microdialysis has become a frequently used method to study extracellular levels of GABA and glutamate in the central nervous system. However, the fact that the major part of GABA and glutamate as measured by microdialysis does not fulfill the classical criteria for exocytotic release questions the vesicular origin of the amino acids in dialysates. Glial metabolism or reversal of the (re)uptake sites has been suggested to be responsible for the pool of nonexocytotically released amino‐acid transmitters that seem to predominate over the neuronal exocytotic pool. The origin of extracellular GABA and glutamate levels and, as a consequence, the implications of changes in these levels upon manipulations are therefore obscure. This review critically analyzes what microdialysis data signify, i.e., whether amino‐acid neurotransmitters sampled by microdialysis represent synaptic release, carrier‐mediated release, or glial metabolism. The basal levels of GABA and glutamate are virtually tetrodotoxin‐ and calcium‐independent. Given the fact that evidence for nonexocytotic release mediated by reversal of the uptake sites as a release mechanism relevant for normal neurotransmission is so far limited to conditions of “excessive stimulation,” basal levels most likely reflect a nonneuronal pool of amino acids. Extracellular GABA and glutamate concentrations can be enhanced by a wide variety of pharmacological and physiological manipulations. However, it is presently impossible to ascertain that the stimulated GABA and glutamate in dialysates are of neuronal origin. On the other hand, under certain stimulatory conditions, increases in amino‐acid transmitters can be obtained in the presence of tetrodotoxin, again suggesting that aspecific factors not directly related to neurotransmission underlie these changes in extracellular levels. It is concluded that synaptic transmission of GABA and glutamate is strictly compartmentalized and as a result, these amino acids can hardly leak out of the synaptic cleft and reach the extracellular space where the dialysis probe samples. Synapse 27:242–261, 1997.

β-Amyloid neurotoxicity is mediated by a glutamate-triggered excitotoxic cascade in rat nucleus basalis

Whereas a cardinal role for β‐amyloid protein (Aβ) has been postulated as a major trigger of neuronal injury in Alzheimers disease, the pathogenic mechanism by which Aβ deranges nerve cells remains largely elusive. Here we report correlative in vitro and in vivo evidence that an excitotoxic cascade mediates Aβ neurotoxicity in the rat magnocellular nucleus basalis (MBN). In vitro application of Aβ to astrocytes elicits rapid depolarization of astroglial membranes with a concomitant inhibition of glutamate uptake. In vivo Aβ infusion by way of microdialysis in the MBN revealed peak extracellular concentrations of excitatory amino acid neurotransmitters within 20–30 min. Aβ‐triggered extracellular elevation of excitatory amino acids coincided with a significantly enhanced intracellular accumulation of Ca2+ in the Aβ injection area, as was demonstrated by 45Ca2+ autoradiography. In consequence of these acute processes delayed cell death in the MBN and persistent loss of cholinergic fibre projections to the neocortex appear as early as 3 days following the Aβ‐induced toxic insult. Such a sequence of Aβ toxicity was effectively antagonized by the N‐methyl‐d‐aspartate (NMDA) receptor ligand dizocilpine maleate (MK‐801). Moreover, Aβ toxicity in the MBN decreases with advancing age that may be associated with the age‐related loss of NMDA receptor expression in rats. In summary, the present results indicate that Aβ compromises neurons of the rat MBN via an excitotoxic pathway including astroglial depolarization, extracellular glutamate accumulation, NMDA receptor activation and an intracellular Ca2+ overload leading to cell death.

Do neurotransmitters sampled by brain microdialysis reflect functional release

Abstract Brain microdialysis is an invasive sampling technique and will always cause damage to nervous tissue. For proper interpretation of the results, possible sources of interference need to be identified. The present review discusses the possible artefacts of the microdialysis technique and evaluates methods used to investigate the possible neuronal origin of the sampled transmitters. Various neurotransmitter systems that are currently sampled by brain microdialysis are critically evaluated here. Three criteria to validate the neuronal origin of transmitters are discussed: 1. TTX- and calcium-dependency, 2. the detection of autoreceptors, and 3. the outcome of behavioural studies. The question whether neurotransmitters sampled by the brain microdialysis technique reflect functional release can be positively answered in case of dopamine, noradrenaline, serotonin and acetylcholine. However, in the case of glutamate and GABA, the relationship between neuronal release and dialysate content is not convincingly demonstrated. It is likely that synaptic transmission of glutamate and GABA is very strictly compartmentalized, and as a result, these amino acids can hardly leak out of the synaptic cleft into the extracellular space where the dialysis probe samples.

MICRODIALYSIS AND STRIATAL DOPAMINE RELEASE - STEREOSELECTIVE ACTIONS OF THE ENANTIOMERS OF N-0437

An intracerebral dialysis method was used to test both enantiomers of the very potent and selective dopamine (DA) D-2 agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin, N-0437, for their actions on DA receptors in the striatum of the rat. (-)N-0437 induced a 60% decrease in DA release, which was independent of the presence or absence of a kainic acid lesion placed unilaterally in the striatum. Stereotyped behaviour was apparent following administration of the (-) enantiomer. Thus, (-)N-0437 displayed an agonistic action on both pre- and postsynaptic D-2 receptors. (+)N-0437 did not induce any effect in the release model after peripheral administration nor did it induce any form of stereotypy. A comparison between the effects of (-)N-0437 after oral (10 mumol/kg) and transdermal (10 mumol/kg) administration showed the advantages of the latter mode of administration. Transdermal application induced a much longer duration of action of the drug (13 h) in comparison with the oral mode (5 h). Thus, transdermal administration may be a very useful method of drug application for therapeutic use.

EXTRACELLULAR GAMMA-AMINOBUTYRIC-ACID IN THE SUBSTANTIA-NIGRA RETICULATA MEASURED BY MICRODIALYSIS IN AWAKE RATS - EFFECTS OF VARIOUS STIMULANTS

The gamma-aminobutyric acid (GABA)-ergic system in the substantia nigra reticulata (SNR) was challenged by local infusion of various receptor-specific agents to obtain additional information on the physiological significance of extracellular GABA levels as measured by microdialysis in awake rats. Notwithstanding in vitro results, basal extracellular GABA levels were not affected by local infusion of the GABA-A agonist muscimol or by infusion of the GABA-B agonist baclofen. Upon a dopaminergic challenge, the D2 agonist LY 171555 was equally ineffective, but the D1 agonist induced an increase in extracellular GABA levels, which persisted in the presence of tetrodotoxin (TTX). Using excitatory amino acids, kainic acid was ineffective in modulating GABA levels, whereas N-methyl-D-aspartate induced an increase in extracellular GABA levels, again persisting when co-infused with TTX. The functional significance of TTX-independent changes in extracellular GABA levels is discussed.

The significance of extracellular GABA in the substantia nigra of the rat during seizures and anticonvulsant treatments

The effects of the anti-epileptic drugs valproic acid and gamma-vinyl-GABA (vigabatrin) on the extracellular content of GABA was determined by microdialysis. Probes were implanted in the substantia nigra reticulata (SNR) of rats. It was found that gamma-vinyl-GABA (1000 mg/kg) induced a 4-6-fold increase in the extracellular content of GABA. This increase lasted for at least 72 h. PTZ-induced convulsions were partly antagonized by the GVG treatment. The increase of extracellular GABA after gamma-vinyl-GABA was not affected by infusion of tetrodotoxin. In contrast valproic acid (200 mg/kg), although effective in preventing pentylenetetrazol (PTZ)-induced convulsions, did not affect extracellular GABA in the SNR. PTZ-induced convulsions did not modify extracellular GABA, neither in control rats nor in valproic acid or gamma-vinyl-GABA pretreated animals. The results do not support the idea that extracellular GABA in the SNR plays a significant role in anti-convulsive treatment. However, the present data can also be interpreted that extracellular GABA, as sampled by microdialysis, is not a reliable marker for GABA release.

Electrical stimulation of the substantia nigra reticulata: Detection of neuronal extracellular GABA in the ventromedial thalamus and its regulatory mechanism using microdialysis in awake rats

A combination of electrical stimulation and microdialysis was used to study the nigrothalamic gamma aminobutyric acid (GABA)ergic system and its regulatory mechanisms in awake rats. Extracellular GABA levels in the ventromedial nucleus of the thalamus were detected in 3‐min fractions collected before, during and after a 10‐min stimulation period of the substantia nigra reticulata. Electrical stimulation of the substantia nigra reticulata increased the GABA levels to 155% of basal values in the ventromedial thalamus only during the first 3‐min interval upon stimulation. The increase in GABA levels was tetrodotoxin‐dependent, implicating an exocytotic origin. The basal levels of extracellular GABA in the ventromedial thalamus were of nonexocytotic origin. To study the mechanism underlying the fast compensatory response in neuronal GABA release after nigral stimulation, local infusions into the ventromedial thalamus of reuptake inhibitors and GABA antagonists were performed and the effect of nigral stimulation was examined under the various applications. Local infusion of the reuptake inhibitors nipecotic acid (500 μM) and SKF 89976‐A (20 and 50 μM) increased extracellular GABA levels to 350%, 180% and 600%, respectively, of basal values in the ventromedial thalamus tetrodotoxin‐independently. Under these conditions, the increase in extracellular GABA was absent (nipecotic acid) or suppressed (20% of basal values; SKF 89976‐A for both doses), leaving it unsolved whether or not the uptake system was responsible for the fast compensation in neuronal GABA after stimulation. The GABA‐A antagonist bicucilline (50 μM) was ineffective when infused locally in the ventromedial thalamus, but prolonged the increase in neuronal GABA release after nigral stimulation; the GABA levels were increased during two 3‐min samples to ∼165%, indicating a functional role for GABA‐A receptors in regulating the release of GABA from nigrothalamic GABAergic neurons. The GABA‐B receptor antagonist CGP 35348 (50 μM) did not affect GABA levels when infused locally in the ventromedial thalamus and neither affected the response in neuronal GABA after stimulation. This finding does not support a role for GABA‐B receptors in controlling the release from the nigrothalamic neurons. Synapse 26:62–71, 1997.

Characterization of the effect of dopamine D3 receptor stimulation on locomotion and striatal dopamine levels

By examining the effect of dopamine (DA) D3 receptor stimulation on locomotor activity and extracellular levels of DA in striatum we show that inhibition of locomotor activity induced by DA D3 receptor-selective agonists is mediated by two interacting mechanisms: (1) directly via the stimulation of DA D3 receptors that inhibit locomotor activity, and (2) indirectly via a decrease in extracellular levels of DA. Thus, the moderately DA D3 receptor-selective agonist R-(+)-7-OH- DPAT (R-(+)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin) decreased locomotor activity after administration of 10 nmol/kg and extracellular DA levels in accumbens and striatum after administration of 30 nmol/kg. A decrease in locomotor activity that coincided with a decrease in extracellular DA levels in striatum was observed after administration of 100 nmol/kg of the DA D3 receptor-selective agonist PD128907 ((+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3 b]-1,4-oxasin-9-ol. In combination with the partial, DA D3 receptor-selective agonist PD151328 (2-[4[3-(4-phenyl)-1- piperazinyl)propoxy]phenyl]-benzamidazole), a reversal of the attenuating effect of PD128907 on locomotor activity was observed, without an effect on extracellular levels of DA. In combination with a low--10 nmol/kg--dose of haloperidol, a reversal of the inhibitory effect of PD128907 on locomotor activity was observed that coincided with an increase in extracellular levels of DA. In the presence of 0.5 mg/kg amphetamine, PD128907 decreased amphetamine-induced locomotor activity. This effect could be reversed by PD151328.

In Vivo Evidence for the Existence of Autoreceptors on Dopaminergic, Serotonergic, and Cholinergic Neurons in the Brain

Intrastriatal infusions as well as systemic administration of the selective D-2 antagonist (-)-sulpiride caused similar increases in the dialysate levels of dopamine (DA) to about 180% of controls. A similar conclusion was drawn when the selective D-2 agonist (-)-N-0437 was infused intrastriatally or administered systemically: both routes of administration caused a decrease in the release of DA to about 40-50% of controls. In order to evaluate the properties of synthesis-modulating autoreceptors on dopaminergic and serotonergic neurons we have estimated the synthesis rate of serotonin (5-HT) or DA by monitoring the 5-HTP or DOPA formation in the dialysates during infusion of a decarboxylase inhibitor. Infusion of (-)-N-0437 decreased the DOPA formation, whereas infusion of (-)-sulpiride increased the dialysate levels of DOPA; these results indicate that the D-2 receptors controlling the synthesis of DA are localized on nerve terminals. Administration of the selective 5-HT-IA agonist 8-hydroxy-dipropyl-aminotetraline (8-OH-DPAT) resulted in a decrease in the synthesis rate of 5-HT. When 8-OH-DPAT was infused via the dialysis membrane, the agonist was unable to modify the release of 5-HT. The effects of infusion of the muscarinic agonist oxotremorine and the muscarinic antagonist atropine were dependent on the presence of the esterase inhibitor neostigmine in the perfusion fluid. In the absence of neostigmine, oxotremorine caused a pronounced decrease in the output of acetylcholine (ACh), whereas atropine was without effect. In the presence of neostigmine oxotremorine was without effect but infusion of atropine or other anticholinergics caused a pronounced increase in the dialysate levels of ACh. It is concluded that the autoreceptor controlling the release of ACh is of the M3-type and that the receptor is not fully occupied during normal conditions. In conclusion, microdialysis of neurotransmitters is a valuable tool for the study of autoreceptors in vivo. The presented studies provided evidence for the existence of autoreceptors controlling the synthesis and/or release of DA, 5-HT, as well as ACh in the striatum.

Effects of D-2 agonists on the release of dopamine: localization of the mechanism of action

SummaryBrain microdialysis was used to localize the mechanism of action of the effect induced by the D-2 agonists (−)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin [(−)-N-0437] and (+)-4-propyl-9-hydroxynaphtoxazine [(+)-PHNO], on the release of DA in the striatum. Both agonists induced a stronger decrease in the release of DA when administered systemically in comparison to local administration. This suggests that the action of D-2 agonists is not exclusively mediated by autoreceptors regulating the release of DA at the level of the nerve terminals. By co-infusing nomifensine (10 µM) the effect of intrastriatally administered D-2 agonists on the release of DA could be completely abolished. As both agonists were effective when administered systemically in normal rats and in rats with kainic acid lesions performed in the striatum during nomifensine infusion, the effects induced by D-2 agonists seem to be partly mediated by autoreceptors situated on cell bodies, regulating the impulse flow of the neuron. In addition, D-2 receptors located on postsynaptic structures participating in the striatonigral feedback loops were suggested to be involved. (−)-N-0437 and (+)-PHNO were less effective after systemic administration when kainic acid lesioned rats were used in comparison with normal rats. Thus, D-2 agonists interact in a complex way with D-2 receptors for displaying their effect on the release of DA: autoreceptors situated on nerve terminals and on cell bodies as well as D-2 receptors located on postsynaptic structures participating in the striatonigral neuronal loops may all be involved to a certain extent in the mechanism of action of D-2 agonists.