Potassium-induced Release Research Articles

Basal and Evoked Neurotransmitter Levels in Parkin, DJ-1, PINK1 and LRRK2 Knockout Rat Striatum

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of neurons in the substantia nigra that project to the striatum and release dopamine (DA), which is required for normal movement. Common non-motor symptoms likely involve abnormalities with other neurotransmitters, such as serotonin, norepinephrine, acetylcholine, glycine, glutamate and gamma-aminobutyric acid (GABA). As part of a broad effort to provide better PD research tools, the Michael J. Fox Foundation for Parkinson’s Research funded the generation and characterization of knockout (KO) rats for genes with PD-linked mutations, including PINK1, Parkin, DJ-1 and LRRK2. Here we extend the phenotypic characterization of these lines of KO rats to include in vivo microdialysis to measure both basal and potassium-induced release of the above neurotransmitters and their metabolites in the striatum of awake and freely moving rats at ages 4, 8 and 12 months compared to wild-type (WT) rats. We found age-dependent abnormalities in basal DA, glutamate and acetylcholine in PINK1 KO rats and age-dependent abnormalities in basal DA metabolites in Parkin and LRRK2 KO rats. Parkin KO rats had increased glycine release while DJ-1 KO rats had decreased glutamate release and increased acetylcholine release compared to WT rats. All lines except DJ-1 KO rats showed age-dependent changes in release of one or more neurotransmitters. Our data suggest these rats may be useful for studies of PD-related synaptic dysfunction and neurotransmitter dynamics as well as studies of the normal and pathogenic functions of these genes with PD-linked mutations.

Effect of parasitic infection on dopamine biosynthesis in dopaminergic cells

Infection by the neurotropic agent Toxoplasma gondii alters rodent behavior and can result in neuropsychiatric symptoms in humans. Little is understood regarding the effects of infection on host neural processes but alterations to dopaminergic neurotransmission are implicated. We have previously reported elevated levels of dopamine (DA) in infected dopaminergic cells however the involvement of the host enzymes and fate of the produced DA were not defined. In order to clarify the effects of infection on host DA biosynthetic enzymes and DA packaging we examined enzyme levels and activity and DA accumulation and release in T. gondii-infected neurosecretory cells. Although the levels of the host tyrosine hydroxylase (TH) and DOPA decarboxylase and AADC (DDC) did not change significantly in infected cultures, DDC was found within the parasitophorous vacuole (PV), the vacuolar compartment where the parasites reside, as well as in the host cytosol in infected dopaminergic cells. Strikingly, DDC was found within the intracellular parasite cysts in infected brain tissue. This finding could provide some explanation for observations of DA within tissue cysts in infected brain as a parasite-encoded enzyme with TH activity was also localized within tissue cysts. In contrast, cellular DA packaging appeared unchanged in single-cell microamperometry experiments and only a fraction of the increased DA was accessible to high potassium-induced release. This study provides some understanding of how this parasite produces elevated DA within dopaminergic cells without the toxic ramifications of free cytosolic DA. The mechanism for synthesis and packaging of DA by T. gondii-infected dopaminergic cells may have important implications for the effects of chronic T. gondii infection on humans and animals.

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.

All polymer chip for amperometric studies of transmitter release from large groups of neuronal cells

We present an all polymer electrochemical chip for simple detection of transmitter release from large groups of cultured PC 12 cells. Conductive polymer PEDOT:tosylate microelectrodes were used together with constant potential amperometry to obtain easy-to-analyze oxidation signals from potassium-induced release of transmitter molecules. The nature of the resulting current peaks is discussed, and the time for restoring transmitter reservoirs is studied. The relationship between released transmitters and potassium concentration was found to fit to a sigmoidal dose-response curve. Finally, we demonstrate how the presented device can be used for simple drug screening purposes, by measuring the increase of transmitter release due to short-term treatment with L-DOPA.

Inhibition of glucosylceramide synthase reversibly decreases the capsaicin-induced activation and TRPV1 expression of cultured dorsal root ganglion neurons

Recent studies have demonstrated significant changes in the neuronal ganglioside status associated with altered functional states of nociceptive primary sensory neurons. In the present study, therefore, the effects of the inhibition of glucosylceramide synthase, the key enzyme of ganglioside synthesis, were studied on chemically defined populations and on the activation of TRPV1 of cultured adult rat sensory ganglion neurons. In control cultures, capsaicin resulted in the activation of TRPV1 in 29.7 ± 2.5% of the neurons, as assessed with the cobalt uptake assay. Pretreatment of the cultures for 4 days with an inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( d-PDMP), significantly decreased the proportion of capsaicin-activated neurons to 11.6 ± 1.2%. Immunohistochemistry demonstrated that, in control cultures, 37.5 ± 1.4% of the neurons displayed TRPV1 immunoreactivity, whereas in d-PDMP-treated cultures the proportion of TRPV1-immunoreactive neurons was diminished to 18.2 ± 2.1%. Further experiments disclosed that these effects of d-PDMP were reversible. The capsaicin-, but not the high potassium-induced release of CGRP, was also significantly reduced after d-PDMP treatment, as measured with ELISA. The proportions of IB4- and CGRP-positive neurons were not significantly affected by d-PDMP. The present observations demonstrate that inhibition of neuronal ganglioside synthesis profoundly modulates the expression of the TRPV1 receptor, apparently leaving other markers of nociceptive neurons, such as CGRP and IB4, unaffected. The findings indicate that as yet unidentified ganglioside(s) synthesized by the glucosylceramide synthase pathway may be essential for nociception through mechanisms which may implicate membrane lipid raft function and/or altered nerve growth factor signaling, which are essential for the TRPV1 receptor function.

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [ 3 H ]glutamate by synaptosomes, Na +-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na +-dependent glutamate uptake into astrocytes, respectively. GA (1–100 nM) significantly stimulated [ 3 H ]glutamate binding to brain plasma membranes (40–70%) in the absence of extracellular Na + concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [ 3 H ]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na +-dependent [ 3 H ]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 μM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.

In vitro effects of d-2-hydroxyglutaric acid on glutamate binding, uptake and release in cerebral cortex of rats

Neurological dysfunction is common in patients with d-2-hydroxyglutaric aciduria (DHGA). However, the mechanisms underlying the neuropathology of this disorder are far from understood. In the present study, we investigated the in vitro effects of d-2-hydroxyglutaric acid (DGA) at various concentrations (0.1–1.0 mM) on various parameters of the glutamatergic system, namely the basal and potassium-induced release of l-[ 3H]glutamate by synaptosomal preparations, Na +-dependent l-[ 3H]glutamate uptake by synaptosomal preparations and Na +-independent l-[ 3H]glutamate uptake by synaptic vesicles, as well as of Na +-independent and dependent l-[ 3H]glutamate binding to synaptic plasma membranes from cerebral cortex of male adult Wistar rats. We observed that DGA significantly increased synaptosomal l-[ 3H]glutamate uptake, without altering the other parameters. Although these findings do not support a direct excitotoxic action for DGA since the metabolite did not affect important parameters of the main neurotransmission system, they do not exclude a direct action of DGA on NMDA or other glutamate receptors. More comprehensive studies are therefore necessary to evaluate the exact role of DGA on neurotransmission.

3-Hydroxyglutaric acid enhances glutamate uptake into astrocytes from cerebral cortex of young rats

A predominantly neurological presentation is common in patients with glutaric acidemia type I (GA-I). 3-Hydroxyglutaric acid (3-OHGA), which accumulates in affected patients, has recently been demonstrated to play a central role in the neuropathogenesis of this disease. In the present study, we investigated the in vitro effects of 3-OHGA at concentrations ranging from 10 to 1000 μM on various parameters of the glutamatergic system, such as the basal and potassium-induced release of [ 3H]glutamate by synaptosomes, as well as on Na +-dependent [ 3H]glutamate uptake by synaptosomes and astrocytes and Na +-independent [ 3H]glutamate uptake by synaptic vesicles from cerebral cortex of 30-day-old Wistar rats. First, we observed that exposure of cultured astrocytes to 3-OHGA for 20 h did not reduce their viability. Furthermore, 3-OHGA significantly increased Na +-dependent [ 3H]glutamate uptake by astrocytes by up to 80% in a dose-dependent manner at doses as low as 30 μM. This effect was not dependent on the presence of the metabolite during the uptake assay, since it occurred even when 3-OHGA was withdrawn from the medium after cultured cells had been exposed to the acid for approximately 1 h. All other parameters investigated were not influenced by this organic acid, indicating a selective action of 3-OHGA on astrocyte transporters. Although the exact mechanisms involved in 3-OHGA-stimulatory effect on astrocyte glutamate uptake are unknown, the present findings contribute to the understanding of the pathophysiology of GA-I, suggesting that astrocytes may protect neurons against excitotoxic damage caused by 3-OHGA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.

Effects of L-2-hydroxyglutaric acid on various parameters of the glutamatergic system in cerebral cortex of rats.

L-2-Hydroxyglutaric acid (LGA) accumulates and is the biochemical hallmark of the neurometabolic disorder L-2-hydroxyglutaric aciduria (LHGA). Although this disease is predominantly characterized by severe neurological findings and pronounced cerebral atrophy, the pathomechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of LGA (0.1-1 mM) on various parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomal preparations, Na(+)-dependent L-[3H]glutamate uptake by synaptosomal preparations and Na(+)-independent L-[3H]glutamate uptake by synaptic vesicles, as well as of L-[3H]glutamate binding to synaptic plasma membranes from cerebral cortex of male adult Wistar rats. We observed that LGA significantly increased L-[3H]glutamate uptake into synaptosomes and synaptic vesicles, without altering synaptosomal glutamate release and glutamate binding to synaptic plasma membranes. Although more comprehensive studies are necessary to evaluate the exact role of LGA on neurotransmission, our findings do not support a direct excitotoxic action for LGA. Therefore, other abnormalities should be searched for to explain neurodegeneration of LHGA.

Mechanisms of the Release of Anterogradely Transported Neurotrophin-3 from Axon Terminals

Neurotrophins have profound effects on synaptic function and structure. They can be derived from presynaptic, as well as postsynaptic, sites. To date, it has not been possible to measure the release of neurotrophins from axon terminals in intact tissue. We implemented a novel, extremely sensitive assay for the release and transfer of anterogradely transported neurotrophin-3 (NT-3) from a presynaptic to a postsynaptic location that uses synaptosomal fractionation after introduction of radiolabeled NT-3 into the retinotectal projection of chick embryos. Release of the anterogradely transported NT-3 in intact tissue was assessed by measuring the amount remaining in synaptosomal preparations after treatment of whole tecta with pharmacological agents. Use of this assay reveals that release of NT-3 from axon terminals is increased by depolarization, calcium influx via N-type calcium channels, and cAMP analogs, and release is most profoundly increased by excitation with kainic acid or mobilization of calcium from intracellular stores. NT-3 release depends on extracellular sodium, CaM kinase II activity, and requires intact microtubules and microfilaments. Dantrolene inhibits the high potassium-induced release of NT-3, indicating that release of calcium from intracellular stores is required. Tetanus toxin also inhibits NT-3 release, suggesting that intact synaptobrevin or synaptobrevin-like molecules are required for exocytosis. Ultrastructural autoradiography and immunolabel indicate that NT-3 is packaged in presumptive large dense-core vesicles. These data show that release of NT-3 from axon terminals depends on multiple regulatory proteins and ions, including the mobilization of local calcium. The data provide insight in the mechanisms of anterograde neurotrophins as synaptic modulators.

Effects of methylmalonic and propionic acids on glutamate uptake by synaptosomes and synaptic vesicles and on glutamate release by synaptosomes from cerebral cortex of rats

Neurological dysfunction is common in patients with methylmalonic and propionic acidemias. However, the mechanisms underlying the neuropathology of these disorders are far from understood. In the present study we investigated the in vitro effects of methylmalonic (MMA) and propionic (PA) acids at various concentrations (1 μM–5 mM) on three parameters of the glutamatergic system, namely the basal and potassium-induced release of l-[ 3H]glutamate by synaptosomes, Na +-dependent l-[ 3H]glutamate uptake by synaptosomes and Na +-independent l-[ 3H]glutamate uptake by synaptic vesicles from cerebral cortex of male adult Wistar rats. The results showed that MMA significantly increased potassium-induced but not basal l-[ 3H]glutamate release from synaptosomes with no alteration in synaptosomal l-[ 3H]glutamate uptake. A significant reduction of l-[ 3H]glutamate incorporation into vesicles caused by MMA was also detected. In contrast, PA had no effect on these parameters. These findings indicate that MMA alters the glutamatergic system. Although additional studies are necessary to evaluate the importance of these observations for the neuropathology of methylmalonic acidemia, it is possible that the effects elicited by MMA may lead to excessive glutamate concentrations at the synaptic cleft, a fact that may explain previous in vivo and in vitro findings associating MMA with excitotoxicity.

Changes in spinal cholecystokinin release after peripheral axotomy.

The gene expression of cholecystokinin (CCK), a neuropeptide with anti-opioid properties, has been reported to be upregulated in some primary sensory neurons after a peripheral nerve lesion. We have recently demonstrated that the upregulation of CCK mRNA is not accompanied by an increased potassium-evoked release CCK-like immunoreactivity (CCK-LI) 2-4 weeks after a complete transection of the sciatic nerve. The potassium-evoked release of CCK-LI at earlier and later time points has, however, not been studied. The aim of the present in vivo microdialysis study was to monitor how the basal and stimulated extracellular level of CCK in the dorsal horn of the spinal cord is affected at various time points after a complete transection of the sciatic nerve (axotomy). During the first week after transection of the sciatic nerve a tendency towards an elevation of the potassium-induced (100 mM in the perfusion fluid) release of spinal CCK-LI was observed. In contrast, no potassium-induced release of CCK-LI could be detected 2-3 weeks and 2 months after axotomy. No significant effect was observed on the basal extracellular levels of CCK-LI in the dorsal horn. The present study provides further support for the notion that the adaptive changes in the dorsal horn 2 weeks and later after a deafferentiation injury do not include an increased release of CCK.

Peripheral axotomy increases cholecystokinin release in the rat anterior cingulate cortex

The anterior cingulate cortex (ACC) is a limbic region with a high density of cholecystokinin (CCK) immunoreactivity, that has been suggested to be of importance for the affective and emotional component of pain. In the present microdialysis study, performed in the awake rat, we demonstrate a bilateral 4- to 6-fold increase of the potassium-induced release of CCK-like immunoreactivity (CCK-LI) in the ACC 2-3 weeks after a unilateral transection of the sciatic nerve (axotomy), an animal model of phantom limb or deafferentiation pain. Considering the implication of CCK in pain modulation and anxiety, we suggest that an altered activity of CCK containing neurons in the ACC may be of importance for the affective component of certain pain conditions.

Capsaicin-stimulated release of substance P from cultured dorsal root ganglion neurons: involvement of two distinct mechanisms

Capsaicin, the pungent component of “hot” chilli peppers, selectively activates a distinct population of primary sensory neurons responsive to noxious stimuli. Many of these fibres express neuropeptides including the tachykinin, substance P. Using cultured dorsal root ganglion neurons, we found that capsaicin (10 μM) stimulated a 2-fold increase in release of substance P in the absence of extracellular Ca 2+. Elevated potassium (75 mM) was unable to induce release under these conditions. The introduction of Ca 2+ enhanced capsaicin-induced release and brought about a robust response to potassium. Preincubation of cells with botulinum neurotoxin A (100 nM) completely blocked potassium-induced release but the capsaicin response, in the absence of Ca 2+, was unaffected. However, toxin treatment dramatically reduced capsaicin-stimulated release in the presence of Ca 2+. It is concluded that capsaicin induces release of substance P from dorsal root ganglion neurons via two mechanisms, one requiring extracellular Ca 2+ and the intact synaptosomal-associated protein 25 kDa (SNAP-25), and the other independent of extracellular Ca 2+ and not involving SNAP-25.

Differences in release and clearance of extracellular dopamine in the striatum after spontaneous or GM1-ganglioside-stimulated recovery from experimental Parkinsonism

Purpose: This study was designed to assess differences in dopamine clearance rates and potassium chloride (KCl)-stimulated release in the striatum of cats that had either spontaneously recovered from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinsonism or recovered after receiving GM1 ganglioside treatment. Methods: A severe Parkinsonian motor disorder was produced in 17 adult cats by administration of MPTP for seven to ten days. Six MPTP-treated cats received daily GM1 administration (30 mg/kg, i.m.) for 6 weeks and eleven MPTP-treated cats were allowed to spontaneously recover over the same period of time. High-speed chronoamperometric electrochemical measurements were obtained from dorsal and ventral striatal regions in all animals. Dopamine clearance rates were obtained by measuring the clearance of pressure-ejected dopamine from the extracellular space and local potassium-induced release was studied by applying KCl to the tissue. Results: Dopamine clearance rates recorded in all striatal areas in GM1-treated cats were significantly faster than dopamine clearance rates recorded in spontaneously recovered cats. In GM1-treated animals, electrochemical signals recorded in response to KCl stimulation were sig-nificantly greater in all striatal areas compared to spontaneously recovered animals. Reduction/oxidation (redox) ratios recorded in GM1- treated animals indicated dopamine to be the predominant electroactive species released in all striatal areas in response to KCl stimulation. Redox ratios recorded in the ventral striatum of spontaneously recovered cats also indicated dopamine to be the predominant electroactive spe-cies released in response to KCl stimulation. However, redox ratios recorded in the dorsal striatum of spontaneosuly recovered cats indicated serotonin to significantly contribute to the recorded signal. Conclusions: These results support previous observations that volume transmission may predominate dopaminergic signaling in the stria-tum of spontaneously recovered cats and suggest that a greater degree of synaptic transmission is possible in GM1-treated animals. While the functional significance of this partial restoration of dopaminergic synaptic transmission in the striatum remains to be determined, it may under-lie improved behavioral recovery observed following GM1 treatment.

Endogenous GABA potentiates the potassium-induced release of dopamine in striatum of the freely moving rat: a microdialysis study

Using microdialysis, a study was made of the effects of an increase of endogenous GABA on basal and potassium-stimulated release of dopamine in striatum of the awake rat. The dopamine metabolites DOPAC and HVA were also measured. Extracellular concentrations of GABA were increased by inhibiting its uptake with nipecotic acid. TTX (10 μM) reduced basal extracellular concentrations of dopamine, and dopamine metabolites, but not GABA. Nipecotic acid (200, 500, and 1000 μM) produced a dose-related increase in basal extracellular concentrations of GABA, but did not change basal extracellular concentrations of dopamine and dopamine metabolites. However, nipecotic acid significantly enhanced the dopamine release produced by perfusion of potassium (50 mM) and also enhanced the extracellular increase of GABA produced by high potassium. These results suggest that an increase of endogenous GABA is facilitating the stimulated release, but not the basal release, of dopamine in the striatum of the awake rat.

Effects of mild protein prenatal malnutrition and subsequent postnatal nutritional rehabilitation on noradrenaline release and neuronal density in the rat occipital cortex

There is evidence that severe malnutrition started during gestation and continued through lactation affects adversely the morphologic development of the neocortex leading to increased neuronal cell packing density and decreased dendritic branching. Nevertheless, the effect of purely mild protein prenatal malnutrition on neocortical development remains rather unexplored. This study evaluates the effects of mild protein prenatal malnutrition (8% casein diet, calorically compensated by carbohydrates) and subsequent postnatal nutritional rehabilitation (25% casein diet) on: (i) the potassium-induced release of [ 3 H ]-noradrenaline (NA) in occipital cortex slices obtained from 1- and 22-day-old pups; and (ii) the packing density of neurons in lateral, dorso-lateral and dorsal regions of the occipital cortex of 22-day-old pups by using the optical dissector method. The experiments were performed in rats normally fed during gestation and lactation (G +L +), malnourished during gestation but rehabilitated during lactation (G −L +) and malnourished during gestation and lactation (G −L −). At day 1 of age, no significant differences in body and brain weights were observed between prenatally well-nourished and malnourished pups. Nevertheless, at this early age, pups born from mothers submitted to the 8% casein diet had significantly higher cortical net percent NA release than pups born from mothers receiving the 25% casein diet. At weaning (22 days of age) G −L + rats had, compared to the G +L + group, similar body weight, brain weight and [ 3 H ]-NA release values, but significantly higher neuron density scores in the lateral region of the occipital cortex. In contrast, at 22 days of age, G −L − rats exhibited, compared to G +L + animals, significant deficits in body and brain weights as well as significant increases in cortical net percent NA release together with enhanced packing density of neurons in the lateral, dorso-lateral and dorsal regions of the occipital cortex. Moreover, in G −L − animals was not found the laterodorsal histogenetic gradient of neuronal cell packing density observed in G +L +rats. Results suggest that mild prenatal malnutrition per se is able to induce deleterious effects on cortical neuronal density, in spite of nutritional rehabilitation during lactation, through a mechanism involving central NA hyperactivity during gestation. Prosecution of malnutrition after birth magnifies both neurochemical and morphometric disorders.

Synaptic localization of the 67,000 mol. wt isoform of glutamate decarboxylase and transmitter function of GABA in the mouse cerebellum lacking the 65,000 mol. wt isoform

Subcellular localization of the 67,000 mol. wt isoform of glutamate decarboxylase and neurotransmitter function of GABA were investigated in the cerebellum of the mice lacking the 65,000 mol. wt isoform of glutamate decarboxylase. The GABA content decreased by 25% in the cerebellum. Putative GABA-releasing terminals from basket/stellate and Golgi cells were immunostained with glutamate decarboxylase-67 antibody. Basket cell-derived inhibitory postsynaptic currents in Purkinje cells and the high potassium-induced release of GABA were not significantly affected. Although previous investigations have suggested that glutamate decarboxylase-65 is mainly involved in transmitter synthesis and that glutamate decarboxylase-67 is transported to the nerve terminals only after association with glutamate decarboxylase-65, the present results indicate that glutamate decarboxylase-67 is independently concentrated in the nerve terminals and provides GABA for synaptic transmission in the absence of glutamate decarboxylase-65.

Mazindol, nomifensine and desmethylimipramine inhibit potassium-induced release of dopamine: effect of stimulus strength.

Catecholamine uptake blockers exhibit a second action: They suppress the potassium (K+)-stimulated release of [3H]-dopamine (DA) from rat striatal tissue in vitro. In the present study, a K+ dose-response curve was obtained for the release of both [3H]-DA and endogenous DA from striatal tissue, in the absence and presence of catecholamine uptake blockers. Three drugs were used, namely, mazindol, nomifensine, and desmethylimipramine. The data showed that diminished release in the presence of mazindol or nomifensine was dependent upon the concentration of K+. Marked inhibition of stimulus-induced release of either 3H-DA or endogenous DA was observed at low concentrations of K+ (15 and 20 mM), but not at higher concentrations of K+ (40 and 60 mM). In contrast, the diminished release of DA in the presence of desmethylimipramine persisted over the K+ range of 20-60 mM. These data show that the drug action observed previously was not restricted to a specific neuronal pool of DA that was labelled with [3H]-DA. Further, the data emphasize the importance of the concentration of the depolarizing stimulus when evaluating the effects of drugs on release of catecholamines.

Peripheral axotomy influences the in vivo release of cholecystokinin in the spinal cord dorsal horn—possible involvement of cholecystokinin-B receptors

An increased expression of cholecystokinin (CCK) messenger RNA (mRNA) as well as CCK-B receptor mRNA in dorsal root ganglion (DRG) cells following peripheral axotomy has previously been demonstrated. In the present in vivo microdialysis study, the effect of unilateral sciatic nerve section on basal and potassium-induced release of CCK-like (CCK-LI) immunoreactivity in the rat dorsal horn was investigated. We also compared the effects of the CCK-B receptor antagonist CI988 on basal and potassium-stimulated CCK-LI release in intact animals and in chronically axotomized rats. Perfusion of the microdialysis probe with KCl (100 mM) induced a more than 6-fold increase of the extracellular level of CCK-LI in control animals. In contrast, following unilateral sciatic nerve section the same KCl stimulation failed to evoke a release of CCK-LI ipsilaterally. However, after systemic administration of CI988 (1 mg kg −1, i.v.), 100 mM KCl induced a significant increase of the extracellular CCK-LI level in axotomized rats, similar to that observed in control animals. In control animals no effect of CI988 on KCl-stimulated CCK-LI release could be detected. CI988 by itself had no influence on the extracellular CCK-LI level in either nerve injured or control animals. The present data suggest that axotomy reduces the release of CCK-like immunoreactivity in the spinal cord by a mechanism involving the CCK-B receptor binding site.