Effects of subanesthetic ketamine and (2R,6R) hydroxynorketamine on working memory and synaptic transmission in the nucleus reuniens in mice.

RATIONALE: Acute cognitive impairment and abuse potential of ketamine incentivizes the search for alternatives to ketamine for clinical management of treatment-resistant depression. Recently, (2R,6R) hydroxynorketamine ((2R,6R)-HNK), a metabolite of ketamine, has shown promise due to its reported lack of ketamine-like reinforcing properties. Nonetheless, the effect of (2R,6R)-HNK on cognition has not been reported. METHOD: Adult male mice were placed in a Y-maze to measure spatial working memory (SWM) 24 h after treatment with either a single or repeated subanesthetic dose of (2R,6R)-HNK or ketamine. To determine the effect of the drug regimens on synaptic mechanisms in neural circuits deemed critical for SWM, we conducted patch-clamp electrophysiological recordings from neurons in the midline thalamic nucleus reuniens (RE) in response to optogenetic stimulation of medial prefrontal cortex (mPFC) inputs in acutely prepared brain slices. RESULTS: Single or repeated treatment with a 10 mg/kg dose of either drug did not impact performance in a Y-maze. However, single administration of a ½-log higher dose (32 mg/kg) of ketamine significantly reduced SWM. The same dose of (2R,6R)-HNK did not produce SWM deficits. Interestingly, repeated administration of either drugs at the 32 mg/kg had no effect on SWM performances. Concomitant to these effects on SWM, only single injection of 32 mg/kg of ketamine was found to increase the mPFC-driven action potential firing activity in the RE neurons. Conversely, both single and repeated administration of the 32 mg/kg dose of (2R,6R)-HNK but not ketamine, increased the input resistance of the RE neurons. CONCLUSION: Our results indicate that acute treatment of ketamine at 32 mg/kg increases mPFC-driven firing activity of RE neurons, and this contributes to the ketamine-mediated cognitive deficit. Secondly, sub-chronic treatment with the same dose of ketamine likely induces tolerance. Although single or repeated administration of the 32 mg/kg dose of (2R,6R)-HNK can alter intrinsic properties of RE neurons, this dose does not produce cognitive deficit or changes in synaptic mechanism in the RE. This article is part of the special Issue on 'Stress, Addiction and Plasticity'.

Nucleus Reuniens Afferents in Hippocampus Modulate CA1 Network Function via Monosynaptic Excitation and Polysynaptic Inhibition.

The thalamic midline nucleus reuniens modulates hippocampal CA1 and subiculum function via dense projections to the stratum lacunosum-moleculare (SLM). Previously, anatomical data has shown that reuniens inputs in the SLM form synapses with dendrites of both CA1 principal cells and inhibitory interneurons. However, the ability of thalamic inputs to excite the CA1 principal cells remains controversial. In addition, nothing is known about the impact of reuniens inputs on diverse subpopulations of interneurons in CA1. Therefore, using whole cell patch-clamp electrophysiology in ex vivo hippocampal slices of wild-type and transgenic mice, we measured synaptic responses in different CA1 neuronal subtypes to optogenetic stimulation of reuniens afferents. Our data shows that reuniens inputs mediate both excitation and inhibition of the CA1 principal cells. However, the optogenetic excitation of the reuniens inputs failed to drive action potential firing in the majority of the principal cells. While the excitatory postsynaptic currents were mediated via direct monosynaptic activation of the CA1 principal cells, the inhibitory postsynaptic currents were generated polysynaptically via activation of local GABAergic interneurons. Moreover, we demonstrate that optogenetic stimulation of reuniens inputs differentially recruit at least two distinct and non-overlapping subpopulations of local GABAergic interneurons in CA1. We show that neurogliaform cells located in SLM, and calretinin-containing interneuron-selective interneurons at the SLM/stratum radiatum border can be excited by stimulation of reuniens inputs. Together, our data demonstrate that optogenetic stimulation of reuniens afferents can mediate excitation, feedforward inhibition, and disinhibition of the postsynaptic CA1 principal cells via multiple direct and indirect mechanisms.

The Entorhinal Cortical Alvear Pathway Differentially Excites Pyramidal Cells and Interneuron Subtypes in Hippocampal CA1.

The entorhinal cortex alvear pathway is a major excitatory input to hippocampal CA1, yet nothing is known about its physiological impact. We investigated the alvear pathway projection and innervation of neurons in CA1 using optogenetics and whole cell patch clamp methods in transgenic mouse brain slices. Using this approach, we show that the medial entorhinal cortical alvear inputs onto CA1 pyramidal cells (PCs) and interneurons with cell bodies located in stratum oriens were monosynaptic, had low release probability, and were mediated by glutamate receptors. Optogenetic theta burst stimulation was unable to elicit suprathreshold activation of CA1 PCs but was capable of activating CA1 interneurons. However, different subtypes of interneurons were not equally affected. Higher burst action potential frequencies were observed in parvalbumin-expressing interneurons relative to vasoactive-intestinal peptide-expressing or a subset of oriens lacunosum-moleculare (O-LM) interneurons. Furthermore, alvear excitatory synaptic responses were observed in greater than 70% of PV and VIP interneurons and less than 20% of O-LM cells. Finally, greater than 50% of theta burst-driven inhibitory postsynaptic current amplitudes in CA1 PCs were inhibited by optogenetic suppression of PV interneurons. Therefore, our data suggest that the alvear pathway primarily affects hippocampal CA1 function through feedforward inhibition of select interneuron subtypes.

Photodynamic Modification of Native HCN Channels Expressed in Thalamocortical Neurons.

The photodynamic process requires three elements: light, oxygen, and photosensitizer, and involves the formation of singlet oxygen, the molecular oxygen in excited electronic states. Previously, we reported that heterologously expressed hyperpolarization-activated cAMP-gated (HCN) channels in excised membrane patches are sensitive to photodynamic modification (PDM). Here we extend this study to native HCN channels expressed in thalamocortical (TC) neurons in the ventrobasal (VB) complex of the thalamus and dopaminergic neurons (DA) of the ventral tegmental area (VTA). To do this, we introduced the photosensitizer FITC-cAMP into TCs or DAs of rodent brain slices via a whole-cell patch-clamp recording pipette. After illumination with blue light pulses, we observed an increase in the voltage-insensitive, instantaneous Iinst component, accompanied by a long-lasting decrease in the hyperpolarization-dependent Ih component. Both Ih and the increased Iinst after PDM could be blocked by the HCN blockers Cs+ and ZD7288. When FITC and cAMP were dissociated and loaded into neurons as two separate chemicals, light application did not result in any long-lasting changes of the HCN currents. In contrast, light pulses applied to HCN2-/- neurons loaded with FITC-cAMP generated a much greater reduction in the Iinst component compared to that of WT neurons. Next, we investigated the impact of the long-lasting increases in Iinst after PDM on the cellular physiology of VB neurons. Consistent with an upregulation of HCN channel function, PDM elicited a depolarization of the resting membrane potential (RMP). Importantly, Trolox-C, an effective quencher for singlet oxygen, could block the PDM-dependent increase in Iinst and depolarization of the RMP. We propose that PDM of native HCN channels under physiological conditions may provide a photodynamic approach to alleviate HCN channelopathy in certain pathological conditions.

Acetylcholine Release Inhibits Distinct Excitatory Inputs Onto Hippocampal CA1 Pyramidal Neurons via Different Cellular and Network Mechanisms.

In hippocampal CA1, muscarinic acetylcholine (ACh) receptor (mAChR) activation via exogenous application of cholinergic agonists has been shown to presynaptically inhibit Schaffer collateral (SC) glutamatergic inputs in stratum radiatum (SR), and temporoammonic (TA) and thalamic nucleus reuniens (RE) glutamatergic inputs in stratum lacunosum-moleculare (SLM). However, steady-state uniform mAChR activation may not mimic the effect of ACh release in an intact hippocampal network. To more accurately examine the effect of ACh release on glutamatergic synaptic efficacy, we measured electrically evoked synaptic responses in CA1 pyramidal cells (PCs) following the optogenetic release of ACh in genetically modified mouse brain slices. The ratio of synaptic amplitudes in response to paired-pulse SR stimulation (stimulus 2/stimulus 1) was significantly reduced by the optogenetic release of ACh, consistent with a postsynaptic decrease in synaptic efficacy. The effect of ACh release was blocked by the M3 receptor antagonist 4-DAMP, the GABAB receptor antagonist CGP 52432, inclusion of GDP-ß-S, cesium, QX314 in the intracellular patch clamp solution, or extracellular barium. These observations suggest that ACh release decreased SC synaptic transmission through an M3 muscarinic receptor-mediated increase in inhibitory interneuron excitability, which activate GABAB receptors and inwardly rectifying potassium channels on CA1 pyramidal cells. In contrast, the ratio of synaptic amplitudes in response to paired-pulse stimulation in the SLM was increased by ACh release, consistent with presynaptic inhibition. ACh-mediated effects in SLM were blocked by the M2 receptor antagonist AF-DX 116, presumably located on presynaptic terminals. Therefore, our data indicate that ACh release differentially modulates excitatory inputs in SR and SLM of CA1 through different cellular and network mechanisms.

Restoration of Kv7 Channel-Mediated Inhibition Reduces Cued-Reinstatement of Cocaine Seeking.

Cocaine addicts display increased sensitivity to drug-associated cues, due in part to changes in the prelimbic prefrontal cortex (PL-PFC). The cellular mechanisms underlying cue-induced reinstatement of cocaine seeking remain unknown. Reinforcement learning for addictive drugs may produce persistent maladaptations in intrinsic excitability within sparse subsets of PFC pyramidal neurons. Using a model of relapse in male rats, we sampled >600 neurons to examine spike frequency adaptation (SFA) and afterhyperpolarizations (AHPs), two systems that attenuate low-frequency inputs to regulate neuronal synchronization. We observed that training to self-administer cocaine or nondrug (sucrose) reinforcers decreased SFA and AHPs in a subpopulation of PL-PFC neurons. Only with cocaine did the resulting hyperexcitability persist through extinction training and increase during reinstatement. In neurons with intact SFA, dopamine enhanced excitability by inhibiting Kv7 potassium channels that mediate SFA. However, dopamine effects were occluded in neurons from cocaine-experienced rats, where SFA and AHPs were reduced. Pharmacological stabilization of Kv7 channels with retigabine restored SFA and Kv7 channel function in neuroadapted cells. When microinjected bilaterally into the PL-PFC 10 min before reinstatement testing, retigabine reduced cue-induced reinstatement of cocaine seeking. Last, using cFos-GFP transgenic rats, we found that the loss of SFA correlated with the expression of cFos-GFP following both extinction and re-exposure to drug-associated cues. Together, these data suggest that cocaine self-administration desensitizes inhibitory Kv7 channels in a subpopulation of PL-PFC neurons. This subpopulation of neurons may represent a persistent neural ensemble responsible for driving drug seeking in response to cues.SIGNIFICANCE STATEMENT Long after the cessation of drug use, cues associated with cocaine still elicit drug-seeking behavior, in part by activation of the prelimbic prefrontal cortex (PL-PFC). The underlying cellular mechanisms governing these activated neurons remain unclear. Using a rat model of relapse to cocaine seeking, we identified a population of PL-PFC neurons that become hyperexcitable following chronic cocaine self-administration. These neurons show persistent loss of spike frequency adaptation, reduced afterhyperpolarizations, decreased sensitivity to dopamine, and reduced Kv7 channel-mediated inhibition. Stabilization of Kv7 channel function with retigabine normalized neuronal excitability, restored Kv7 channel currents, and reduced drug-seeking behavior when administered into the PL-PFC before reinstatement. These data highlight a persistent adaptation in a subset of PL-PFC neurons that may contribute to relapse vulnerability.

Arachidonic acid-induced Ca2+ entry and migration in a neuroendocrine cancer cell line.

BACKGROUND: Store-operated Ca2+ entry (SOCE) has been implicated in the migration of some cancer cell lines. The canonical SOCE is defined as the Ca2+ entry that occurs in response to near-maximal depletion of Ca2+ within the endoplasmic reticulum. Alternatively, arachidonic acid (AA) has been shown to induce Ca2+ entry in a store-independent manner through Orai1/Orai3 hetero-multimeric channels. However, the role of this AA-induced Ca2+ entry pathway in cancer cell migration has not been adequately assessed. METHODS: The present study investigated the involvement of AA-induced Ca2+ entry in migration in BON cells, a model gastro-enteropancreatic neuroendocrine tumor (GEPNET) cell line using pharmacological and gene knockdown methods in combination with live cell fluorescence imaging and standard migration assays. RESULTS: We showed that both the store-dependent and AA-induced Ca2+ entry modes could be selectively activated and that exogenous administration of AA resulted in Ca2+ entry that was pharmacologically distinct from SOCE. Also, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-induced Ca2+ entry channel required the expression of Orai3 as well as Orai1. Moreover, we showed that AA treatment enhanced the migration of BON cells and that this migration could be abrogated by selective inhibition of the AA-induced Ca2+ entry. CONCLUSIONS: Taken together, these data revealed that an alternative Orai3-dependent Ca2+ entry pathway is an important signal for GEPNET cell migration.

cAMP-dependent recruitment of acidic organelles for Ca2+ signaling in the salivary gland.

Autonomic neural activation of intracellular Ca2+ release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca2+ signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca2+ release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca2+ from acidic organelles following elevation of cAMP. Our data are consistent with the "trigger" hypothesis where localized release of Ca2+ sensitizes canonical intracellular Ca2+ channels to enhance signals from the endoplasmic reticulum. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands.

Gastro-Enteropancreatic Neuroendocrine Tumor Cell Dynamics in Liver Microvasculature.

For many cancers, liver metastasis is common and usually indicates poor prognosis. Gastro-enteropancreatic neuroendocrine tumors (GEPNETs) of the midgut are a heterogeneous group of cancers that typically remain asymptomatic until they metastasize to the liver. However, the mechanisms by which these usually indolent cancers establish distal metastasis remain unclear. To begin to elucidate this process, we performed standard in vitro assays to assess cell motility, transendothelial migration, and invasion using BON cells, a widely used model GEPNET cell line. In addition, transmission electron microscopy was used in combination with a novel ex vivo organ slice xenograft model to reveal ultrastructural details of the initial events of BON cell extravasation and re-distribution within the liver. The ultrastructural resolution of the extravasation process revealed the route, sequence, and time course by which tumor cells migrated from the sinusoidal lumen into the hepatic parenchyma in this organ slice model. Both standard in vitro assays and our organ slice model indicated that tumor cells migrated through the discontinuous sinusoidal endothelium to invade the liver parenchyma.