Elena E. Bagley

Cellular Actions Of Opioids And Other Analgesics: Implications For Synergism In Pain Relief

1. μ‐Opioid receptor agonists mediate their central analgesic effects by actions on neurons within brain regions such as the mid‐brain periaqueductal grey (PAG). Within the PAG, μ‐opioid receptor‐mediated analgesia results from inhibition of GABAergic influences on output projection neurons. We have established that μ‐opioid receptor activation in the PAG causes a presynaptic inhibition of GABA release that is mediated by activation of a voltage‐dependent K+ channel via 12‐lipoxygenase (LOX) metabolites of arachidonic acid.

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.

GABA Transporter Currents Activated by Protein Kinase A Excite Midbrain Neurons during Opioid Withdrawal

Adaptations in neurons of the midbrain periaqueductal gray (PAG) induced by chronic morphine treatment mediate expression of many signs of opioid withdrawal. The abnormally elevated action potential rate of opioid-sensitive PAG neurons is a likely cellular mechanism for withdrawal expression. We report here that opioid withdrawal in vitro induced an opioid-sensitive cation current that was mediated by the GABA transporter-1 (GAT-1) and required activation of protein kinase A (PKA) for its expression. Inhibition of GAT-1 or PKA also prevented withdrawal-induced hyperexcitation of PAG neurons. Our findings indicate that GAT-1 currents can directly increase the action potential rates of neurons and that GAT-1 may be a target for therapy to alleviate opioid-withdrawal symptoms.

Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine

The midbrain periaqueductal gray (PAG) is a major site of opioid analgesic action, and a significant site of cellular adaptations to chronic morphine treatment (CMT). We examined μ‐opioid receptor (MOP) regulation of voltage‐gated calcium channel currents (ICa) and G‐protein‐activated K channel currents (GIRK) in PAG neurons from CMT mice. Mice were injected s.c. with 300 mg kg−1 of morphine base in a slow release emulsion three times over 5 days, or with emulsion alone (vehicles). This protocol produced significant tolerance to the antinociceptive effects of morphine in a test of thermal nociception. Voltage clamp recordings were made of ICa in acutely isolated PAG neurons and GIRK in PAG slices. The MOP agonist DAMGO (Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol enkephalin) inhibited ICa in neurons from CMT mice (230 nM) with a similar potency to vehicle (150 nM), but with a reduced maximal effectiveness (37% inhibition in vehicle neurons, 27% in CMT neurons). Inhibition of ICa by the GABAB agonist baclofen was not altered by CMT. Met‐enkephalin‐activated GIRK currents recorded in PAG slices were significantly smaller in neurons from CMT mice than vehicles, while GIRK currents activated by baclofen were unaltered. These data demonstrate that CMT‐induced antinociceptive tolerance is accompanied by homologous reduction in the effectiveness of MOP agonists to inhibit ICa and activate GIRK. Thus, a reduction in MOP number and/or functional coupling to G proteins accompanies the characteristic cellular adaptations to CMT previously described in PAG neurons.

Inhibition by adenosine receptor agonists of synaptic transmission in rat periaqueductal grey neurons

1 The actions of selective adenosine A1 and A2 receptor agonists were examined on synaptic currents in periaqueductal grey (PAG) neurons using patch‐clamp recordings in brain slices. 2 The A1 receptor agonist 2‐chloro‐N‐cyclopentyladenosine (CCPA), but not the A2 agonist, 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS21680), inhibited both electrically evoked inhibitory (eIPSCs) and excitatory (eEPSCs) postsynaptic currents. The actions of CCPA were reversed by the A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX). 3 In the absence or presence of forskolin, DPCPX had no effect on eIPSCs, suggesting that concentrations of tonically released adenosine are not sufficient to inhibit synaptic transmission in the PAG. 4 CCPA decreased the frequency of spontaneous miniature action potential‐independent IPSCs (mIPSCs) but had no effect on their amplitude distributions. Inhibition persisted in nominally Ca2+‐free, high Mg2+ solutions and in 4‐aminopyridine. 5 The CCPA‐induced decrease in mIPSC frequency was partially blocked by the non‐selective protein kinase inhibitor staurosporine, the specific protein kinase A inhibitor 8‐para‐chlorophenylthioadenosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐CPT‐cAMPS), and by 8‐bromoadenosine cyclic 3′,5′ monophosphate (8‐Br‐cAMP). 6 These results suggest that A1 adenosine receptor agonists inhibit both GABAergic and glutamatergic synaptic transmission in the PAG. Inhibition of GABAergic transmission is mediated by presynaptic mechanisms that partly involve protein kinase A.

Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors

Neurotransmitter transporters can affect neuronal excitability indirectly via modulation of neurotransmitter concentrations or directly via transporter currents. A physiological or pathophysiological role for transporter currents has not been described. We found that GABA transporter 1 (GAT-1) cation currents directly increased GABAergic neuronal excitability and synaptic GABA release in the periaqueductal gray (PAG) during opioid withdrawal in rodents. In contrast, GAT-1 did not indirectly alter GABA receptor responses via modulation of extracellular GABA concentrations. Notably, we found that GAT-1–induced increases in GABAergic activity contributed to many PAG-mediated signs of opioid withdrawal. Together, these data support the hypothesis that GAT-1 activity directly produces opioid withdrawal signs through direct hyperexcitation of GABAergic PAG neurons and nerve terminals, which presumably enhances GABAergic inhibition of PAG output neurons. These data provide, to the best of our knowledge, the first evidence that dysregulation of a neurotransmitter transporter current is important for the maladaptive plasticity that underlies opiate withdrawal.

Cellular actions of opioids on periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR-1

Patch clamp recordings were made from periaqueductal grey (PAG) neurons in vitro to investigate the cellular actions of opioids in wild‐type C57B16/J mice and mutant mice lacking the first exon of the μ‐opioid (MOP) receptor. In wild‐type mice, the κ‐(KOP) agonist U‐69593 (300 nM) and the mixed μ/δ‐opioid agonist met‐enkephalin (10 μM), but not the δ‐(DOP) agonist deltorphin (300 nM), reduced the amplitude of evoked GABAA‐mediated inhibitory postsynaptic currents (IPSCs). Met‐enkephalin and U‐69593 also reduced the rate of spontaneous miniature IPSCs, but had no effect on their amplitude and kinetics. In μ‐receptor‐deleted mice, only U‐69593 (300 nM) reduced the amplitude of evoked IPSCs. In wild‐type mice, the MOP agonist DAMGO (3 μM) produced an outward current in 76% of the neurons. Deltorphin and U‐69593 produced outward currents in 24 and 32% of the neurons, respectively. In μ‐receptor‐deleted mice, deltorphin and U‐69593 produced similar outward currents in 32 and 27% of the neurons, respectively, while DAMGO was without effect. All neurons in both the wild‐type and μ‐receptor‐deleted mice responded with similar outward currents to either the GABAB receptor agonist baclofen (10 μM), or the opioid‐like receptor ORL1 (NOP) agonist nociceptin (300 nM). The DAMGO‐, deltorphin‐, U‐69593‐, baclofen‐ and nociceptin‐induced currents displayed inward rectification and reversed polarity at −109 to −116 mV. These findings indicate that μ‐, δ‐ and κ‐opioid receptor activation has complex pre‐ and postsynaptic actions within the mouse PAG. This differs to the rat PAG where only μ‐opioid receptor actions have been observed.

Cellular actions of somatostatin on rat periaqueductal grey neurons in vitro

Functional studies indicate that the midbrain periaqueductal grey (PAG) is involved in the analgesic actions of somatostatin; however, the cellular actions of somatostatin in this brain region are unknown. In the present study, whole‐cell patch clamp recordings were made from rat PAG neurons in vitro. In 93% of acutely isolated neurons, somatostatin inhibited Ca2+‐channel currents. This effect was mimicked by the sst‐2 selective agonist BIM‐23027, but not by the sst‐1 and sst‐5 selective agonists CH‐275 and L‐362855. In brain slices, 81% of neurons responded to somatostatin (300 nM) with an increase in K+ conductance that reversed polarity at −114 mV. A greater proportion of somatostatin‐sensitive neurons (93%) than somatostatin‐insensitive neurons (53%) responded to the opioid agonist met‐enkephalin (10 μM). Somatostatin also reduced the amplitude of evoked GABAA‐mediated inhibitory postsynaptic currents (IPSCs). The actions of somatostatin in brain slices were mimicked by BIM‐23027, but not by CH‐275. Somatostatin had a variable effect on the rate of spontaneous miniature IPSCs in normal external potassium solutions. In high external potassium solutions, somatostatin reduced the rate of miniature IPSCs in all neurons, and this inhibition was abolished by addition of Cd2+ (30 μM). Somatostatin had no effect on the amplitude of miniature IPSCs. These results indicate that somatostatin acts via sst‐2 receptors to directly inhibit a subpopulation of PAG neurons by activating a potassium conductance and inhibits GABA release within PAG via a presynaptic Ca2+‐dependent mechanism. Thus, like opioids, somatostatin has the potential to exert pre‐ and postsynaptic disinhibitory effects within the PAG.

Disrupted Prediction Error Links Excessive Amygdala Activation to Excessive Fear.

Basolateral amygdala (BLA) is critical for fear learning, and its heightened activation is widely thought to underpin a variety of anxiety disorders. Here we used chemogenetic techniques in rats to study the consequences of heightened BLA activation for fear learning and memory, and to specifically identify a mechanism linking increased activity of BLA glutamatergic neurons to aberrant fear. We expressed the excitatory hM3Dq DREADD in rat BLA glutamatergic neurons and showed that CNO acted selectively to increase their activity, depolarizing these neurons and increasing their firing rates. This chemogenetic excitation of BLA glutamatergic neurons had no effect on the acquisition of simple fear learning, regardless of whether this learning led to a weak or strong fear memory. However, in an associative blocking task, chemogenetic excitation of BLA glutamatergic neurons yielded significant learning to a blocked conditioned stimulus, which otherwise should not have been learned about. Moreover, in an overexpectation task, chemogenetic manipulation of BLA glutamatergic neurons prevented use of negative prediction error to reduce fear learning, leading to significant impairments in fear inhibition. These effects were not attributable to the chemogenetic manipulation enhancing arousal, increasing asymptotic levels of fear learning or fear memory consolidation. Instead, chemogenetic excitation of BLA glutamatergic neurons disrupted use of prediction error to regulate fear learning. SIGNIFICANCE STATEMENT Several neuropsychiatric disorders are characterized by heightened activation of the amygdala. This heightened activation has been hypothesized to underlie increased emotional reactivity, fear over generalization, and deficits in fear inhibition. Yet the mechanisms linking heightened amygdala activation to heightened emotional learning are elusive. Here we combined chemogenetic excitation of rat basolateral amygdala glutamatergic neurons with a variety of behavioral approaches to show that, although simple fear learning is unaffected, the use of prediction error to regulate this learning is profoundly disrupted, leading to formation of inappropriate fear associations and impaired fear inhibition.

β‐Arrestin‐2 knockout prevents development of cellular μ‐opioid receptor tolerance but does not affect opioid‐withdrawal‐related adaptations in single PAG neurons

Tolerance to the behavioural effects of morphine is blunted in β‐arrestin‐2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β‐arrestin‐2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena.