Homeostatic regulation of excitatory synapses on striatal medium spiny neurons expressing the D2 dopamine receptor

Striatal medium spiny neurons (MSNs) are contacted by glutamatergic axon terminals originating from cortex, thalamus and other regions. The striatum is also innervated by dopaminergic (DAergic) terminals, some of which release glutamate as a co-transmitter. Despite evidence for functional DA release at birth in the striatum, the role of DA in the establishment of striatal circuitry is unclear. In light of recent work suggesting activity-dependent homeostatic regulation of glutamatergic terminals on MSNs expressing the D2 DA receptor (D2-MSNs), we used primary co-cultures to test the hypothesis that stimulation of DA and glutamate receptors regulates the homeostasis of glutamatergic synapses on MSNs. Co-culture of D2-MSNs with mesencephalic DA neurons or with cortical neurons produced an increase in spines and functional glutamate synapses expressing VGLUT2 or VGLUT1, respectively. The density of VGLUT2-positive terminals was reduced by the conditional knockout of this gene from DA neurons. In the presence of both mesencephalic and cortical neurons, the density of synapses reached the same total, compatible with the possibility of a homeostatic mechanism capping excitatory synaptic density. Blockade of D2 receptors increased the density of cortical and mesencephalic glutamatergic terminals, without changing MSN spine density or mEPSC frequency. Combined blockade of AMPA and NMDA glutamate receptors increased the density of cortical terminals and decreased that of mesencephalic VGLUT2-positive terminals, with no net change in total excitatory terminal density or in mEPSC frequency. These results suggest that DA and glutamate signaling regulate excitatory inputs to striatal D2-MSNs at both the pre- and postsynaptic level, under the influence of a homeostatic mechanism controlling functional output of the circuit.     

Colocalization of neurokinin-1, N-methyl-D-aspartate, and AMPA receptors on neurons of the rat nucleus tractus solitarii

Substance P (SP) and glutamate are implicated in cardiovascular regulation by the nucleus tractus solitarii (NTS). Our earlier studies suggest that SP, which acts at neurokinin 1 (NK1) receptors, is not a baroreflex transmitter while glutamate is. On the other hand, our recent studies showed that loss of NTS neurons expressing NK1 receptors leads to loss of baroreflex responses and increased blood pressure lability. Furthermore, studies have suggested that SP may interact with glutamate in the NTS. In this study, we sought to test the hypothesis that NK1 receptors colocalize with glutamate receptors, either N-methyl-d-aspartate (NMDA) receptors or AMPA receptors or both in the NTS. We performed double-label immunofluorescent staining for NK1 receptors and either N-methyl-d-aspartate receptor subunit 1 (NMDAR1) or AMPA specific glutamate receptor subunit 2 (GluR2) in the rat NTS. Because vesicular glutamate transporter 2 (VGLUT2) containing fibers are prominent in portions of the NTS where cardiovascular afferent fibers terminate, we also performed double-label immunofluorescent staining for NK1 receptors and VGLUT2. Confocal microscopic images showed that NK1 receptors-immunoreactivity (IR) and NMDAR1-IR colocalized in the same neurons in many NTS subnuclei. Almost all NTS neurons positive for NK1 receptor-IR also contained NMDAR1-IR, but only 53.4% to 74.8% of NMDAR1-IR positive neurons contained NK1 receptors-IR. NK1 receptor-IR and GluR2-IR also colocalized in many neurons in NTS subnuclei. A majority of NK1 receptor-IR positive NTS neurons also contained GluR2-IR, but only 45.8% to 73.9% of GluR2-IR positive NTS neurons contained NK1 receptors-IR. Our results also showed that fibers labeled for VGLUT2-IR were in close apposition to fibers and neurons labeled for NK1 receptor-IR. The data support our hypothesis, provide an anatomical framework for glutamate and SP interactions, and may explain the loss of baroreflexes when NTS neurons, which could respond to glutamate as well as SP, are killed.     

Coexistence of NMDA and AMPA receptor subunits with nNOS in the nucleus tractus solitarii of rat

We previously showed that most neuronal nitric oxide synthase (nNOS)-containing neurons in the nucleus tractus solitarii (NTS) contain NMDAR1, the fundamental subunit for functional N-methyl-D-aspartate (NMDA) receptors. Likewise, we found that almost all nNOS-containing neurons in the NTS contain GluR1, the calcium permeable AMPA receptor subunit. These data suggest that AMPA and NMDA receptors may colocalize in NTS neurons that contain nNOS. However, other investigators have suggested that non-NMDA receptors are located primarily on second-order neurons and NMDA receptors are located predominantly on higher-order neurons in NTS. We now seek to test the hypothesis that NMDA receptors, AMPA receptors and nNOS are colocalized in NTS cells. We performed triple fluorescent immunohistochemical staining of nNOS, NMDAR1 and GluR1, and performed confocal laser scanning microscopic analysis of the NTS. The distributions of nNOS immunoreactivity (IR), NMDAR1-IR and GluR1-IR in the NTS were similar to those we reported earlier. Superimposed images revealed that almost all NMDAR1-IR cells contained GluR1-IR and almost all GluR1-IR cells contained NMDAR1-IR. Some double-labeled cells were additionally labeled for nNOS-IR. All nNOS-IR neurons contained both GluR1-IR and NMDAR1-IR. These studies support our hypothesis that NMDA and AMPA receptors are colocalized in NTS neurons and are consistent with a role of both types of ionotropic receptors in transmission of afferent signals in NTS. In addition, these data provide support for an anatomical link between ionotropic glutamate receptors and nitric oxide in the NTS.     

The effect of heliox treatment in a rat model of focal transient cerebral ischemia

At nerve terminals G protein coupled receptors modulate neurotransmitter release probability. We recently showed that prolonged activation of metabotropic glutamate receptor 7, mGlu7 receptor, potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein, the hydrolysis of phosphatidylinositol (4,5)-bisphosphate, and the subsequent activation of the non-kinase diacylglycerol binding protein Munc13-1 which primes synaptic vesicle for exocytosis at the active zone. Here we found that inhibitors of diacylglycerol metabolism (diacylglycerol kinase inhibitor II and diacylglycerol lipase inhibitor RHC80267) remarkably reduce the time of mGlu7 receptor stimulation required for glutamate release potentiation in mice cerebrocortical nerve terminals. We conclude that changes in diacylglycerol levels at nerve terminals control the efficiency of the exocytotic release machinery.     

Unique responses to mitochondrial complex I inhibition in tuberoinfundibular dopamine neurons may impart resistance to toxic insult

Tuberoinfundibular dopamine (TIDA) neurons are spared in Parkinson's disease (PD), a disorder that causes degeneration of midbrain nigrostriatal dopamine (NSDA) and mesolimbic dopamine (MLDA) neurons. This pattern of susceptibility has been demonstrated in acute complex I inhibitor-induced models of PD, and extrinsic factors such as toxin distribution, bioactivation, entry into the cell and sequestration into vesicles are postulated to underlie the resistance of TIDA neurons. In the present experiments, direct exposure to rotenone or 1-methyl-4-phenylpyridinium (MPP+) had no effect on mediobasal hypothalamic TIDA neurons, but significantly increased the percentage of apoptag immunoreactive neurons in midbrain primary NSDA and MLDA cultures. In vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure caused an initial decrease (by 4 h) in dopamine (DA) in brain regions containing axon terminals of TIDA (median eminence [ME]), NSDA (striatum [ST]) and MLDA (nucleus accumbens [NA]) neurons. By 16 h after MPTP treatment, DA concentrations in ME returned to control levels, while ST and NA DA levels remained low up to 32 h after treatment with MPTP. When mice and rats were chronically treated with MPTP and rotenone, respectively, the same pattern of susceptibility emerged. TIDA neurons were unaffected while NSDA neurons suffered loss of cell bodies and axon terminal DA. These experiments demonstrate that the resistance of hypothalamic TIDA neurons is not likely to be due to extrinsic factors, and that further examination of the intrinsic properties of these neurons may elucidate mechanisms that can be translated into neuroprotective strategies in PD.     

Kainic acid lesion-induced nigral neuronal death

Parkinson's disease (PD) is characterized by progressive death of dopamine (DA) neurons in the substantia nigra pars compacta. We report a rat model that exhibits progressive death of nigral neurons following unilateral injection of kainic acid in the striatum. In situ end-labeling revealed significant numbers of dying nigral neurons ipsilateral to the lesion during the first 3 weeks following injection. An indication of the gradual nature of death was that similar small numbers of cells were detected at each time point. These early morphological markers of neuronal death led to a significant reduction (20%) at 5 months of tyrosine hydroxylase-positive neurons and total number of neurons in the ipsilateral substantia nigra compared with the contralateral control. To examine the role of nigrostriatal DA metabolism in the observed nigral neuronal death, we manipulated DA metabolism during the initial 2 weeks following kainic acid lesion. Neurons in the ventral tier of the substantia nigra pars compacta were protected from death by treatment with 2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP cyclohydrolase, the initial enzyme in the synthesis of the tyrosine hydroxylase co-substrate, tetrahydrobiopterin (BH(4)). Neurons in both the dorsal and ventral tier of substantia nigra pars compacta were protected from death by treatment with DAHP and L-DOPA. These experiments suggest that intrastriatal kainic acid lesion is an in vivo model of trophic support withdrawal. This experimental procedure is useful for studying mechanisms underlying protracted death of nigral DA neurons and may provide valuable mechanistic information relevant to understanding the etiology of PD.     

Glutamatergic neurons say NO in the nucleus tractus solitarii

Both glutamate and nitric oxide (NO) may play an important role in cardiovascular reflex and respiratory signal transmission in the nucleus tractus solitarii (NTS). Pharmacological and physiological data have shown that glutamate and NO may be linked in mediating cardiovascular regulation by the NTS. Through tract tracing, multiple-label immunofluorescent staining, confocal microscopic, and electronic microscopic methods, we and other investigators have provided anatomical evidence that supports a role for glutamate and NO as well as an interaction between glutamate and NO in cardiovascular regulation in the NTS. This review article focuses on summarizing and discussing these anatomical findings. We utilized antibodies to markers of glutamatergic neurons and to neuronal NO synthase (nNOS), the enzyme that synthesizes NO in NTS neurons, to study the anatomical relationship between glutamate and NO in rats. Not only were glutamatergic markers and nNOS both found in similar subregions of the NTS and in vagal afferents, they were also frequently colocalized in the same neurons and fibers in the NTS. In addition, glutamatergic markers and nNOS were often present in fibers that were in close apposition to each other. Furthermore, N-methyl-d-aspartate (NMDA) type glutamate receptors and nNOS were often found on the same NTS neurons. Similarly, alpha-amino-3-hydroxy-5-methylisoxozole-proprionic acid (AMPA) type glutamate receptors also frequently colocalized with nNOS in NTS neurons. These findings support the suggestion that the interaction between glutamate and NO may be mediated both through NMDA and AMPA receptors. Finally, by applying tracer to the cut aortic depressor nerve (ADN) to identify nodose ganglion (NG) neurons that transmit cardiovascular signals to the NTS, we observed colocalization of vesicular glutamate transporters (VGluT) and nNOS in the ADN neurons. Thus, taken together, these neuroanatomical data support the hypothesis that glutamate and NO may interact with each other to regulate cardiovascular and likely other visceral functions through the NTS.     

Dopamine D1 receptors co-distribute with N-methyl-D-aspartic acid type-1 subunits and modulate synaptically-evoked N-methyl-D-aspartic acid currents in rat basolateral amygdala

Activation of dopamine D1 or glutamate, N-methyl-d-aspartic acid (NMDA) receptors in the basolateral amygdala (BLA) can potently influence affective behaviors and associative learning. Physical protein-protein interactions also can occur between C-terminal peptides of D1 receptors and the NMDA-receptor subunit-1 (NR1), suggesting intracellular associations of direct relevance to dopaminergic modulation of NMDA currents. We examined this possibility by combining electron microscopic immunolabeling of the D1 and NR1 C-terminal peptides with in vitro patch-clamp recording in the rat BLA. In the in vivo preparations, D1 and NR1 were localized to the surface or endomembranes of many of the same somata and dendrites as well as a few axon terminals, including those forming asymmetric, excitatory-type synapses. In vitro analysis of physiologically characterized projection neurons revealed an excitatory response to bath application of either dopamine or the preferential D1 receptor agonist, dihydrexidine. In these neurons, dopamine also selectively reduced stimulation-evoked isolated NMDA receptor-mediated currents, but not isolated non-NMDA receptor-mediated currents or the response to exogenous NMDA application. The selective reduction of the NMDA receptor-mediated currents suggests that this effect occurs at a postsynaptic locus. Moreover, both D1 and NR1 were localized to postsynaptic surfaces of biocytin-filled and physiologically characterized projection neurons. Our results provide ultrastructural evidence for D1/NR1 endomembrane associations that may dynamically contribute to the attenuation of NMDA receptor-mediated currents following prior activation of D1 receptors in BLA projection neurons. The potential for postsynaptic cross-talk between D1 and NMDA receptors in BLA projection neurons as well as a similar interaction in presynaptic terminals could have important implications for the formation and extinction of affective memories.     

Collateral damage and compensatory changes after injection of a toxin targeting neurons with the neurokinin-1 receptor in the nucleus tractus solitarii of rat

Injection into the nucleus tractus solitarii (NTS) of toxins that target substance P (SP) receptors ablates neurons that express neurokinin-1 (NK1) receptors, attenuates baroreflexes, and results in increased lability of arterial pressure. We and others have shown that the toxin leads to loss of neurons containing SP receptors and loss of GABAergic neurons in the NTS; but given that neither type neuron is thought to be integral to baroreflex transmission in NTS, mechanisms responsible for the cardiovascular changes remained unclear. Because NK1 receptors colocalize with N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in NTS and because glutamate transmission may be integral to baroreflex transmission in the NTS we hypothesized that the toxic lesions may interrupt mechanisms for glutamate transmission. Interruption of those mechanisms could be responsible for the cardiovascular effects. We tested the hypothesis by performing fluorescent immunohistochemistry, confocal microscopy and image analysis after injecting stabilized SP-SAP (SSP-SAP) unilaterally into the NTS. We assessed changes in immunoreactivity (IR) of NMDA receptor subunit 1 (NMDAR1), AMPA receptor subunit 2 (GluR2), and 3 types of vesicular glutamate transporters (VGluT) as well as IR of gamma-aminobutyric acid receptors type b (GABAb), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and protein gene product 9.5 (PGP 9.5), a neuronal marker, in the NTS. When compared to that of the same section of the un-injected NTS, IR decreased significantly in the injected side for NMDAR1 (p<0.01), GluR2 (p<0.01), VGluT3 (p<0.01), GABAb (p<0.001), and PGP9.5 (p<0.001). In contrast, IR for VGluT1 (p<0.001), VGluT2 (p<0.001), nNOS (p<0.001), and TH (p<0.001) increased significantly. We conclude that pathologic effects following ablation of neurons with NK1 receptors in NTS may result from interruption of neurotransmission through other neurochemical systems associated with NK1 receptors-containing neurons.     

Dopamine enhancement of NMDA currents in dissociated medium-sized striatal neurons: role of D1 receptors and DARPP-32

Dopamine (DA), via activation of D1 receptors, enhances N-methyl-D-aspartate (NMDA)-evoked responses in striatal neurons. The present investigation examined further the properties of this enhancement and the potential mechanisms by which this enhancement might be effected. Dissociated medium-sized striatal neurons were obtained from intact rats and mice or mutant mice lacking the DA and cyclic adenosine 3',5' monophosphate (cAMP)-regulated phosphoprotein of M(R) 32,000 (DARPP-32). NMDA (10-1,000 microM) induced inward currents in all neurons. In acutely dissociated neurons from intact rats or mice, activation of D1 receptors with the selective agonist, SKF 81297, produced a dose-dependent enhancement of NMDA currents. This enhancement was reduced by the selective D1 receptor antagonist SKF 83566. Quinpirole, a D2 receptor agonist alone, produced small reductions of NMDA currents. However, it consistently and significantly reduced the enhancement of NMDA currents by D1 agonists. In dissociated striatal neurons, in conditions that minimized the contributions of voltage-gated Ca(2+) conductances, the D1-induced potentiation was not altered by blockade of L-type voltage-gated Ca(2+) conductances in contrast to results in slices. The DARPP-32 signaling pathway has an important role in D1 modulation of NMDA currents. In mice lacking DARPP-32, the enhancement was significantly reduced. Furthermore, okadaic acid, a protein phosphatase 1 (PP-1) inhibitor, increased D1-induced potentiation, suggesting that constitutively active PP-1 attenuates D1-induced potentiation. Finally, activation of D1 receptors produced differential effects on NMDA and gamma aminobutyric acid (GABA)-induced currents in the same cells, enhancing NMDA currents and inhibiting GABA currents. Thus simultaneous activation of D1, NMDA, and GABA receptors could predispose medium-sized spiny neurons toward excitation. Taken together, the present findings indicate that the unique potentiation of NMDA receptor function by activation of the D1 receptor signaling cascade can be controlled by multiple mechanisms and has major influences on neuronal function.     

Motor Impairments,Striatal Degeneration,and Altered Dopamine-Glutamate Interplay in Mice Lacking PSD-95

Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32–positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor–mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95–dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.     

Glutamate measurement in Parkinson's disease using MRS at 3 T field strength

Loss of nigral dopamine neurons in Parkinson's disease induces abnormal activation of glutamate systems in the basal ganglia. The purpose of this study was to assess these changes in the lentiform nucleus using MRS with optimized glutamate sensitivity (TE-averaged method). Ten patients with Parkinson's disease and 10 healthy controls were examined. Compared with healthy controls, no significant differences in glutamate were measured in patients, but a trend to lower total creatine was observed.     

A tale of two endings: modulation of satiation by NMDA receptors on or near central and peripheral vagal afferent terminals

Glutamate is the neurotransmitter responsible for fast excitatory transmission from vagal afferents to second order neurons in the NTS. Antagonism of NMDA-type glutamate receptors in the NTS increases food intake and attenuates reduction of food intake by vagally mediated satiation signals, such as cholecystokinin. Although, the cellular location(s) of NMDA receptors that participate in satiation is uncertain, recent findings suggest that attenuation of satiation by NMDA receptor antagonists is due, at least in part, to their action on primary vagal afferents themselves. While evidence is accumulating that NMDA receptors located on vagal afferent endings in the hindbrain are involved in control of food intake, there also is preliminary evidence that peripheral NMDA receptors also may influence vagal control of food intake. Hence, NMDA receptor expression on central and perhaps peripheral vagal afferent endings could provide a parsimonious mechanism for modulation of satiation signals by endogenously released glutamate.     

Presence of glutamate receptor subtypes on barosensitive neurons in the nucleus tractus solitarius of the dog

Afferent baroreceptor information is transmitted to the nucleus tractus solitarius (NTS) in the dorsal medulla where glutamate is thought to be the primary neurotransmitter. However, the subtypes of glutamate receptors involved in the baroreflex remain to be established. The present study compared the distribution of immunohistochemically labeled ionotropic receptor subtypes to the distribution of physiologically stimulated barosensitive neurons in the NTS of the dog and also identified ionotropic receptor subtypes located on barosensitive neurons. Both NMDA and non-NMDA receptors were located in barosensitive areas and on barosensitive neurons, suggesting that both may be involved in the baroreflex.     

Adenosine A2A receptors and basal ganglia physiology

Adenosine A2A receptors are highly enriched in the basal ganglia system. They are predominantly expressed in enkephalin-expressing GABAergic striatopallidal neurons and therefore are highly relevant to the function of the indirect efferent pathway of the basal ganglia system. In these GABAergic enkephalinergic neurons, the A2A receptor tightly interacts structurally and functionally with the dopamine D2 receptor. Both by forming receptor heteromers and by targeting common intracellular signaling cascades, A2A and D2 receptors exhibit reciprocal antagonistic interactions that are central to the function of the indirect pathway and hence to basal ganglia control of movement, motor learning, motivation and reward. Consequently, this A2A/D2 receptors antagonistic interaction is also central to basal ganglia dysfunction in Parkinson's disease. However, recent evidence demonstrates that, in addition to this post-synaptic site of action, striatal A2A receptors are also expressed and have physiological relevance on pre-synaptic glutamatergic terminals of the cortico-limbic-striatal and thalamo-striatal pathways, where they form heteromeric receptor complexes with adenosine A1 receptors. Therefore, A2A receptors play an important fine-tuning role, boosting the efficiency of glutamatergic information flow in the indirect pathway by exerting control, either pre- and/or post-synaptically, over other key modulators of glutamatergic synapses, including D2 receptors, group I metabotropic mGlu5 glutamate receptors and cannabinoid CB1 receptors, and by triggering the cAMP-protein kinase A signaling cascade.     

Glutamate and ATP signalling in white matter pathology

Excessive signalling by excitatory neurotransmitters like glutamate and ATP can be deleterious to neurons and oligodendroglia, and cause disease. In particular, sustained activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) receptors damages oligodendrocytes, a feature that depends entirely on Ca(2+) overload of the cytoplasm and that can be initiated by disruption of glutamate homeostasis. Thus, inhibition of glutamate uptake by activated microglia can compromise glutamate homeostasis and induce oligodendrocyte excitotoxicity. Moreover, non-lethal, brief activation of kainate receptors in oligodendrocytes rapidly sensitizes these cells to complement attack as a consequence of oxidative stress. In addition to glutamate, ATP signalling can directly trigger oligodendrocyte excitotoxicity via activation of Ca(2+) -permeable P2X7 purinergic receptors, which mediates ischaemic damage to white matter (WM) and causes lesions that are reminiscent of multiple sclerosis (MS) plaques. Conversely, blockade of P2X7 receptors attenuates post-ischaemic injury to WM and ameliorates chronic experimental autoimmune encephalomyelitis, a model of MS. Importantly, P2X7 expression is elevated in normal-appearing WM in patients with MS, suggesting that signalling through this receptor in oligodendrocytes may be enhanced in this disease. Altogether, these observations reveal novel mechanisms by which altered glutamate and ATP homeostasis can trigger oligodendrocyte death. This review aims at summarizing current knowledge about the mechanisms leading to WM damage as a consequence of altered neurotransmitter signalling, and their relevance to disease. This knowledge will generate new therapeutic avenues to treat more efficiently acute and chronic WM pathology.     

Cortico-accumbens fiber stimulation does not induce dopamine release in the nucleus accumbens in vitro

Interactions between dopamine (DA) and glutamate in the nucleus accumbens (NA) are important for a variety of cognitive and limbic functions. Although, there is strong evidence that DA controls glutamate responses, the converse (glutamate affecting DA release) is controversial. To determine whether endogenous glutamate released from corticostriatal terminals can evoke DA release by local interactions in the NA, we measured DA release with amperometry simultaneously with whole cell recordings from NA medium spiny neurons (MSNs) in a slice preparation preserving DA terminals (but not cell bodies) and cortico-accumbens fibers. MSNs responded to cortical stimulation with a postsynaptic potential that was blocked by the AMPA antagonist CNQX, but no DA overflow was detected with the carbon fiber electrode. This absence of DA release cannot be accounted for by a deterioration of the DA terminals in this slice preparation since DA release was evoked with a caudal stimulation in the same slices. The DA signal was modulated as expected by bath application of a DA transporter blocker. The data show that cortico-striatal activation does not induce DA release by local interactions, suggesting that observations of glutamate-evoked DA release previously reported in vivo may be taking place via an extra-NA circuit.     

Expression and regulation of NMDA receptor subunit R1 and neuronal nitric oxide synthase in cortical neuronal cultures: correlation with cytochrome oxidase

Our previous studies showed a differential distribution of the glutamatergic terminals in cytochrome oxidase-rich and -poor regions of the visual cortex. The NMDA type of glutamate receptors have been proposed to be involved in the activation of nitric oxide synthase to produce nitric oxide, the neurotransmitter. In the present study, we hypothesized that the expressions of glutamate receptor, NMDA receptors (NMDAR1) and neuronal nitric oxide synthase (nNOS) were colocalized and were also correlated with that of cytochrome oxidase (CO) in a subset of neurons. We used primary cultures of postnatal rat visual cortical neurons as a model system, so that we could examine both the somatic and dendritic expressions of these neurochemicals in individual neurons. We found a difference in the sequence of developmental expressions of NMDAR1, nNOS, CO, and Na⁺/K⁺ ATPase. Triple labeling showed that all nNOS-positive neurons were immunoreactive for NMDAR1, and a subpopulation of them had high CO activity. The expression of NMDAR1 was positively correlated with CO activity. This is consistent with our previous finding that CO activity is strongly governed by excitatory glutamatergic synapses. After 40 hours of depolarizing potassium chloride treatment, CO activity was increased, and NMDAR1and nNOS levels were up-regulated in parallel. One week of tetrodotoxin significantly decreased the expression of NMDAR1, nNOS, and CO activity. Our results demonstrate that NMDA receptors and nNOS do co-exist in a subset of neurons that have high CO activity and their expressions are under the control of neuronal activity.     

Glutamate-induced free radical formation in rat brain synaptosomes is not dependent on intrasynaptosomal mitochondria membrane potential

Glutamate induces reactive oxygen species formation (ROS) in neurons. Free radicals can potentially be synthesized by NADPH oxidase or mitochondria. The primary source of ROS origin has yet to be identified. In addition, pro-oxidant action of glutamate receptors on neuronal presynaptic terminals is still not characterized. We investigated the influence of glutamate and agonists of its ionotropic receptors on ROS formation detected by fluorescent dye DCFDA in rat brain synaptosomes. Glutamate in concentration 10 and 100μM led to an increase of probe fluorescence pointing to free radical accumulation. This effect was mimicked by 100μM of NMDA or 100μM of kainate. Glutamate-induced ROS formation was sensitive to NMDA inhibitors MK-801 (10μM), NO synthase (NOS) inhibitor l-NAME (100μM) and NADPH oxidase inhibitors DPI (30μM) and not affected by mitochondrial uncoupler CCCP (10μM) and mitochondrial toxins rotenone (10μM)+oligomycin (5μg/ml). We also showed that 100μM of glutamate leads to a decrease of intrasynaptosomal mitochondrial potential monitored by fluorescent dye Rhodamine-123. Hence, the depolarization of intrasynaptosomal mitochondria is not a primary cause of glutamate-induced ROS formation in neuronal presynaptic terminals. Activation of NMDA receptors might be responsible for a certain part of glutamate pro-oxidant action. Most likely, sources of glutamate-induced ROS formation in neuronal presynaptic terminals are NADPH oxidase and NOS activation.