[Show abstract][Hide abstract] ABSTRACT: Although MPP(+) (1-methyl-4-phenylpyridinium) has been widely used to damage dopaminergic neurons of the Substantia Nigra pars compacta (SNc) and produce animal and cellular models of Parkinson's disease, the action of this toxin on ion channels and electrophysiological properties of these neurons remains controversial. Previous work has attributed the early effects of MPP(+) on the membrane potential and firing frequency of SNc neurons either to block of hyperpolarisation-activated (Ih) current, or to activation of ATP-sensitive K(+) (KATP) channels. Using a combination of electrophysiological and pharmacological techniques, we investigated the acute effects of MPP(+) (20μM) on SNc neurons in rat midbrain slices. Our results show that MPP(+) inhibits the activity of these neurons in distinct stages involving different mechanisms. The early phase of inhibition was dependent on D2 autoreceptors, but [(3)H]raclopride membrane binding and cAMP production assays demonstrated that the toxin (0.001-100μM) did not directly bind to these receptors nor activated the Gi-linked signalling pathway. Depletion of vesicular dopamine with Ro4-1284 attenuated the early inhibitory effect, indicating that D2 autoreceptors were activated by dopamine released from the somato-dendritic region. After longer exposure (>10-20min), MPP(+) produced a late phase of inhibition which mainly involved activation of KATP channels, and required uptake of the toxin via dopamine transporter. Although Ih current mediated by hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels was reduced by MPP(+), neither inhibition of firing nor membrane potential hyperpolarisation were significantly attenuated by blocking HCN channels with ZD7288. Our results indicate that the initial cellular events that lead to activation of cell death pathways by MPP(+) are complex and include KATP, and dopamine-dependent components, and show that the inhibitory effect of the toxin is independent of Ih block.
[Show abstract][Hide abstract] ABSTRACT: Prior to the "onset of hearing," developing cochlear inner hair cells (IHCs) and primary auditory neurons undergo experience-independent activity, which is thought to be important in retaining and refining neural connections in the absence of sound. One of the major hypotheses regarding the origin of such activity involves a group of columnar epithelial supporting cells forming Kölliker's organ, which is only present during this critical period of auditory development. There is strong evidence for a purinergic signalling mechanism underlying such activity. ATP released through connexin hemichannels may activate P2 purinergic receptors in both Kölliker's organ and the adjacent IHCs, leading to generation of electrical activity throughout the auditory system. However, recent work has suggested an alternative origin, by demonstrating the ability of IHCs to generate this spontaneous activity without activation by ATP. Regardless, developmental abnormalities of Kölliker's organ may lead to congenital hearing loss, considering that mutations in ion channels (hemichannels, gap junctions, and calcium channels) involved in Kölliker's organ activity share strong links with such types of deafness.
BioMed Research International 08/2014; 2014:367939. DOI:10.1155/2014/367939 · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies suggested that amyloid β (Aβ)-induced disruption of astrocytic Ca(2+) signalling and oxidative stress play a major role in the progression towards neuronal and glial death in Alzheimer's disease. We have recently demonstrated that Ca(2+)-permeable TRPV4 channels are highly expressed in rat hippocampal astrocytes and are involved in oxidative stress-induced cell damage. The aim of this study was to test the hypothesis that TRPV4 channels also contribute to hippocampal damage evoked by Aβ. Synthetic Aβ40 evoked cell death in hippocampal slice cultures in a concentration (0-20μM) and time (12-48 hr) dependent manner, after cultures were preconditioned with sublethal concentration of buthionine sulfoximine (1.5μM) which enhanced endogenous ROS production. As demonstrated by propidium iodide fluorescence, damage was observed in the granule cell layer of the dentate gyrus and to a smaller degree in pyramidal neurons of the CA1-CA3 region, as well as in glia cells mainly at the edge of the slice. Immunocytochemistry revealed an altered pattern of TRPV4 and GFAP protein expression, and reactive astrogliosis surrounding pyramidal CA1-CA3 neurons. Neuronal and astrocytic damage was attenuated by the antioxidant Trolox, TRPV4 channel blockers Gd(3+) and ruthenium red (RR), and a specific inhibitor of the redox and Ca(2+)-sensitive phospholipase A2 enzyme (MAFP). In disassociated co-cultures of hippocampal neurons and astrocytes without BSO preconditioning, Aβ40 evoked pronounced neuronal damage, enhanced the expression of TRPV4 and GFAP proteins (indicative of reactive astrogliosis), and increased intracellular free Ca(2+) concentration in astrocytes. The latter effect was attenuated by RR and in Ca(2+)-free media. These data show that Aβ40 can activate astrocytic TRPV4 channels in the hippocampus, leading to neuronal and astrocytic damage in a Ca(2+) and oxidative stress-dependent manner.
[Show abstract][Hide abstract] ABSTRACT: Endocannabinoids (eCBs) modulate synaptic transmission in the brain, but little is known of their regulatory role in nigral dopaminergic neurons, and whether transmission to these neurons is tonically inhibited by eCBs as seen in some other brain regions. Using whole-cell recording in midbrain slices, we observed potentiation of evoked IPSCs (eIPSCs) in these neurons after blocking CB1 receptors with rimonabant or LY-320,135, indicating the presence of an eCB tone reducing inhibitory synaptic transmission. Increased postsynaptic calcium buffering and block of mGluR1 or postsynaptic G-protein coupled receptors prevented this potentiation. Increasing spillover of endogenous glutamate by inhibiting uptake attenuated eIPSC amplitude, while enhancing the potentiation by rimonabant. Group I mGluR activation transiently inhibited eIPSCs, which could be prevented by GDP-β-S, increased calcium buffering or rimonabant. We explored the possibility that the dopamine-derived eCB N-arachidonoyl dopamine (NADA) is involved. The eCB tone was abolished by preventing dopamine synthesis, and enhanced by L-DOPA. It was not detected in adjacent non-dopaminergic neurons. Preventing 2-AG synthesis did not affect the tone, while inhibition of NADA production abolished it. Quantification of ventral midbrain NADA suggested a basal level that increased following prolonged depolarization or mGluR activation. Since block of the tone was not always accompanied by attenuation of depolarization-induced suppression of inhibition (DSI) and vice versa, our results indicate DSI and the eCB tone are mediated by distinct eCBs. This study provides evidence that dopamine modulates the activity of SNc neurons not only by conventional dopamine receptors, but also by CB1 receptors, potentially via NADA.
[Show abstract][Hide abstract] ABSTRACT: Spreading depression (SD) is a profound depolarization of neurons and glia that propagates in a wave-like manner across susceptible brain regions, and can develop during periods of compromised cellular energy such as ischemia, when it influences the severity of acute neuronal damage. Although SD has been well characterised in the cerebral cortex and hippocampus, little is known of this event in the Substantia Nigra (SN), a brainstem nucleus engaged in motor control and reward-related behaviour. Transverse brain slices (250μm; P21-23 rats) containing the SN were subject to oxygen and glucose deprivation (OGD) tests, modelling brain ischemia. SD developed in lateral aspects of the SN within 3.3±0.2min of OGD onset, and spread through the Substantia Nigra pars reticulata (SNr), as indicated by fast-occurring and propagating increased tissue light transmittance and negative shift of extracellular DC potential. These events were associated with profound mitochondrial membrane depolarization (ΔΨm) throughout the SN, as demonstrated by increased Rhodamine 123 fluorescence. Extracellular recordings from individual SNr neurons indicated rapid depolarization followed by depolarizing block, while dopaminergic neurons in the Substantia Nigra pars compacta (SNc) showed inhibition of firing associated with hyperpolarization. SD evoked in the SNr was similar to OGD-induced SD in the CA1 region in hippocampal slices. In the hippocampus, SD also developed during anoxia or aglycemia alone (associated with less profound ΔΨm than OGD), while these conditions rarely led to SD in the SNr. Our results demonstrate that OGD consistently evokes SD in the SN, and that this phenomenon only involves the SNr. It remains to be established whether nigral SD contributes to neuronal damage associated with a sudden-onset form of Parkinson's disease known as 'vascular parkinsonism'.
Brain research 06/2013; 1527. DOI:10.1016/j.brainres.2013.06.016 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: l-DOPA (Levodopa) remains the gold standard for the treatment of motor symptoms of Parkinson's disease (PD), despite indications that the drug may have detrimental effects in cell culture. Classically, l-DOPA increases the production of dopamine (DA) in nigral dopaminergic neurons, while paradoxically inhibiting the firing of these neurons due to activation of D2 autoreceptors by extracellularly released DA. Using a combination of electrophysiology and calcium microfluorometry in brain slices, we have identified a novel effect of l-DOPA on dopaminergic neurons when D2 receptors were blocked. Under these conditions, l-DOPA (0.03-3mM) evoked an excitatory effect consisting of two components. The 'early' component observed during and immediately after application of the drug, was associated with increased firing, membrane depolarization and inward current. This excitatory response was strongly attenuated by CNQX (10μM), pointing to the involvement of TOPA quinone, an auto-oxidation product of l-DOPA and a potent activator of AMPA/kainate receptors. The 'late' phase of excitation persisted >30min after brief l-DOPA application and was not mediated by ionotropic glutamate receptors, nor by D1, α1-adrenergic, mGluR1 or GABAB receptors. It was eliminated by carbidopa, demonstrating its dependence on conversion of l-DOPA to DA. Exogenous DA (50μM) also evoked a glutamate-receptor independent increase in firing and an inward current when D2 receptors were blocked. In voltage-clamped neurons, both l-DOPA and DA produced a long-lasting increase in [Ca2+]i which was unaffected by block of ionotropic glutamate receptors. These results demonstrate that l-DOPA has dual, inhibitory and excitatory, effects on nigral dopaminergic neurons, and suggest that the excitation and calcium rise may have long-lasting consequences for the activity and survival of these neurons when the expression or function of D2 receptors is impaired.
[Show abstract][Hide abstract] ABSTRACT: Purpose: While the major hallmark of Parkinson’s disease (PD) is
relatively selective degeneration of dopaminergic neurons in the
Substantia Nigra pars compacta (SNc), there is also substantial loss of
noradrenergic Locus Coeruleus (LC) neurons. The loss of these neurons
may contribute to non-motor symptoms of PD which often occur earlier
in the disease than characteristic motor symptoms. Based on the theory
that LC neurons are damaged earlier in the progression of PD, we
hypothesize that these neurons are more vulnerable to neurotoxic insult
than SNc neurons. Methods: Using electrophysiological techniques,
we have directly compared the responses of LC and SNc neurons in
acute brain slices to rotenone, a mitochondrial complex I inhibitor, widely
used to produce animal models of PD. Results: Rotenone (0.1-5 μM)
produced a dose-dependent (p<0.05) decrease in the spontaneous
firing of LC and SNc neurons recorded extracellularly, associated with
cell membrane hyperpolarisation and a tolbutamide (100 μM)-sensitive
outward current in whole cell patch clamp recordings. These effects were
largely mediated by ATP-sensitive K+
(KATP) channels, the activation of
which was greater in SNc neurons than LC neurons (p<0.01). When KATP channels were blocked with tolbutamide, rotenone (1 μM) increased the
firing of LC and SNc neurons. This effect which was associated with an
inward current, unmasked by intracellular Cs+
, which effectively blocks
conductances. Conclusion: Rotenone activates KATP channels
more strongly in SNc neurons than in the LC, potentially protecting
these neurons from detrimental effects of mitochondrial toxins such as
rotenone. Thus LC neurons may be more susceptible to neurotoxininduced damage than SNc neurons.
[Show abstract][Hide abstract] ABSTRACT: Transient receptor potential melastatin 2 (TRPM2) channels are sensitive to oxidative stress, and their activation can lead to cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remains unknown. In this study, we assessed the expression and functional properties of TRPM2 channels in rat dopaminergic SNc neurons, using acute brain slices. RT-PCR analysis revealed TRPM2 mRNA expression in the SNc region. Immunohistochemistry demonstrated expression of TRPM2 protein in tyrosine hydroxylase-positive neurons. Channel function was tested with whole cell patch-clamp recordings and calcium (fura-2) imaging. Intracellular application of ADP-ribose (50-400 μM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)](i)) rise. These responses were strongly inhibited by the nonselective TRPM2 channel blockers clotrimazole and flufenamic acid. Exogenous application of H(2)O(2) (1-5 mM) evoked a rise in [Ca(2+)](i) and an outward current mainly due to activation of ATP-sensitive potassium (K(ATP)) channels. Inhibition of K(+) conductance with Cs(+) and tetraethylammonium unmasked an inward current. The inward current and/or [Ca(2+)](i) rise were partially blocked by clotrimazole and N-(p-amylcinnamoyl)anthranilic acid (ACA). The H(2)O(2)-induced [Ca(2+)](i) rise was abolished in "zero" extracellular Ca(2+) concentration and was enhanced at higher baseline [Ca(2+)](i), consistent with activation of TRPM2 channels in the cell membrane. These results provide evidence for the functional expression of TRPM2 channels in dopaminergic SNc neurons. Given the involvement of oxidative stress in degeneration of SNc neurons in Parkinson's disease, further studies are needed to determine the pathophysiological role of these channels in the disease process.
Journal of Neurophysiology 09/2011; 106(6):2865-75. DOI:10.1152/jn.00994.2010 · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is consensus that amelioration of the motor symptoms of Parkinson's disease is most effective with L-DOPA (levodopa). However, this necessary therapeutic step is biased by an enduring belief that L-DOPA is toxic to the remaining substantia nigra dopaminergic neurons by itself, or by specific metabolites such as dopamine. The concept of L-DOPA toxicity originated from pre-clinical studies conducted mainly in cell culture, demonstrating that L-DOPA or its derivatives damage dopaminergic neurons due to oxidative stress and other mechanisms. However, the in vitro data remain controversial as some studies showed neuroprotective, rather than toxic action of the drug. The relevance of this debate needs to be considered in the context of the studies conducted on animals and in clinical trials that do not provide convincing evidence for L-DOPA toxicity in vivo. This review presents the current views on the pathophysiology of Parkinson's disease, focusing on mitochondrial dysfunction and oxidative/proteolytic stress, the factors that can be affected by L-DOPA or its metabolites. We then critically discuss the evidence supporting the two opposing views on the effects of L-DOPA in vitro, as well as the animal and human data. We also address the problem of inadequate experimental models used in these studies. L-DOPA remains the symptomatic 'hero' of Parkinson's disease. Whether it contributes to degeneration of nigral dopaminergic neurons, or is a 'scapegoat' for explaining undesirable or unexpected effects of the treatment, remains a hotly debated topic.
Progress in Neurobiology 06/2011; 94(4):389-407. DOI:10.1016/j.pneurobio.2011.06.005 · 9.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Organotypic cultures (OCs) have been widely used to investigate the midbrain dopaminergic system, but only a few studies focused on the functional properties of dopaminergic neurons and their synaptic inputs from dopaminergic and non-dopaminergic neurons also contained in such cultures. In addition, it is not clear whether the culturing process affects the intrinsic neuronal properties and the expression of specific receptors and transporters. We performed patch-clamp recordings from dopaminergic neurons in mesencephalic-striatal co-cultures obtained from transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. Some (10/44) GFP+ neurons displayed a bursting activity that renders the firing of these cells similar to that of the dopaminergic neurons in vivo. The culturing process reduced the hyperpolarization-activated current (I(h) ) and the expression of D₂ receptors. Downregulation of D₂ receptor mRNA and protein was confirmed with reverse transcriptase polymerase chain reaction and Western blotting. Immunocytochemistry revealed that many synaptic terminals, most likely originating from dopaminergic neurons, co-expressed the dopamine (DA) transporter and the vesicular glutamate transporter-2, suggesting a co-release of DA and glutamate. Interestingly, exogenous DA decreased glutamate release in young cultures [days in vitro (DIV)<20] by acting on pre-synaptic D₂ receptors, while in older cultures (DIV>26) DA increased glutamate release by acting on α-1 adrenoreceptors. The facilitatory effect of DA on glutamatergic transmission to midbrain dopaminergic neurons may be important in conditions when the expression of D₂ receptors is compromised, such as long-term treatment with antipsychotic drugs. Our data show that midbrain OCs at DIV>26 may provide a suitable model of such conditions.
European Journal of Neuroscience 03/2011; 33(9):1622-36. DOI:10.1111/j.1460-9568.2011.07659.x · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A reliable model system of epileptiform insult would facilitate investigation into the underlying biological mechanisms. Epileptiform insult was induced in hippocampal slice cultures by lowering extracellular Mg(2+), (+)-bicuculline, or (-)-bicuculline methochloride, a stable salt form of bicuculline (both forms block GABA(A) receptors). Cell death was assessed by propidium iodide uptake. Low Mg(2+) or (+)-bicuculline did not produce cell death regardless of dose or incubation period. Exposure to 100 microM (-)-bicuculline methochloride for 48 hr resulted in prominent CA1 cell death. These findings demonstrate that not all pro-epileptic drugs/ion changes used routinely for electrophysiological recording of seizure activity lead to cell death in hippocampal slice cultures and that treatment with bicuculline methochloride can be used as a reliable model for epileptiform insult.
The International journal of neuroscience 10/2010; 120(12):752-9. DOI:10.3109/00207454.2010.520378 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TRPM2 and TPPV4 channels, two members of TRP channel family, are known to be widely expressed in the brain but their exact expression pattern and function are not well understood. Due to their high Ca(2+) permeability and gating by reactive oxygen species (TRPM2), or cell swelling, low pH and high temperature (TRPV4), they are likely to be involved in cell damage associated with various brain pathologies. The aim of this study was to investigate the expression of these channels and their potential role in oxidative stress-induced cell damage in organotypic hippocampal slice cultures, a model that retains the complex interaction between neurons and astrocytes. Channel expression was confirmed with RT-PCR and western blotting, while immunocytochemistry demonstrated TRPM2 in CA1-CA3 pyramidal neurons and TRPV4 in astrocytes. Oxidative stress induced by exogenous application of H(2)O(2) (600 microM) caused preferential damage of pyramidal neurons, while oxidative stress evoked with mercaptosuccinate (MCS; 400 microM) or buthionine sulfoximine (BSO; 4 microM) mainly damaged astrocytes, as identified by propidium iodide fluorescence. Antioxidants (Trolox 500 microM; MitoE 2 microM) reduced both neuronal and astrocytic cell death. Blockers of TRPV4 channels (Gd(3+) 500 microM; Ruthenium red 1 microM) increased the viability of astrocytes following MCS or BSO treatments, consistent with the expression pattern of these channels. Blockers of TRPM2 channels clotrimazole (20 microM), N-(p-amylcinnomoyl)anthranilic acid (ACA, 25 microM) or flufenamic acid (FFA, 200 microM) failed to protect pyramidal neurons from damage caused by exogenous H(2)O(2), and increased damage of these neurons caused by MCS and BSO. The differential expression of stress-sensitive TRPM2 and TRPV4 channels in hippocampal neurons and astrocytes that show distinct differences in vulnerability to different forms of oxidative stress suggests the specific involvement of these channels in oxidative stress-induced cell damage. However, the exact relationship between TRPM2 channel activation and cell death still remains to be determined due to the lack of protective effects of TRPM2 channel blockers.
[Show abstract][Hide abstract] ABSTRACT: Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05-1 microm) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 +/- 15 pA) associated with increases in intracellular [Ca(2+)] ([Ca(2+)](i)) (73.8 +/- 7.7 nm) and intracellular [Na(+)] (3.1 +/- 0.6 mm) (all with 1 microm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca(2+)](i) rise was abolished by removing extracellular Ca(2+), and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N-(p-amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca(2+)](i) rise resulted in a large (46.6 +/- 25.3 nm) Ca(2+) response when baseline [Ca(2+)](i) was increased by a 'priming' protocol that activated voltage-gated Ca(2+) channels. There was also a positive correlation between 'naturally' occurring variations in baseline [Ca(2+)](i) and the rotenone-induced [Ca(2+)](i) rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K(+) channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca(2+)](i) rise by a small increase in baseline [Ca(2+)](i).
European Journal of Neuroscience 11/2009; 30(10):1849-59. DOI:10.1111/j.1460-9568.2009.06990.x · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pauses in the tonic firing of striatal cholinergic interneurons emerge during reward-related learning and are triggered by neutral cues which develop behavioural significance. In a previous in vivo study we have proposed that these pauses in firing may be due to intrinsically generated afterhyperpolarisations (AHPs) evoked by excitatory synaptic inputs, including those below the threshold for action potential firing. The aim of this study was to investigate the mechanism of the AHPs using a brain slice preparation which preserved both cerebral hemispheres. Augmenting cortically evoked postsynaptic potentials (PSPs) by repetitive stimulation of cortical afferents evoked AHPs that were unaffected by blocking either GABA(A) receptors with bicuculline, or GABA(B) receptors with saclofen or CGP55845. Apamin (a blocker of small conductance Ca(2+)-activated K(+) channels) had minimal effects, while chelation of intracellular Ca(2+) with BAPTA reduced the AHP by about 30%. In contrast, blocking hyperpolarisation and cyclic nucleotide activated (HCN) cation current (I(H)) with ZD7288 or Cs(+) diminished the size of the AHPs by 60% and reduced the proportion of episodes that contained this hyperpolarisation. The reversal potential (20 mV) and voltage dependence of the AHPs were consistent with the hypothesis that a transient deactivation of I(H) caused most of the AHP at hyperpolarised potentials, while the slow AHP-type Ca(2+)-activated K(+) channels increasingly contributed at more depolarised membrane potentials. Subthreshold somatic current injections yielded similar AHPs with a median duration of approximately 700 ms that were not affected by firing of a single action potential. These results indicate that transient deactivation of HCN channels evokes pauses in tonic firing of cholinergic interneurons, an event likely to be elicited by augmentation of afferent synaptic inputs during learning.
The Journal of Physiology 11/2009; 587(Pt 24):5879-97. DOI:10.1113/jphysiol.2009.177600 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: P2 receptor (R) signalling plays an important role in the central ventilatory response to hypoxia. The frequency increase that results from activation of P2Y(1)Rs in the preBötzinger complex (preBötC; putative site of inspiratory rhythm generation) may contribute, but neither the cellular nor ionic mechanism(s) underlying these effects are known. We applied whole-cell recording to rhythmically-active medullary slices from neonatal rat to define, in preBötC neurones, the candidate cellular and ionic mechanisms through which ATP influences rhythm, and tested the hypothesis that putative rhythmogenic preBötC neurones are uniquely sensitive to ATP. ATP (1 mm) evoked inward currents in all non-respiratory neurones and the majority of respiratory neurons, which included inspiratory, expiratory and putative rhythmogenic inspiratory neurones identified by sensitivity to substance P (1 microM) and DAMGO (50 microM) or by voltage-dependent pacemaker-like activity. ATP current densities were similar in all classes of preBötC respiratory neurone. Reversal potentials and input resistance changes for ATP currents in respiratory neurones suggested they resulted from either inhibition of a K(+) channel or activation of a mixed cationic conductance. The P2YR agonist 2MeSADP (1 mm) evoked only the latter type of current in inspiratory and pacemaker-like neurones. In summary, putative rhythmogenic preBötC neurones were sensitive to ATP. However, this sensitivity was not unique; ATP evoked similar currents in all types of preBötC respiratory neurone. The P2Y(1)R-mediated frequency increase is therefore more likely to reflect activation of a mixed cationic conductance in multiple types of preBötC neurone than excitation of one, highly sensitive group.
The Journal of Physiology 04/2008; 586(5):1429-46. DOI:10.1113/jphysiol.2007.143024 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calpains are Ca(2+)-activated enzymes which cleave cytoskeletal and other proteins, contributing to neuronal damage in conditions of pathological intracellular Ca(2+) elevation, including stroke. However, the consequences of calpain overactivation have typically been observed hours after insult. To identify the earliest events attributable to calpain activation, and thus potentially isolate calpain substrates involved in acute neuronal damage, we dynamically recorded the effects of calpain inhibition in an in vitro model of stroke. Extracellular DC potentials and fEPSPs were monitored together with changes of light transmittance (as a measure of cell and mitochondrial swelling) and Rh 123 fluorescence (to monitor mitochondrial membrane potential; DeltaPsi(m)) in hippocampal slices obtained from P12-P17 rats. No differences were observed in the latencies of fEPSP disruption or onset of extracellular DC shifts associated with hypoxic spreading depression (HSD) evoked by oxygen-glucose deprivation (OGD) under control conditions or in the presence of calpain inhibitor III (MDL 28170). However, a significant difference was observed in transmitted light signals during OGD with calpain inhibition. Given the potential contribution of mitochondrial swelling to changes in light transmittance, these experiments were also conducted in the presence of cyclosporin A to block opening of the mitochondrial permeability transition pore (MPTP). Our results indicate that differences in OGD-induced changes of light transmittance in the presence of MDL 28170 are not likely the result of MPTP blockade or changes in dendritic beading. We propose that calpain inhibition may alter changes in light transmittance by limiting conformational changes of mitochondria.
Brain Research 03/2008; 1196:121-30. DOI:10.1016/j.brainres.2007.12.006 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Astrocytic glutamate transporters are considered an important target for neuroprotective therapies as the function of these transporters is abnormal in stroke and other neurological disorders associated with excitotoxicity. Recently, Rothstein et al., [Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB (2005) Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433:73-77] reported that beta-lactam antibiotics (including ceftriaxone, which easily crosses the blood-brain barrier) increase glutamate transporter 1 (GLT-1) expression and reduce cell death resulting from oxygen-glucose deprivation (OGD) in dissociated embryonic cortical cultures. To determine whether a similar neuroprotective mechanism operates in more mature neurons, which show a different pattern of response to ischemia than primary cultures, we exposed acute hippocampal slices obtained from rats treated with ceftriaxone for 5 days (200 mg/kg; i.p.) to OGD. Whole-cell patch clamp recording of glutamate-induced N-methyl-d-aspartate (NMDA) currents from CA1 pyramidal neurons showed a larger potentiation of these currents after application of 15 microM dl-threo-beta-benzyloxyaspartic acid (TBOA; a potent blocker of glutamate transporters) in ceftriaxone-injected animals than in untreated animals, indicating increased glutamate transporter activity. Western blot analysis did not reveal GLT-1 upregulation in the hippocampus. Delay to OGD-induced hypoxic spreading depression (HSD) recorded in slices obtained from ceftriaxone-treated rats was longer (6.3+/-0.2 vs. 5.2+/-0.2 min; P<0.001) than that in the control group, demonstrating a neuroprotective action of the antibiotic in this model. The effect of ceftriaxone was also tested in organotypic hippocampal slices obtained from P7-9 rats (>14 days in vitro). OGD or glutamate (3.5-5.0 mM) damaged CA1 pyramidal neurons as assessed by propidium iodide (PI) fluorescence. Similar damage was observed after pre-treatment with ceftriaxone (10-200 microM; 5 days) and ceftriaxone exposure did not result in GLT-1 upregulation as assayed by Western blot. Treatment of slice cultures with dibutyryl cAMP (100-250 microM; 5 days) increased GLT-1 expression but did not reduce cell damage induced by OGD or glutamate. Thus we confirm the neuroprotective effect of antibiotic exposure on OGD-induced injury, but suggest that this action is related to independent modulation of transporter activity rather than to the level of GLT-1 protein expression. In addition, our results indicate that the protective effects of beta-lactam antibiotics are highly dependent on the experimental model.
[Show abstract][Hide abstract] ABSTRACT: ATP is released during hypoxia from the ventrolateral medulla (VLM) and activates purinergic P2 receptors (P2Rs) at unknown loci to offset the secondary hypoxic depression of breathing. In this study, we used rhythmically active medullary slices from neonatal rat to map, in relation to anatomical and molecular markers of the pre-Bötzinger complex (preBötC) (a proposed site of rhythm generation), the effects of ATP on respiratory rhythm and identify the P2R subtypes responsible for these actions. Unilateral microinjections of ATP in a three-dimensional grid within the VLM revealed a "hotspot" where ATP (0.1 mM) evoked a rapid 2.2 +/- 0.1-fold increase in inspiratory frequency followed by a brief reduction to 0.83 +/- 0.02 of baseline. The hotspot was identified as the preBötC based on histology, overlap of injection sites with NK1R immunolabeling, and potentiation or inhibition of respiratory frequency by SP ([Sar9-Met(O2)11]-substance P) or DAMGO ([D-Ala2,N-MePhe4,Gly-ol5]-enkephalin), respectively. The relative potency of P2R agonists [2MeSADP (2-methylthioadenosine 5'-diphosphate) approximately = 2MeSATP (2-methylthioadenosine 5'-triphosphate) approximately = ATPgammas (adenosine 5'-[gamma-thio]triphosphate tetralithium salt) approximately = ATP > UTP approximately = alphabeta meATP (alpha,beta-methylene-adenosine 5'-triphosphate)] and attenuation of the ATP response by MRS2179 (2'-deoxy-N6-methyladenosine-3',5'-bisphosphate) (P2Y1 antagonist) indicate that the excitation is mediated by P2Y1Rs. The post-ATP inhibition, which was never observed in response to ATPgammas, is dependent on ATP hydrolysis. These data establish in neonatal rats that respiratory rhythm generating networks in the preBötC are exquisitely sensitive to P2Y1R activation, and suggest a role for P2Y1Rs in respiratory motor control, particularly in the P2R excitation of rhythm that occurs during hypoxia.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 01/2007; 27(5):993-1005. DOI:10.1523/JNEUROSCI.3948-06.2007 · 6.34 Impact Factor