Peter S. Freestone

Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices.

During a period of acute ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 mum) obtained from Wistar rats were submerged in ACSF at 36 degrees C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La3+, Gd3+, 2-APB) or lowering temperature by 3 degrees C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 degrees C) and La3+ or Gd3+ (100 microM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.

Acute action of rotenone on nigral dopaminergic neurons – involvement of reactive oxygen species and disruption of Ca2+ homeostasis

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 μm) 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 [Ca2+] ([Ca2+]i) (73.8 ± 7.7 nm) and intracellular [Na+] (3.1 ± 0.6 mm) (all with 1 μm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca2+]i rise was abolished by removing extracellular Ca2+, 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 [Ca2+]i rise resulted in a large (46.6 ± 25.3 nm) Ca2+ response when baseline [Ca2+]i was increased by a ‘priming’ protocol that activated voltage‐gated Ca2+ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca2+]i and the rotenone‐induced [Ca2+]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 [Ca2+]i rise by a small increase in baseline [Ca2+]i.

Glutamate spillover drives endocannabinoid production and inhibits GABAergic transmission in the Substantia Nigra pars compacta

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.

Properties of dopaminergic neurons in organotypic mesencephalic-striatal co-cultures - evidence for a facilitatory effect of dopamine on the glutamatergic input mediated by α-1 adrenergic receptors

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 (Ih) 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.

Effects of the Parkinsonian toxin MPP + on electrophysiological properties of nigral dopaminergic neurons

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 Parkinsons 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(+) (20 μM) 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 (0.001-100 μM) 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 (>10-20 min), 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 was 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.

Tonabersat Prevents Inflammatory Damage in the Central Nervous System by Blocking Connexin43 Hemichannels

The cis benzopyran compound tonabersat (SB-220453) has previously been reported to inhibit connexin26 expression in the brain by attenuating the p38-mitogen-activated protein kinase pathway. We show here that tonabersat directly inhibits connexin43 hemichannel opening. Connexin43 hemichannels have been called “pathological pores” based upon their role in secondary lesion spread, edema, inflammation, and neuronal loss following central nervous system injuries, as well as in chronic inflammatory disease. Both connexin43 hemichannels and pannexin channels released adenosine triphosphate (ATP) during ischemia in an in vitro ischemia model, but only connexin43 hemichannels contributed to ATP release during reperfusion. Tonabersat inhibited connexin43 hemichannel-mediated ATP release during both ischemia and reperfusion phases, with direct channel block confirmed using electrophysiology. Tonabersat also reduced connexin43 gap junction coupling in vitro, but only at higher concentrations, with junctional plaques internalized and degraded via the lysosomal pathway. Systemic delivery of tonabersat in a rat bright-light retinal damage model (a model for dry age-related macular degeneration) resulted in significantly improved functional outcomes assessed using electroretinography. Tonabersat also prevented thinning of the retina, especially the outer nuclear layer and choroid, assessed using optical coherence tomography. We conclude that tonabersat, already given orally to over 1000 humans in clinical trials (as a potential treatment for, and prophylactic treatment of, migraine because it was thought to inhibit cortical spreading depression), is a connexin hemichannel inhibitor and may have the potential to be a novel treatment of central nervous system injury and chronic neuroinflammatory disease.

Pulse-Width Modulation of Optogenetic Photo-Stimulation Intensity for Application to Full-Implantable Light Sources

Optogenetics allows control of neuronal activity with unprecedented spatiotemporal precision, and has enabled both significant advances in neuroscience and promising clinical prospects for some neurological, cardiac, and sensory disorders. The ability to chronically stimulate light-sensitive excitable cells is crucial for developing useful research tools and viable long-term treatment strategies. Popular optogenetic stimulation devices often rely on bench-top light-sources tethered via an optical fibre to the research animal, or significant componentry protruding externally from animal. These approaches are prone to infection, vulnerable to damage and restrict the experimental approaches that can be conducted. An ideal optogenetic stimulator would be contained entirely within the animal and provide precisely controlled optical output. However, existing prototypes of fully implantable devices rely on amplitude tuning of wireless power, which can vary strongly with environmental conditions. Here we show that pulse-width modulation (PWM) of the intensity of a light-emitting diode (LED) can enable control of photo-stimulation intensity equivalent to direct amplitude modulation. This result has significant implications for fully implantable light delivery tools, as PWM can be implemented with simple and miniaturized circuit architectures. We have modified a telemeter device previously developed by our group to include a small form-factor LED capable of generating sufficient optical power with manageable electrical power requirements and minimal heat generation. We have tested key device components in an in vitro mouse brain slice preparation and shown that pulse-width-modulation is an alternative method to modulate photo-stimulation intensity using a miniature circuit and providing easy control.

Paradoxical lower sensitivity of Locus Coeruleus than Substantia Nigra pars compacta neurons to acute actions of rotenone.

&NA; Parkinsons disease (PD) is not only associated with degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra, but also with profound loss of noradrenergic neurons in the Locus Coeruleus (LC). Remarkably, LC degeneration may exceed, or even precede the loss of nigral DAergic neurons, suggesting that LC neurons may be more susceptible to damage by various insults. Using a combination of electrophysiology, fluorescence imaging and electrochemistry, we directly compared the responses of LC, nigral DAergic and nigral non‐dopaminergic (non‐DAergic) neurons in rat brain slices to acute application of rotenone, a mitochondrial toxin used to create animal and in vitro models of PD. Rotenone (0.01–5.0 &mgr;M) dose‐dependently inhibited the firing of all three groups of neurons, primarily by activating KATP channels. The toxin also depolarised mitochondrial potential (&PSgr;m) and released reactive oxygen species (H2O2). When KATP channels were blocked, rotenone (1 &mgr;M) increased the firing of LC neurons by activating an inward current associated with dose‐dependent increase of cytosolic free Ca2 + ([Ca2 +]i). This effect was attenuated by blocking oxidative stress‐sensitive TRPM2 channels, and by pre‐treatment of slices with anti‐oxidants. These results demonstrate that rotenone inhibits the activity of LC neurons mainly by activating KATP channels, and increases [Ca2 +]i via TRPM2 channels. Since the responses of LC neurons were smaller than those of nigral DAergic neurons, our study shows that LC neurons are paradoxically less sensitive to acute effects of this parkinsonian toxin. Graphical abstract: Figure. No caption available. HighlightsRotenone inhibited the firing of NAergic LC neurons by activating KATP channels.The toxin also increased intracellular Ca2 + by activating TRPM2 channels.Rotenone depolarised mitochondria and released H2O2 from these neurons.Responses of LC neurons were compared to nigral DAergic and non‐DAergic neurons.LC neurons were less sensitive than nigral DAergic neurons to effects of rotenone.

Excitatory drive from the Subthalamic nucleus attenuates GABAergic transmission in the Substantia Nigra pars compacta via endocannabinoids.

Endocannabinoids (eCBs) are cannabis-like substances produced in the brain where their primary function is to regulate synaptic transmission by inhibiting neurotransmitter release in a retrograde fashion. We have recently demonstrated a novel mechanism regulating GABAergic transmission from neurons in the Substantia Nigra pars reticulata (SNr) to dopaminergic neurons in the Substantia Nigra pars compacta (SNc) mediated by eCBs. Production of eCBs was initiated by spillover of glutamate, yet the source of the glutamate was not determined (Freestone et al., 2014; Neuropharmacology 79 p467). The present study aimed at elucidating the potential role of glutamatergic terminals arising from neurons in the Subthalamic nucleus (STN) in driving the eCB-mediated modulation of this inhibitory transmission. GABAergic IPSCs or IPSPs evoked in SNc neurons by electrical stimuli delivered to the SNr region were transiently inhibited by electrical or pharmacological (U-tube application of muscarinic agonist carbachol [100 µM]) stimulation of the STN (to 74±5% and 69±4% respectively). In both stimulation protocols, the attenuation of GABAergic transmission was abolished by cannabinoid receptor 1 antagonist rimonabant (3 µM), and reduced by group 1 metabotropic glutamate receptor antagonist CPCCOEt (100 µM), consistent with a glutamate-initiated and eCB-mediated mechanism. The carbachol-induced attenuation of GABAergic transmission was abolished by M3 muscarinic receptor antagonist 4-DAMP (10 µM), confirming a specific activation of STN neurons. These results demonstrate that glutamatergic projection from the STN to dopaminergic SNc neurons underlies an eCB-mediated inhibition of GABAergic input to these neurons.

Action potential and calcium dependence of tonic somatodendritic dopamine release in the Substantia Nigra pars compacta

Despite the importance of somatodendritic dopamine (DA) release in the Substantia Nigra pars compacta (SNc), its mechanism remains poorly understood. Using a novel approach combining fast‐scan controlled‐adsorption voltammetry (FSCAV) and single‐unit electrophysiology, we have investigated the mechanism of somatodendritic release by directly correlating basal (non‐stimulated) extracellular DA concentration ([DA]out), with pharmacologically‐induced changes of firing of nigral dopaminergic neurons in rat brain slices. FSCAV measurements indicated that basal [DA]out in the SNc was 40.7 ± 2.0 nM (at 34 ± 0.5°C), which was enhanced by amphetamine, cocaine, and L‐DOPA, and reduced by VMAT2 inhibitor, Ro4‐1284. Complete inhibition of firing by TTX decreased basal [DA]out, but this reduction was smaller than the effect of D2 receptor agonist, quinpirole. Despite similar effects on neuronal firing, the larger decrease in [DA]out evoked by quinpirole was attributed to cell membrane hyperpolarization and greater reduction in cytosolic free Ca2+ ([Ca2+]in). Decreasing extracellular Ca2+ also reduced basal [DA]out, despite increasing firing frequency. Furthermore, inhibiting L‐type Ca2+ channels decreased basal [DA]out, although specific Cav1.3 channel inhibition did not affect firing rate. Inhibition of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) also decreased [DA]out, demonstrating the importance of intracellular Ca2+ stores for somatodendritic release. Finally, in vivo FSCAV measurements showed that basal [DA]out in the SNc was 79.8 ± 10.9 nM in urethane‐anesthetized rats, which was enhanced by amphetamine. Overall, our findings indicate that although tonic somatodendritic DA release is largely independent of action potentials, basal [DA]out is strongly regulated by voltage‐dependent Ca2+ influx and release of intracellular Ca2+.