Potassium-evoked Dopamine Research Articles

Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra.

Glial cell line-derived neurotrophic factor (GDNF) improves motor dysfunction associated with aging in rats and non-human primates, in animal models of Parkinson's disease, and may improve motoric function in patients with advanced Parkinson's disease. These improvements are associated with increased dopamine function in the nigrostriatal system, but the molecular events associated with this increase are unknown. In these studies, 100 micro g of GDNF was injected into the striatum of normal aged (24-month-old) male Fischer 344 rats. The protein levels and phosphorylation of TH, ERK1/2, and related proteins were determined by blot-immunolabeling of striatum and substantia nigra harvested 30 days after injection. In GDNF-treated rats, TH phosphorylation at Ser31 increased approximately 40% in striatum and approximately 250% in the substantia nigra. In the substantia nigra, there was a significant increase in ERK1 phosphorylation. In striatum, there was a significant increase in ERK2 phosphorylation. Microdialysis studies in striatum showed that both amphetamine- and potassium-evoked dopamine release in GDNF recipients were significantly increased. These data show that GDNF-induced increases in dopamine function are associated with a sustained increase in TH phosphorylation at Ser31, which is greatest in the substantia nigra and maintained for at least one month following a single striatal administration of GDNF. These findings, taken from the nigrostriatal system of normal aged rats, may help explain the long lasting effects of GDNF on dopamine function and prior studies supporting that a major effect of GDNF involves its effects on dopamine storage and somatodendritic release of dopamine in the substantia nigra.

Prenatal cocaine exposure alters potassium-evoked dopamine release dynamics in rat striatum

The emerging profile for the effects of prenatal cocaine exposure presents two prominent features in the exposed offspring: cognitive/attention deficits and an age-associated trend toward motor/tone abnormalities up to 2 years of age. One candidate mechanism underlying these clinical features is long-lasting alterations to dopamine (DA) neuron function. However, the impact of prenatal cocaine exposure on DA release in dopaminergic terminal fields in vivo in mature offspring is poorly understood. Long-Evans female rats were implanted with an i.v. access port, bred, and given saline or cocaine-HCl (3 mg/kg/ml) for gestational days (GD) 8–14 (1×/day), GD 15–21 (2×/day), or GD 8–21 (1×/day-GD 8–14, 2×/day-GD 15–21). Using in vivo high-speed chronoamperometric recordings, potassium-stimulated DA release was measured in striatum of anesthetized male offspring 90–150 days after birth. There was a trend toward increased potassium-evoked DA signal amplitudes in offspring exposed to cocaine at any time period examined. In offspring exposed to cocaine during GD 8–21 and GD 15–21, but not at GD 8–14, there were significant decreases in the clearance capacity of the potassium-evoked DA signal compared with control offspring. The time required to clear 80% of the evoked DA signal (T 80) in striatum for DA was significantly prolonged (approximately 150% of control) and this effect was further increased in the mean-evoked DA concentration range for these two groups. We also measured total dopamine transporter (DAT) and tyrosine hydroxylase protein levels in these offspring by blot immunolabeling and found a small, but significant, decrease in DAT protein in striatum from offspring exposed at GD 8–21 and GD 15–21. Collectively, these data demonstrate that prenatal cocaine exposure during dopamine neuron neurogenesis has long-lasting effects on DA neuron function lasting into early adulthood which may be related in part to steady state DAT protein levels. These molecular events may be associated with established cognitive deficits and perhaps the trends seen in altered motor behavior.

Ceramic-based multisite microelectrode array for rapid choline measures in brain tissue

A ceramic-based multisite microelectrode array to measure choline in vivo in brain tissues is described. The microelectrodes were linear to 200 μM choline ( R 2=0.999±0.001) with a detection limit of approximately 0.4 μM (S/N of 3) in both single microelectrode and self-referencing amperometric recording modes. The 90% rise time of the sensor was 1.4 s, allowing for rapid measures of choline. Good selectivity (>300:1) was observed over interferents such as ascorbic acid, uric acid, and DOPAC in the single microelectrode mode. However, a self-referencing recording mode was needed to remove potassium-evoked dopamine signals in rat striatum. In vivo measurements of choline in the rat brain are presented.

Methodology for coupling local application of dopamine and other chemicals with rapid in vivo electrochemical recordings in freely-moving rats

Methodology is presented for constructing and using an electrode/microcannulae assembly that allows in vivo electrochemical measurements coupled with local application of dopamine (DA) and other chemicals in the unanesthetized freely-moving rat. Rats were implanted with a voltammetric electrode constructed of a carbon fiber sealed in fused silica tubing attached to a pair of stainless steel guide cannulae, into which fused silica injection cannulae were inserted for local application of DA and other chemicals. Precise delivery of nanoliter volumes was accomplished using a syringe drive combined with a fluid swivel to deliver the solutions to the injection cannulae. A newly-designed miniature potentiostat connected to a commutator via a modular telephone jack assembly allowed for high-speed chronoamperometric electrochemical recordings in freely-moving rats. Initial experiments characterized the in vitro electrochemical recording characteristics of the voltammetric electrode. In vivo studies were also carried out to study clearance of locally-applied DA and of potassium-evoked endogenous DA in the striatum and nucleus accumbens of freely-moving rats. In addition, the effects of chloral hydrate anesthesia on DA clearance signals in the nucleus accumbens were investigated. Moreover, the stability and reproducibility of this recording technique for measuring exogenous DA clearance was verified over a period of 5 days. Finally, the concurrent effects of systemic cocaine injection on DA uptake in nucleus accumbens and locomotor activity were examined. These studies support the conclusion that the methodology described herein allows for rapid chronoamperometric electrochemical recordings in freely-moving rats with precise microapplications of DA and other chemicals combined with concurrent measures of animal behavior.

Recovery of high potassium-evoked dopamine release after depolarization challenge in the striatum of young and old male rats

The aim of this study was to assess the recovery of high potassium-evoked dopamine (DA) release after depolarization challenge in young (3–4 months) and old (21–25 months) male Wistar rats. Recovery of DA release was evaluated by comparison of the peak responses of DA release induced by two serial high potassium stimulations. Concentric microdialysis probes were stereotaxically implanted in the lateral striatum of rats, and microdialysis was commenced 24 h after surgery. Using a low flow rate of perfusion (1 μl/min), all rats received 2×20 min infusions of 100 mM potassium solution separated by either 60 or 140 min. No difference in the basal DA concentration or the potassium-evoked DA release or its recovery was seen between the two groups. Our results suggest that the vesicular DA store recovers rapidly after high potassium challenge in both young and old rats.

Isolation Rearing in Rats: Pre- and Postsynaptic Changes in Striatal Dopaminergic Systems

Isolation rearing of rats produces a behavioral syndrome indicative of altered dopamine (DA) function in the nucleus accumbens (NAC). The present experiments extend these findings by investigating: (a) interactions between isolation rearing and repeated handling/testing on presynaptic DA function in the NAC using in vivo microdialysis; (b) the dose–response curve for the effects of d-amphetamine, and the responses elicited by high potassium, using in vivo microdialysis; and (c) postsynaptic function in isolates as indexed by DA receptor-linked cAMP production. Experiment 1 showed that both isolation rearing and repeated handling/testing had effects on monoamine function in the NAC. However, while both manipulations enhanced DA release evoked by d-amphetamine, only isolated rats had elevated basal DA levels. Opposite neurochemical changes were observed with respect to the serotonin metabolite 5-HIAA, isolates having lower, and repeatedly handled/tested animals having higher, extracellular levels. Experiment 2 provided evidence for enhanced d-amphetamine–evoked DA release in isolated animals, while potassium-evoked DA release was reduced. Experiment 3 provided evidence that the isolation rearing induced changes in presynaptic DA function were accompanied by postsynaptic changes. Specifically, the inhibitory influence of the D 2 receptor on D 1 receptor-stimulated cAMP production was attenuated in ventral striatal slices taken from isolates, suggesting a functional downregulation of D 2 receptors.

Morphological and Functional Evidence for Enhanced Growth and Potassium-Evoked Dopamine Release in Striatal Grafts Innervated with a Patchy Growth Pattern. An In Oculo Nigrostriatal Cograft Study

During development of the nigrostriatal dopamine system, a patchy and a diffuse type of striatal innervation pattern can be seen. It has been suggested that when fetal dopaminergic neurons, obtained from the ventral mesencephalon (VM), are grafted adjacent to mature striatal tissue, only the diffuse growth is induced. Intraocular grafting studies have indicated that the dopaminergic growth pattern might be influenced by the age of the target area, the lateral ganglionic eminence (LGE). In this study VM grafts were allowed to innervate LGE grafts of different ages. Fetal VM was implanted next to 2-wk-old or 26-day-old striatal in oculo grafts, and the resulting dopaminergic innervation of the striatal grafts was studied using tyrosine hydroxylase (TH) immunohistochemistry. In striatal grafts receiving innervation at the age of 2 wk in oculo, a patchy TH-immunoreactive growth pattern was found, while in striatal grafts innervated at the age of 26 days mainly the diffuse growth pattern was seen. This implies that grafted striatum reached maturity at approximately 1 mo of age. The age of the dopaminergic neurons at dissection and grafting was also studied concerning the ability to induce patchy growth into mature striatum. Thus, VM dissected from 13- and 18-mm fetuses was implanted to either 4-mo-old LGE (grafted in sequence) or to LGE from the same fetus (grafted simultaneously) as controls. TH-positive innervation of striatal tissue, evaluated 4 wk after implantation of VM, revealed a patchy growth pattern in LGE grafted simultaneously with 13- and 18-mm VM. However, when the striatum was mature at the time of innervation, diffuse growth was observed in striatum innervated by VM dissected from 13-mm fetuses. Interestingly, patchy growth was noted in striatal areas close to VM grafts when the dopaminergic neurons were derived from older fetuses (CRL 18 mm). Furthermore, potassium-induced dopamine release was greater in striatal grafts exhibiting the patchy growth than those showing the diffuse pattern of innervation. In conclusion, patchy dopaminergic growth can be induced in mature striatal tissue by grafting VM from older fetuses. Functionally, potassium-evoked dopamine release is enhanced in dopaminergic patches. These results have implications in terms of finding ways to induce patchy growth when grafting to the mature striatum of patients suffering from Parkinson’s disease.

Morphological and functional evidence for enhanced growth and potassium-evoked dopamine release in striatal grafts innervated with a patchy growth pattern. An in oculo nigrostriatal cograft study.

During development of the nigrostriatal dopamine system, a patchy and a diffuse type of striatal innervation pattern can be seen. It has been suggested that when fetal dopaminergic neurons, obtained from the ventral mesencephalon (VM), are grafted adjacent to mature striatal tissue, only the diffuse growth is induced. Intraocular grafting studies have indicated that the dopaminergic growth pattern might be influenced by the age of the target area, the lateral ganglionic eminence (LGE). In this study VM grafts were allowed to innervate LGE grafts of different ages. Fetal VM was implanted next to 2-wk-old or 26-day-old striatal in oculo grafts, and the resulting dopaminergic innervation of the striatal grafts was studied using tyrosine hydroxylase (TH) immunohistochemistry. In striatal grafts receiving innervation at the age of 2 wk in oculo, a patchy TH-immunoreactive growth pattern was found, while in striatal grafts innervated at the age of 26 days mainly the diffuse growth pattern was seen. This implies that grafted striatum reached maturity at approximately 1 mo of age. The age of the dopaminergic neurons at dissection and grafting was also studied concerning the ability to induce patchy growth into mature striatum. Thus, VM dissected from 13- and 18-mm fetuses was implanted to either 4-mo-old LGE (grafted in sequence) or to LGE from the same fetus (grafted simultaneously) as controls. TH-positive innervation of striatal tissue, evaluated 4 wk after implantation of VM, revealed a patchy growth pattern in LGE grafted simultaneously with 13- and 18-mm VM. However, when the striatum was mature at the time of innervation, diffuse growth was observed in striatum innervated by VM dissected from 13-mm fetuses. Interestingly, patchy growth was noted in striatal areas close to VM grafts when the dopaminergic neurons were derived from older fetuses (CRL 18 mm). Furthermore, potassium-induced dopamine release was greater in striatal grafts exhibiting the patchy growth than those showing the diffuse pattern of innervation. In conclusion, patchy dopaminergic growth can be induced in mature striatal tissue by grafting VM from older fetuses. Functionally, potassium-evoked dopamine release is enhanced in dopaminergic patches. These results have implications in terms of finding ways to induce patchy growth when grafting to the mature striatum of patients suffering from Parkinson's disease.

In vivo microdialysis studies on somatodendritic dopamine release in the rat substantia nigra: effects of unilateral 6-OHDA lesions and GDNF.

Dopamine (DA) release and metabolism within the substantia nigra (SN) were studied in normal rats, rats with unilateral 6-hydroxydopamine (6-OHDA) lesions, and 6-OHDA-lesioned rats treated with glial cells line-derived neurotrophic factor (GDNF). Animals with > 99% DA depletions, as determined by apomorphine-induced circling behavior, also showed significant deficits in several measures of spontaneous motor activity. In vivo microdialysis recordings in the SN were carried out in normal and unilaterally 6-OHDA-lesioned rats. Basal levels of DNA were detectable only in the dialysates of normal animals, and basal levels of t he primary DA metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were found to be significantly reduced in the SN of 6-OHDA-lesioned animals. In the presence of d-amphetamine, either alone or in combination with potassium, significant reductions in DA release were observed in the SN of 6-OHDA-lesioned animals compared to normal animals. Potassium-evoked DA release alone was not significantly different between the groups. A single intranigral administration of GDNF into 6-OHDA-lesioned animals elicited a significant reduction in apomorphine-induced rotation behavior and a significant increase in spontaneous motor activities. These behavioral changes were apparent at 1 week and persisted through 4 weeks following treatment. In vivo microdialysis showed that, although DA metabolism was altered 1 week following GDNF treatment, DA release was not significantly affected until 4 weeks following treatment.

Effects of drugs interfering with sodium channels and calcium channels on the release of endogenous dopamine from superfused substantia nigra slices.

The importance of voltage-dependent sodium channels and different types of voltage-sensitive calcium channels for depolarisation-induced release of endogenous dopamine from dendrites and cell bodies in superfused guinea pig substantia nigra slices was investigated. The stimulatory effect of veratridine (10 microM) on dopamine release was only marginally attenuated in Ca(2+)-free medium but was completely blocked by tetrodotoxin (1 microM) and by the dopamine reuptake inhibitor GBR 12909 (10 microM). Low extracellular concentration of Na+ stimulated the dopamine release. Potassium-evoked dopamine release was completely Ca(2+)-dependent, not blocked by GBR 12909 and partially blocked by tetrodotoxin. Nifedipine (20 microM), omega-conotoxin GVIA (0.5 microM), penfluridol (5 microM), and Ni2+ (20 microM) had no effect, amiloride (1 mM) attenuated and neomycin (350 microM), and omega-agatoxin IVA (1 microM) almost totally blocked the potassium-induced dopamine release. The results suggest that veratridine released dopamine mostly by reversing the dopamine transporter. High concentrations of potassium induced release of nigral dopamine by opening of voltage-sensitive calcium channels of P/Q type but not L-type, N-type and probably not T-type. The depolarisation evoked by high concentrations of potassium seems to open voltage-sensitive calcium channels both by the depolarisation induced by potassium per se and by the secondary depolarisation induced by opening of voltage-dependent sodium channels.

Continued presence of intrastriatal but not intraventricular polymer-encapsulated PC12 cells is required for alleviation of behavioral deficits in Parkinsonian rodents

To date, few studies have systematically evaluated the most appropriate location for grafting catecholaminergic cells as a potential treatment for Parkinson's disease (PD). The following study was conducted to determine 1) if placement of catecholamine-secreting encapsulated PC12 cells into the lateral ventricle of 6-OHDAtreated rats is as effective as intrastriatal implants on reducing apomorphine-induced rotational behavior, and 2) to determine if the survival of encapsulated PC12 cells is differentially affected by the implant site. Polymer-encapsulated PC12 cells were implanted into either the striatum or lateral ventricle of unilateral 6-OHDA-lesioned rats. Animals were tested for apomorphine-induced rotations over a 6-wk period. Only those animals that received intrastriatal implants of encapsulated PC12 cells showed a reduction in rotation behavior. Moreover, removal of the devices from the striatum resulted in a return to preimplant rotation levels. Postexplant neurochemical analyses demonstrated that the potassium-evoked L-dopa device output increased in vivo while the potassium-evoked dopamine output from the devices decreased over time in vivo. The location of the implant significantly affected catecholamine output from the PC12 cell-loaded devices. The increase in potassium-evoked L-dopa output was greatest, as was the decrease in potassium-evoked dopamine output, from those devices implanted in the striatum. Basal output of dopamine and DOPAC was also significantly higher from devices explanted from the lateral ventricle. These results demonstrate that the continued presence of intrastriatal implants of encapsulated PC12 cells is required to maintain the behavioral effects in 6-OHDA-lesioned rats. In addition, the site of implantation appears to affect device output. These results provide additional support for intrapa-renchymal delivery of L-dopa and dopamine via polymer encapsulation as a possible treatment for PD.

Continued presence of intrastriatal but not intraventricular polymer-encapsulated PC12 cells is required for alleviation of behavioral deficits in Parkinsonian rodents.

To date, few studies have systematically evaluated the most appropriate location for grafting catecholaminergic cells as a potential treatment for Parkinson's disease (PD). The following study was conducted to determine 1) if placement of catecholamine-secreting encapsulated PC12 cells into the lateral ventricle of 6-OHDA-treated rats is as effective as intrastriatal implants on reducing apomorphine-induced rotational behavior, and 2) to determine if the survival of encapsulated PC12 cells is differentially affected by the implant site. Polymerencapsulated PC12 cells were implanted into either the striatum or lateral ventricle of unilateral 6-OHDA-lesioned rats. Animals were tested for apomorphine-induced rotations over a 6-wk period. Only those animals that received intrastriatal implants of encapsulated PC12 cells showed a reduction in rotation behavior. Moreover, removal of the devices from the striatum resulted in a return to preimplant rotation levels. Postexplant neurochemical analyses demonstrated that the potassium-evoked L-dopa device output increased in vivo while the potassium-evoked dopamine output from the devices decreased over time in vivo. The location of the implant significantly affected catecholamine output from the PC12 cell-loaded devices. The increase in potassium-evoked L-dopa output was greatest, as was the decrease in potassium-evoked dopamine output, from those devices implanted in the striatum. Basal output of dopamine and DOPAC was also significantly higher from devices explanted from the lateral ventricle. These results demonstrate that the continued presence of intrastriatal implants of encapsulated PC12 cells is required to maintain the behavioral effects in 6-OHDA-lesioned rats. In addition, the site of implantation appears to affect device output. These results provide additional support for intraparenchymal delivery of L-dopa and dopamine via polymer encapsulation as a possible treatment for PD.

In VivoElectrochemical Studies of the Dynamic Effects of Locally Applied Excitatory Amino Acids in the Striatum of the Anesthetized Rat

The actions of locally applied excitatory amino acids (EAAs) on dopamine nerve terminals in the striatum of the urethane-anesthetized rat were investigated. Rapid (5 Hz)in vivoelectrochemical recording was used to measure the amplitude and duration of dopamine (DA) overflow elicited by the local application of glutamate,N-methyl-d-aspartate (NMDA), kainate, quisqualate, quinolinate anddl-α-amino-3-hydroxy-5-methylisoxazolepropionic acid. EAAs were pressure ejected into the striatum via a pipette positioned 300 ± 30 μm away from the electrochemical working electrode. Brief (5 s) applications of the EAA agonists directly elicited DA-like electrochemical signals with amplitudes averaging about 2 μM.In some instances, putative increases in ascorbic acid-like signals were detected. Repeated applications of glutamate agonists in the same brain site resulted in diminished electrochemical responses, compared to the complete reproducibility seen after repeated applications of 120 mMpotassium. Low doses of NMDA (10 μMbarrel conc), which did not cause a detectable increase in the electrochemical baseline signal, significantly potentiated (50%) potassium-evoked DA overflow. These results indicate that low levels of endogenously released glutamate may modulate overflow when DA nerve terminals are depolarized, while higher concentrations of glutamate may directly elicit increases in extracellular levels of DA and/or ascorbic acid.

Nitric oxide inhibits the release of norepinephrine and dopamine from the medial basal hypothalamus of the rat.

Previous research indicates that norepinephrine and dopamine stimulate release of luteinizing hormone (LH)-releasing hormone (LHRH), which then reaches the adenohypophysis via the hypophyseal portal vessels to release LH. Norepinephrine exerts its effect via alpha 1-adrenergic receptors, which stimulate the release of nitric oxide (NO) from nitricoxidergic (NOergic) neurons in the medial basal hypothalamus (MBH). The NO activates guanylate cyclase and cyclooxygenase, thereby inducing release of LHRH into the hypophyseal portal vessels. We tested the hypothesis that these two catecholamines modulate NO release by local feedback. MBH explants were incubated in the presence of sodium nitroprusside (NP), a releaser of NO, and the effect on release of catecholamines was determined. NP inhibited release of norepinephrine. Basal release was increased by incubation of the tissue with the NO scavenger hemoglobin (20 micrograms/ml). Hemoglobin also blocked the inhibitory effect of NP. In the presence of high-potassium (40 mM) medium to depolarize cell membranes, norepinephrine release was increased by a factor of 3, and this was significantly inhibited by NP. Hemoglobin again produced a further increase in norepinephrine release and also blocked the action of NP. When constitutive NO synthase was inhibited by the competitive inhibitor NG-monomethyl-L-arginine (NMMA) at 300 microM, basal release of norepinephrine was increased, as was potassium-evoked release, and this was associated in the latter instance with a decrease in tissue concentration, presumably because synthesis did not keep up with the increased release in the presence of NMMA. The results were very similar with dopamine, except that reduction of potassium-evoked dopamine release by NP was not significant. However, the increase following incubation with hemoglobin was significant, and hemoglobin, when incubated with NP, caused a significant elevation in dopamine release above that with NP alone. In this case, NP increased tissue concentration of dopamine along with inhibiting release, suggesting that synthesis continued, thereby raising the tissue concentration in the face of diminished release. When the tissue was incubated with NP plus hemoglobin, which caused an increase in release above that obtained with NP alone, the tissue concentration decreased significantly compared with that in the absence of hemoglobin, indicating that, with increased release, release exceeded synthesis, causing a fall in tissue concentration. When NO synthase was blocked by NMMA, the release of dopamine, under either basal or potassium-evoked conditions, was increased. Again, in the latter instance the tissue concentration declined significantly, presumably because synthesis did not match release. Therefore, the results were very similar with both catecholamines and indicate that NO acts to suppress release of both amines. Since both catecholamines activate the release of LHRH, the inhibition of their release by NO serves as an ultra-short-loop negative feedback by which NO inhibits the release of the catecholamines, thereby reducing the activation of the NOergic neurons and decreasing the release of LHRH. This may be an important means for terminating the pulses of release of LHRH, which generate the pulsatile release of LH that stimulates gonadal function in both male and female mammals.

Effects of Glial Cell Line-Derived Neurotrophic Factor on Developing and Mature Ventral Mesencephalic Grafts in Oculo

The search for trophic factors that can support injured dopaminergic neurons and can enhance dopaminergic graft survival and outgrowth for therapeutic uses in Parkinson's disease has lately focused on members of the transforming growth factor (TGF) β superfamily. In this paper we have studied the effects of a member of the TGBβ family, glial cell line-derived neurotrophic factor (GDNF), on immature and mature ventral mesencephalic tissue grafted to the anterior chamber of the eye. The results confirm that GDNF increases survival of TH-positive neurons and enhances TH-immunoreactive nerve fiber formation when the grafts are treated during their development. The distribution of nerve terminals is densest within the area of TH-immunoreactive neurons and at the surface of the grafts. However, there is no change in the number of calcium-binding protein (CaBP)-positive neurons, suggesting that the subpopulation of TH-positive neurons that is increased are the CaBP-negative neurons of the ventral tier of pars compacta. Terminals from those neurons form the striatal patches during normal development. When the grafts are treated with GDNF after maturation, no change in TH-positive cell survival is seen but an increase of nerve terminals is still found within the cell dense area of the graft. Potassium-evoked dopamine release, measured using in vivo chronoamperometry, revealed significantly increased extracellular overflow in transplants treated with GDNF during development. The dopamine uptake blocker nomifensine significantly increased the time for clearance of the released dopamine. These data suggest that GDNF treatment of immature grafts enhances survival of TH-positive neurons, which would have innervated the striatal patches, and also increases TH-immunoreactive nerve fiber formation and dopamine release. Furthermore, GDNF treatment of mature grafts also increases dopamine fiber formation within the TH-positive neuronal area, indicating that adult dopaminergic neurons are also responsive to this agent.

Calcium Dependency and Tetrodotoxin Sensitivity of Neostriatal Dopamine Release in 5‐Day‐Old and Adult Rats as Measured by In Vivo Microdialysis

The calcium dependency and tetrodotoxin sensitivity of extracellular dopamine levels were assessed by microdialysis in the neostriatum of 5-day-old rat pups and were compared with those obtained in adult rats. The removal of calcium from the dialysate reduced spontaneous levels of extracellular dopamine to 20% of normal in the 5-day-old pups and to 10% of normal in the adults. Calcium-free dialysate also decreased potassium-evoked dopamine release to approximately 20% of baseline in both ages. Furthermore, the addition of tetrodotoxin to the dialysate decreased spontaneous levels of extracellular dopamine to 10% of baseline in both ages. The effects of calcium removal and the addition of tetrodotoxin on extracellular levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid were less pronounced. The results of this study demonstrate that extracellular levels of dopamine sampled by microdialysis in rats as young as 5 days of age are both calcium dependent and tetrodotoxin sensitive; thus, they are derived from neuronal activity and not from injury caused by acute implantation of the probe. Other age-related differences support the hypothesis that dopamine release and turnover is greater in immature rats and may represent a form of compensation for incomplete dopamine nerve terminal ingrowth.

Regional effects of aging on dopaminergic function in the Fischer-344 rat

To examine the consequences of aging on nigrostriatal (A9) and mesolimbic (A 10) dopamine (DA) function, neurochemical and behavioral measurements were performed in male Fischer-344 rats 6, 12, 18, 24, and 30 months old. Regional analyses of dopaminergic overflow and uptake processes were examined using high-speed (5 Hz) in vivo electrochemical recordings and local drug application techniques. When potassium was used to elicit the presynaptic overflow of dopamine (DA) in striatal areas predominantly innervated by the substantia nigra, the amplitude of DA overflow was significantly lower in 24- and 30-month-old rats ( p < 0.05 and p < 0.01 vs. 6 months, respectively). Furthermore, in ventral striatum (including the nucleus accumbens) which is innervated primarily by A 10 DA cell bodies, potassium-evoked DA overflow release amplitudes were significantly lower in the 18, 24, and 30 month groups ( p < 0.01 vs. 6 months). In addition, age-related differences between the dorsal and ventral striatum were found in a preliminary investigation of DA diffusion and clearance. Local application of nomifensine, a DA uptake inhibitor, significantly increased ( p < 0.05 vs. control) the amplitude of the signal recorded after local application of 25–30 pmol DA in the ventral striatum of 6 month-old but not 24-month-old rats. High-performance liquid chromatography coupled with electrochemical detection (HPLC-EC) was used to analyze whole striatal DA levels, DA metabolite levels and turnover indices. However, no age-related differences in any of these variables were observed. Finally, a rod walking test was used to measure motor coordination and balance in animals prior to in vivo electrochemical recording. Rats in the 24- and 30-month-old age groups were found to be impaired in this task. In summary, this study identified regional age-dependent changes in presynaptic DA overflow and uptake.

Estradiol Rapidly Stimulates Dopamine Release from the Posterior Pituitary in vitro

Dopamine (DA) from both the posterior pituitary (PP) and stalk-median eminence (SME) inhibits prolactin (PRL) secretion from the anterior pituitary. Estradiol participates in the regulation of PRL release, in part by modulating DA release from the SME. However, little is known concerning the effects of estradiol on the release of DA from the PP. The objective of this study was to examine whether estradiol rapidly affects the potassium-evoked release of endogenous DA from the PP and SME in vitro. Tissues were dissected from ovariectomized rats and allowed to equilibrate in media for 30 min. Two pulses of 28 mM K+, 3 min each, were then given 30 min apart. Test substances were added 20 min before the second stimulus. DA in the media was determined by HPLC. Estradiol, at a concentration of 1 and 10 nM, significantly stimulated the potassium-evoked DA release from the PP by 34 and 47%, respectively. This stimulation was specific since 17 alpha-estradiol, a biologically inactive isomer, and testosterone, were without effects. Estradiol did not alter DA release from either the SME or isolated neural lobes of the PP. Naloxone, an opioid receptor antagonist, abolished the estradiol-induced stimulation of DA release from the PP. In contrast, amphetamine, a DA-releasing agent, significantly increased DA release in the presence of naloxone. In conclusion, (1) estradiol stimulates DA release from the PP but not the SME or neural lobe; this effect is rapid and stereospecific, and (2) the effects of estradiol appear to be mediated via an opioid(s) peptide(s) from the intermediate lobe.

In Vivo Mechanisms Underlying Dopamine Release from Rat Nigrostriatal Terminals: II. Studies Using Potassium and Tyramine

The brain microdialysis technique has been used to examine the in vivo effects of potassium and tyramine on dopamine (DA) release and metabolism in the striatum of halothane-anaesthetised rats. Increasing the concentration of potassium perfusing the dialysis probe (30-120 mM) induced a dose-related efflux of DA. A dose-related release of DA was also observed following addition of tyramine (1-100 microM) to the perfusing buffer. High concentrations of potassium were found to reduce the dialysate content of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid and the serotonin metabolite 5-hydroxyindoleacetic acid. No such effect was observed even when using the highest concentration of tyramine tested. Potassium-evoked DA release was facilitated by pretreatment with the DA uptake inhibitor nomifensine, was inhibited by depletion of extracellular calcium, and was not significantly affected by tetrodotoxin (TTX). The effect of tyramine on DA efflux was inhibited by nomifensine and was insensitive to both TTX and calcium depletion. These data suggest that potassium and tyramine induce release of DA via different mechanisms. Potassium-induced DA release involves a carrier-independent process and may utilise an exocytotic release mechanism. On the other hand, tyramine-induced DA release would appear to involve a carrier-dependent process. Depletion of vesicular stores of DA by pretreatment with reserpine did not significantly affect potassium-induced DA release, whereas a marked inhibition of the effects of tyramine was noted. However, in reserpinised animals the potassium-induced release of DA was inhibited by nomifensine, a result suggesting that a carrier-dependent release mechanism operates in the absence of vesicular DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Dopamine Release in vitro from the Posterior Pituitary by Opioid Peptides

Opioid peptides are present in both the posterior pituitary (PP) and stalk-median eminence (SME). Their effects on the dopaminergic neurons in the SME are well documented, but little is known concerning their role in the regulation of dopamine (DA) release from the PP. The objectives of this study were (1) to develop an in vitro method suitable for examining the regulation of endogenous DA release from PP and SME, and (2) to describe and compare the effects of selected opioid peptides on potassium-evoked DA release from these tissues. Tissues were dissected from ovariectomized rats and incubated in media. After equilibration, two pulses of 28 mM potassium (K+), 3 min each, were delivered 30 min apart. Test substances were administered 20 min before the second K+ stimulus. DA in the media was determined by high-performance liquid chromatography. Potassium at 28 and 56 mM elicited a marked increase in DA release from the PP and SME; this was abolished by the removal of calcium. The opioid receptor antagonist, naloxone, significantly increased the release of DA from both PP and SME by 55%. Dynorphin A elicited a significant inhibition of DA release from PP and SME by 33 and 50%, respectively. In contrast, methionine enkephalinamide decreased DA release from the SME by 50%, but was without effect in the PP. The release of DA from both PP and SME was significantly inhibited by beta-endorphin, and this was reversed by naloxone. However, beta-endorphin was fourfold more effective in the SME. N-acetyl-beta-endorphin did not alter DA release. (1) we have developed a simple and sensitive in vitro method for studying the effects of hormones and drugs on the release of endogenous DA from PP and SME; (2) tuberoinfundibular dopaminergic and tuberohypophyseal dopaminergic nerve terminals are subjected to a similar inhibitory control by endogenous opioid peptides, and (3) exogenously applied opioid peptides exert differential effects on the release of DA from SME and PP which could be attributable to a dissimilar distribution of opioid receptor subtypes in these two tissues.