Billy Chieng

Dendritic Function of Tau Mediates Amyloid-β Toxicity in Alzheimer's Disease Mouse Models

Alzheimers disease (AD) is characterized by amyloid-beta (Abeta) and tau deposition in brain. It has emerged that Abeta toxicity is tau dependent, although mechanistically this link remains unclear. Here, we show that tau, known as axonal protein, has a dendritic function in postsynaptic targeting of the Src kinase Fyn, a substrate of which is the NMDA receptor (NR). Missorting of tau in transgenic mice expressing truncated tau (Deltatau) and absence of tau in tau(-/-) mice both disrupt postsynaptic targeting of Fyn. This uncouples NR-mediated excitotoxicity and hence mitigates Abeta toxicity. Deltatau expression and tau deficiency prevent memory deficits and improve survival in Abeta-forming APP23 mice, a model of AD. These deficits are also fully rescued with a peptide that uncouples the Fyn-mediated interaction of NR and PSD-95 in vivo. Our findings suggest that this dendritic role of tau confers Abeta toxicity at the postsynapse with direct implications for pathogenesis and treatment of AD.

Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro

1 In this study we have examined the effects of nociceptin, an endogenous ligand for the opioid‐like receptor ORL1, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, using intracellular and whole cell patch clamp recording. 2 When locus coeruleus neurones were voltage clamped to −60 mV, application of nociceptin caused an outward current in all cells examined (n = 49), with an EC50 of 90 nM. Neither the potency nor the maximal effect of nociceptin was altered in the presence of the peptidase inhibitors, bestatin (20 μm) or thiorphan (2 μm). 3 The outward currents caused by nociceptin in 2.5 mM extracellular K+ reversed polarity at −123 mV, more negative than the predicted K+ reversal potential of −105 mV. Increasing extracellular K+ to 6.5 mM resulted in a shift of the reversal potential of +25 mV, a shift consistent with a K+ conductance. The conductance activated by nociceptin showed mild inward rectification. 4 Application of a high concentration of nociceptin (3 μm) occluded the current produced by simultaneous application of high concentrations of Met‐enkephalin (10 μm), (3 μm) somatostatin and UK 14304 (3 μm), indicating that nociceptin activated the same conductance as μ‐opioid and somatostatin receptors and α2‐adrenoceptors. 5 The actions of nociceptin were weakly antagonized by the opioid antagonist, naloxone, with pKb′s estimated from 2 cells of −4.23 and −4.33. The μ‐opioid antagonist, CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Pen‐Thr‐NH2, 1 μm), the opioid antagonist, nalorphine (30 μm), or the somatostatin antagonist, CPP (cyclo(7‐aminoheptanoyl‐Phe‐D‐Tip‐Lys‐Thr[Bzl]) 3 μm) did not affect the nociceptin‐induced current. 6 Dynorphin A (3 μm), another putative endogenous ligand for ORL1, caused a robust outward current in locus coeruleus neurones that was, however, completely antagonized by moderate concentrations of naloxone (300 nM‐1 μm). 7 Continuous application of nociceptin (3 μm) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 120s. Desensitization was largely homologous because simultaneous application of Met‐enkephalin (30 μm) during the desensitized period of the nociceptin response resulted in an outward current that was 92% of control responses to Met‐enkephalin in the same cells. Conversely, continuous application of Met‐enkephalin (30 μm) resulted in a decrease of Met‐enkephalin current to a steady level that was 54% of the initial current. During this desensitized period application of nociceptin (3 μm) resulted in a current that was 78% of the control responses to nociceptin in the same cells. 8 Thus nociceptin potently activates an inwardly rectifying K+ conductance in locus coeruleus neurones, with a pharmacological profile consistent with activation of the ORL1 receptor. Dynorphin A does not appear to be a ligand for ORL1 in rat locus coeruleus neurones.

Hyperpolarization by opioids acting on μ-receptors of a sub-population of rat periaqueductal gray neurones in vitro

1 The actions of opioids on membrane properties of rat periaqueductal gray neurones were investigated using intracellular recordings from single neurones in brain slices. Morphological properties and anatomical location of each impaled neurone were characterized by use of intracellular staining with biocytin. The present paper primarily considers neurones which were directly hyperpolarized by opioids. The accompanying paper considers inhibition of synaptic transmission by opioids. 2 Met‐enkephalin (10–30 μm) hyperpolarized 29% (38/130) of neurones. The hyperpolarization was fully antagonised by naloxone (1 μm, n = 3). The response to Met‐enkephalin was not affected by agents which block synaptic neurotransmission (1 μm tetrodotoxin, and 0.1 μm tetrodotoxin + 4 mm Co2+, n = 3). 3 The specific μ‐receptor agonist, d‐ala‐met‐enkephalin‐glyol (3 μm, n = 17) produced hyperpolarizations of similar amplitude to those produced by Met‐enkephalin (10–30 μm). The EC50 of d‐ala‐met‐enkephalin‐glyol was 80 nm and the maximum response was achieved at 1–3 μm. The δ‐receptor (d‐Pen‐d‐pen‐enkephalin, 3 μm, n = 7) and κ‐receptor (U50488H, 3 μm, n = 5) agonists had no effect on the membrane properties of these neurones. 4 The opioid‐induced hyperpolarization was associated with an increased potassium conductance. Hyperpolarizations were accompanied by a significant decrease in membrane resistance between −70 and −80 mV, and a significantly greater decrease between −110 and −140 mV (n = 16). Hyperpolarizations reversed polarity at −111 ± 3 mV (n = 16), close to the expected equilibrium potential for potassium ions. The reversal potential of outward currents increased by 24 mV when the extracellular potassium concentration was raised from 2.5 to 6.5 mm, which is close to the value predicted by the Nernst equation (25 mV) for a potassium conductance. 5 Resting inward rectification (reduced input resistance at potentials more negative than −100 mV in the absence of opioids) was significantly greater in neurones which were hyperpolarized by opioids than in those which were not hyperpolarized. The amplitude of action potential after hyperpolarizations was significantly smaller in neurones which were hyperpolarized by opioids. Other membrane properties did not differ significantly between opioid‐sensitive and ‐insensitive neurones. 6 Neurones hyperpolarized by opioids were multipolar (58%), triangular (21%) or fusiform (5%) in shape with a soma diameter of 22 ± μm (n = 19, longest axis). Dendritic spread was in a large radiating pattern, usually in all directions, with axons usually originating from primary dendrites. The axons were usually branched and projected in several directions. Morphological properties did not differ significantly between opioid‐sensitive and ‐insensitive neurones. 7 Neurones hyperpolarized by opioids were located predominantly in the lateral periaqueductal gray, as well as in the more dorsal areas of the ventrolateral periaqueductal gray, whereas neurones not hyperpolarized by opioids were located in the more ventral areas of the ventrolateral periaqueductal gray. 8 These studies demonstrate that opioids acting on μ‐receptors increase potassium conductance in a sub‐population of large neurones located predominantly in the lateral column of the periaqueductal gray. The neurones hyperpolarized by opioids could be involved in the antinociceptive actions of opioids, but might also be involved in other functions because a large proportion lie outside of the main ‘antinociceptive zone’ of the periaqueductal gray. It is also unlikely that these neurones are GABAergic, suggesting that they might not participate in the postulated antinociceptive action of opioids mediated via disinhibition of neurones which project to the ventral medulla.

The Thalamostriatal Pathway and Cholinergic Control of Goal-Directed Action: Interlacing New with Existing Learning in the Striatum

The capacity for goal-directed action depends on encoding specific action-outcome associations, a learning process mediated by the posterior dorsomedial striatum (pDMS). In a changing environment, plasticity has to remain flexible, requiring interference between new and existing learning to be minimized, yet it is not known how new and existing learning are interlaced in this way. Here we investigated the role of the thalamostriatal pathway linking the parafascicular thalamus (Pf) with cholinergic interneurons (CINs) in the pDMS in this process. Removing the excitatory input from Pf to the CINs was found to reduce the firing rate and intrinsic activity of these neurons and produced an enduring deficit in goal-directed learning after changes in the action-outcome contingency. Disconnection of the Pf-pDMS pathway produced similar behavioral effects. These data suggest that CINs reduce interference between new and existing learning, consistent with claims that the thalamostriatal pathway exerts state control over learning-related plasticity.

Inhibition by opioids acting on μ‐receptors of GABAergic and glutamatergic postsynaptic potentials in single rat periaqueductal gray neurones in vitro

1 Membrane properties of rat periaqueductal gray neurones were investigated by use of intracellular recordings from single neurones in brain slices. Morphological properties and anatomical location of each impaled neurone were characterized by intracellular staining with biocytin. The present paper considers the properties of electrically‐evoked and spontaneous postsynaptic potentials impinging on periaqueductal gray neurones, and the actions of opioids on postsynaptic potentials in neurones which were not directly hyperpolarized by opioids. The preceding paper considers neurones which were hyperpolarized by opioids. 2 Electrical stimulation in the vicinity of impaled neurones evoked postsynaptic potentials having fast (duration at half‐maximal amplitude 37 ± 2 ms, n = 65) and in some cases slow (duration at half‐maximal amplitude 817 ± 187 ms, n = 3) components. Amplitudes of evoked potentials were dependent on stimulus voltage, membrane potential, and were abolished during superfusion with solutions containing tetrodoxotoxin (100 nm to 1 μm, n = 5) or Co2+ (4 mm, n = 2). 3 Fast postsynaptic potentials were mediated predominantly by activation of glutamate and GABAA receptors. The GABAA‐receptor antagonist, bicucuilline (30 μm), inhibited postsynaptic potentials by 44 ± 8% (n = 14). The non‐NMDA‐receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (10 μm), inhibited postsynaptic potentials by 48 ± 6% (n = 16). Combined superfusion of bicuculline (30 μm) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (10 μm) inhibited postsynaptic potentials by 93 ± 1% (n = 8). Additional superfusion of the NMDA‐receptor antagonist, (±)‐2‐amino‐5‐phosphonovaleric acid (50 μm) inhibited synaptic potentials by 94 ± 1% (n = 3). 4 Slow inhibitory postsynaptic potentials were observed in 12% (8/65) of neurones. They reversed polarity between − 100 and − 110 mV, and were abolished by superfusion with spiperone (1 μm, n = 2), but not the α2‐antagonist, idazoxan (3 μm, n = 2). 5 Selective μ‐receptor agonists inhibited fast postsynaptic potentials in all neurones tested which were not directly hyperpolarized by opioids. Met‐enkephalin (30 μm) and Tyr‐d‐Ala‐Gly‐MePhe‐Glyol (3 μm) inhibited postsynaptic potentials by 53 ± 3% and 49 ± 3%, respectively. This effect was completely antagonised by naloxone (1 μm, n = 3). A small inhibition produced by the selective δ‐receptor agonist, Tyr‐d‐Pen‐Gly‐Phe‐d‐Pen‐enkephalin (3 μm, 26 ± 4%, n = 14), was antagonized by naloxone (1 μm), but not by the selective δ‐receptor antagonist, naltrindole (10 nm), suggesting non‐specific μ‐receptor activation by this agonist. The selective κ‐receptor agonist, U50488H (3 μm), also consistently inhibited postsynaptic potentials by 45 ± 15% (n = 4). However, this effect was not fully reversed by naloxone (1 μm) suggesting a non‐specific action. 6 Both glutamatergic and GABAergic components of fast postsynaptic potentials were inhibited by Met‐enkephalin (10 or 30 μm). Met‐enkephalin inhibited postsynaptic potentials by 55 ± 5% (n = 12) in the presence of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (10 μm, predominantly GABAergic component). Met‐enkephalin did not affect the response to GABA applied directly by pressure ejection, indicating that opioids exclusively inhibited presynaptic release of GABA. Met‐enkephalin (10–30 μm) inhibited postsynaptic potentials by 48 ± 6% (n = 11) in the presence of bicuculline (30 μm, predominantly glutamatergic component). In the presence of both bicuculline and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, Met‐enkephalin inhibited the small residual component of the synaptic potential by 42 ± 15% (n = 2). 7 Frequent spontaneous synaptic potentials were also observed in 11% (10/94) of the neurones which were not directly hyperpolarized by opioids. These were reversibly abolished by bicuculline (30 μm, n = 5) and substantially inhibited by Met‐enkephalin (30 μm, n = 6), but were unaffected by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (10 μm, n = 2). 8 In conclusion, fast glutamatergic and GABAergic synaptic potentials were evoked by electrical stimulation throughout the lateral and ventrolateral periaqueductal gray. Slow inhibitory synaptic potentials were also evoked in some neurones. Opioids acting on μ‐receptors inhibited both GABAergic and glutamatergic components of synaptic potentials throughout this brain region.

Induction of δ-Opioid Receptor Function in the Midbrain after Chronic Morphine Treatment

δ-Opioid receptor (DOPr) activation fails to produce cellular physiological responses in many brain regions, including the periaqueductal gray (PAG), despite neural expression of high densities of the receptor. Previous histochemical studies have demonstrated that a variety of stimuli, including chronic morphine treatment, induce the translocation of DOPr from intracellular pools to the surface membrane of CNS neurons. PAG neurons in slices taken from untreated mice exhibited μ-opioid receptor (MOPr) but not DOPr-mediated presynaptic inhibition of GABAergic synaptic currents. In contrast, after 5-6 d of chronic morphine treatment, DOPr stimulation inhibited synaptic GABA release onto most neurons. Shorter exposure to morphine in vitro (upto 4 h) or in vivo (18 h) did not induce functional DOPr responses. DOPr-mediated presynaptic inhibition could not be induced in slices from untreated animals by increasing synaptic activity in vitro using high extracellular potassium concentrations or activation of protein kinase A. Induction of functional DOPr signaling by chronic morphine required MOPr expression, because no DOPr receptor responses were observed in MOPr knock-out mice. DOPr agonists also had no effect on miniature IPSCs in β-arrestin-2 knock-out mice after chronic morphine. These results suggest that induction of DOPr-mediated actions in PAG by chronic morphine requires prolonged MOPr stimulation and expression of β-arrestin-2.

Characterization of neurons in the rat central nucleus of the amygdala: Cellular physiology, morphology, and opioid sensitivity

The central nucleus of the amygdala (CeA) orchestrates autonomic and other behavioral and physiological responses to conditioned stimuli that are aversive or elicit fear. As a related CeA function is the expression of hypoalgesia induced by conditioned stimuli or systemic morphine administration, we examined postsynaptic opioid modulation of neurons in each major CeA subdivision. Following electrophysiological recording, biocytin‐filled neurons were precisely located in CeA regions identified by chemoarchitecture (enkephalin‐immunoreactivity) and cytoarchitecture (DAPI nuclear staining) in fixed adult rat brain slices. This revealed a striking distribution of physiological types, as 92% of neurons in capsular CeA were classified as late‐firing, whereas no neurons in the medial CeA were of this class. In contrast, 60% or more of neurons in the lateral and medial CeA were low‐threshold bursting neurons. Mu‐opioid receptor (MOPR) agonists induced postsynaptic inhibitory potassium currents in 61% of CeA cells, and this ratio was maintained in each subdivision and for each physiological class of neuron. However, MOPR agonists more frequently inhibited bipolar/fusiform cells than triangular or multipolar neurons. A subpopulation of MOPR‐expressing neurons were also inhibited by delta opioid receptor agonists, whereas a separate population were inhibited kappa opioid receptors (KOPR). The MOPR agonist DAMGO inhibited 9/9 CeM neurons with projections to the parabrachial nucleus identified by retrograde tracer injection. These data support models of striatopallidal organization that have identified striatal‐like and pallidal‐like CeA regions. Opioids can directly inhibit output from each subdivision by activating postsynaptic MOPRs or KOPRs on distinct subpopulations of opioid‐sensitive neurons. J. Comp. Neurol. 497:910–927, 2006.

Distinct cellular properties of identified dopaminergic and GABAergic neurons in the mouse ventral tegmental area

Non‐technical summary The lower mid‐brain of rodent is home to addiction and hedonism of substances of abuse. Due to overlapping physiological properties among different types of nerve cell within this brain region, it has been difficult to selectively study the physiology of a single type of nerve cell. Here, using gene technology together with selective antibody detection, we have exclusively identified the two dominant populations of nerve cells and found dopamine‐containing cells five times more abundant. We found clear non‐overlapping cellular characteristics between the two types that are unequivocal predictors for selection of nerve cells, whilst other previously employed cellular criteria were found to be less useful.

Effects of Repeated Cocaine Exposure on Habit Learning and Reversal by N -Acetylcysteine

Exposure to drugs of abuse can result in a loss of control over both drug- and nondrug-related actions by accelerating the transition from goal-directed to habitual control, an effect argued to reflect changes in glutamate homeostasis. Here we examined whether exposure to cocaine accelerates habit learning and used in vitro electrophysiology to investigate its effects on measures of synaptic plasticity in the dorsomedial (DMS) and dorsolateral (DLS) striatum, areas critical for actions and habits, respectively. We then administered N-acetylcysteine (NAC) in an attempt to normalize glutamate homeostasis and hence reverse the cellular and behavioral effects of cocaine exposure. Rats received daily injections of cocaine (30 mg/kg) for 6 days and were then trained to lever press for a food reward. We used outcome devaluation and whole-cell patch-clamp electrophysiology to assess the behavioral and cellular effects of cocaine exposure. We then examined the ability of NAC to reverse the effects of cocaine exposure on these measures. Cocaine treatment produced a deficit in goal-directed action, as assessed by outcome devaluation, and increased the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) in the DMS but not in the DLS. Importantly, NAC treatment both normalized EPSC frequency and promoted goal-directed control in cocaine-treated rats. The promotion of goal-directed control has the potential to improve treatment outcomes in human cocaine addicts.

Increased fos-like immunoreactivity in the periaqueductal gray of anaesthetised rats during opiate withdrawal

Staining of c-fos-like-immunoreactivity (CFIR) in neurones was used to study neuronal activation within subdivisions of periaqueductal gray (PAG), and in locus coeruleus and ventral tegmental area during opiate withdrawal in awake and anaesthetised, morphine-dependent rats. The number of CFIR containing neurones was significantly increased during naloxone-precipitated withdrawal in lateral and ventrolateral, particularly the caudal ventrolateral PAG. No changes were observed in dorsal-intermediate or dorsal-caudal PAG. In awake rats, a similar but more generalised increase in CFIR was observed in PAG following naloxone-precipitated withdrawal. Increases in ventral tegmental area and locus coeruleus during naloxone-precipitated withdrawal under anaesthesia varied greatly between animals. Induction of c-fos in lateral and ventrolateral PAG during withdrawal is consistent with known functions of these regions, involving the integration of autonomic and somatic components of defensive and escape behaviours which are characteristic signs of opiate withdrawal.