Lalitha Kurada

Cholecystokinin facilitates neuronal excitability in the entorhinal cortex via activation of TRPC-like channels

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain, where it interacts with two G protein-coupled receptors (CCK-1 and CCK-2). Activation of both CCK receptors increases the activity of PLC, resulting in increases in intracellular calcium ion (Ca(2+)) release and activation of PKC. Whereas high density of CCK receptors has been detected in the superficial layers of the entorhinal cortex (EC), the functions of CCK in this brain region have not been determined. Here, we studied the effects of CCK on neuronal excitability of layer III pyramidal neurons in the EC. Our results showed that CCK remarkably increased the firing frequency of action potentials (APs). The effects of CCK on neuronal excitability were mediated via activation of CCK-2 receptors and required the functions of G proteins and PLC. However, CCK-mediated facilitation of neuronal excitability was independent of inositol trisphosphate receptors and PKC. CCK facilitated neuronal excitability by activating a cationic channel to generate membrane depolarization. The effects of CCK were suppressed by the generic, nonselective cationic channel blockers, 2-aminoethyldiphenyl borate and flufenamic acid, but potentiated by gadolinium ion and lanthanum ion at 100 μM. Depletion of extracellular Ca(2+) also counteracted CCK-induced increases in AC firing frequency. Moreover, CCK-induced enhancement of neuronal excitability was inhibited significantly by intracellular application of the antibody to transient receptor potential channel 5 (TRPC5), suggesting the involvement of TRPC5 channels. Our results provide a cellular and molecular mechanism to help explain the functions of CCK in vivo.

Activation of neurotensin receptor 1 facilitates neuronal excitability and spatial learning and memory in the entorhinal cortex: beneficial actions in an Alzheimer's disease model.

Neurotensin (NT) is a tridecapeptide distributed in the CNS, including the entorhinal cortex (EC), a structure that is crucial for learning and memory and undergoes the earliest pathological alterations in Alzheimers disease (AD). Whereas NT has been implicated in modulating cognition, the cellular and molecular mechanisms by which NT modifies cognitive processes and the potential therapeutic roles of NT in AD have not been determined. Here we examined the effects of NT on neuronal excitability and spatial learning in the EC, which expresses high density of NT receptors. Brief application of NT induced persistent increases in action potential firing frequency, which could last for at least 1 h. NT-induced facilitation of neuronal excitability was mediated by downregulation of TREK-2 K+ channels and required the functions of NTS1, phospholipase C, and protein kinase C. Microinjection of NT or NTS1 agonist, PD149163, into the EC increased spatial learning as assessed by the Barnes Maze Test. Activation of NTS1 receptors also induced persistent increases in action potential firing frequency and significantly improved the memory status in APP/PS1 mice, an animal model of AD. Our study identifies a cellular substrate underlying learning and memory and suggests that NTS1 agonists may exert beneficial actions in an animal model of AD.

Activation of Group II Metabotropic Glutamate Receptors Inhibits Glutamatergic Transmission in the Rat Entorhinal Cortex via Reduction of Glutamate Release Probability

Glutamate interacts with ionotropic and metabotropic glutamate receptors (mGluRs). Whereas the entorhinal cortex (EC) is a principal structure involved in learning and memory, the roles of mGluRs in synaptic transmission in the EC have not been completely determined. Here, we show that activation of group II mGluRs (mGluR II) induced robust depression of glutamatergic transmission in the EC. The mGluR II-induced depression was due to a selective reduction of presynaptic release probability without alterations of the quantal size and the number of release sites. The mechanisms underlying mGluR II-mediated suppression of glutamate release included the inhibition of presynaptic release machinery and the depression of presynaptic P/Q-type Ca(2+) channels. Whereas mGluR II-induced depression required the function of Gα(i/o) proteins, protein kinase A (PKA) pathway was only involved in mGluR II-mediated inhibition of release machinery and thereby partially required for mGluR II-induced inhibition of glutamate release. Presynaptic stimulation at 5 Hz for 10 min also induced depression of glutamatergic transmission via activation of presynaptic mGluR II suggesting an endogenous role for mGluR II in modulating glutamatergic transmission.

Vasopressin facilitates GABAergic transmission in rat hippocampus via activation of V1A receptors

Whereas vasopressin has been shown to enhance memory possibly by increasing long-term potentiation and direct excitation of the pyramidal neurons in the hippocampus, the effects of vasopressin on GABAergic transmission in the hippocampus remain to be determined. Here we examined the effects of vasopressin on GABAergic transmission onto CA1 pyramidal neurons and our results demonstrate that bath application of [Arg(8)]-vasopressin (AVP) dose-dependently increased the frequency of spontaneous IPSCs (sIPSCs) recorded from CA1 pyramidal neurons via activation of V(1A) receptors. Immunohistological staining and western blot further confirmed that both CA1 pyramidal neurons and interneurons expressed V(1A) receptors. Bath application of AVP altered neither the frequency nor the amplitude of miniature IPSCs in the presence of tetradotoxin and failed to change significantly the amplitude of evoked IPSCs recorded from CA1 pyramidal neurons. AVP increased the firing frequency of action potentials by depolarizing the GABAergic interneurons in the stratum radiatum of CA1 region. AVP-mediated depolarization of interneurons was mediated by inhibition of a background K(+) conductance which was insensitive to extracellular tetraethylammonium, Cs(+), 4-aminopyridine, tertiapin-Q and Ba(2+). AVP-induced depolarization of interneurons was dependent on Gα(q/11) but independent of phospholipase C, intracellular Ca(2+) release and protein kinase C. The inhibitory effects of AVP-mediated modulation of GABA release onto CA1 pyramidal neurons were overwhelmed by its strong excitation of CA1 pyramidal neurons in physiological condition but revealed when its direct excitation of the pyramidal neurons was blocked suggesting that AVP-mediated modulation of GABAergic transmission fine-tunes the excitability of CA1 pyramidal neurons.

Dopaminergic Modulation of GABAergic Transmission in the Entorhinal Cortex: Concerted Roles of α1 Adrenoreceptors, Inward Rectifier K+, and T-Type Ca2+ Channels

Whereas the entorhinal cortex (EC) receives profuse dopaminergic innervations from the midbrain, the effects of dopamine (DA) on γ-Aminobutyric acid (GABA)ergic interneurons in this brain region have not been determined. We probed the actions of DA on GABAA receptor-mediated synaptic transmission in the EC. Application of DA increased the frequency, not the amplitude, of spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) recorded from entorhinal principal neurons, but slightly reduced the amplitude of the evoked IPSCs. The effects of DA were unexpectedly found to be mediated by α1 adrenoreceptors, but not by DA receptors. DA endogenously released by the application of amphetamine also increased the frequency of sIPSCs. Ca(2+) influx via T-type Ca(2+) channels was required for DA-induced facilitation of sIPSCs and mIPSCs. DA depolarized and enhanced the firing frequency of action potentials of interneurons. DA-induced depolarization was independent of extracellular Na(+) and Ca(2+) and did not require the functions of hyperpolarization-activated (Ih) channels and T-type Ca(2+) channels. DA-generated currents showed a reversal potential close to the K(+) reversal potential and inward rectification, suggesting that DA inhibits the inward rectifier K(+) channels (Kirs). Our results demonstrate that DA facilitates GABA release by activating α1 adrenoreceptors to inhibit Kirs, which further depolarize interneurons resulting in secondary Ca(2+) influx via T-type Ca(+) channels.

Adenosinergic depression of glutamatergic transmission in the entorhinal cortex of juvenile rats via reduction of glutamate release probability and the number of releasable vesicles.

Adenosine is an inhibitory neuromodulator that exerts antiepileptic effects in the brain and the entorhinal cortex (EC) is an essential structure involved in temporal lobe epilepsy. Whereas microinjection of adenosine into the EC has been shown to exert powerful antiepileptic effects, the underlying cellular and molecular mechanisms in the EC have not been determined yet. We tested the hypothesis that adenosine-mediated modulation of synaptic transmission contributes to its antiepileptic effects in the EC. Our results demonstrate that adenosine reversibly inhibited glutamatergic transmission via activation of adenosine A1 receptors without effects on GABAergic transmission in layer III pyramidal neurons in the EC. Adenosine-induced depression of glutamatergic transmission was mediated by inhibiting presynaptic glutamate release probability and decreasing the number of readily releasable vesicles. Bath application of adenosine also reduced the frequency of the miniature EPSCs recorded in the presence of TTX suggesting that adenosine may interact with the exocytosis processes downstream of Ca2+ influx. Both Gαi/o proteins and the protein kinase A pathway were required for adenosine-induced depression of glutamatergic transmission. We further showed that bath application of picrotoxin to the EC slices induced stable epileptiform activity and bath application of adenosine dose-dependently inhibited the epileptiform activity in this seizure model. Adenosine-mediated depression of epileptiform activity was mediated by activation of adenosine A1 receptors and required the functions of Gαi/o proteins and protein kinase A pathway. Our results suggest that the depression of glutamatergic transmission induced by adenosine contributes to its antiepileptic effects in the EC.

Corticotropin-Releasing Factor Facilitates Epileptiform Activity in the Entorhinal Cortex: Roles of CRF2 Receptors and PKA Pathway

Whereas corticotropin-releasing factor (CRF) has been considered as the most potent epileptogenic neuropeptide in the brain, its action site and underlying mechanisms in epilepsy have not been determined. Here, we found that the entorhinal cortex (EC) expresses high level of CRF and CRF2 receptors without expression of CRF1 receptors. Bath application of CRF concentration-dependently increased the frequency of picrotoxin (PTX)-induced epileptiform activity recorded from layer III of the EC in entorhinal slices although CRF alone did not elicit epileptiform activity. CRF facilitated the induction of epileptiform activity in the presence of subthreshold concentration of PTX which normally would not elicit epileptiform activity. Bath application of the inhibitor for CRF-binding proteins, CRF6-33, also increased the frequency of PTX-induced epileptiform activity suggesting that endogenously released CRF is involved in epileptogenesis. CRF-induced facilitation of epileptiform activity was mediated via CRF2 receptors because pharmacological antagonism and knockout of CRF2 receptors blocked the facilitatory effects of CRF on epileptiform activity. Application of the adenylyl cyclase (AC) inhibitors blocked CRF-induced facilitation of epileptiform activity and elevation of intracellular cyclic AMP (cAMP) level by application of the AC activators or phosphodiesterase inhibitor increased the frequency of PTX-induced epileptiform activity, demonstrating that CRF-induced increases in epileptiform activity are mediated by an increase in intracellular cAMP. However, application of selective protein kinase A (PKA) inhibitors reduced, not completely blocked CRF-induced enhancement of epileptiform activity suggesting that PKA is only partially required. Our results provide a novel cellular and molecular mechanism whereby CRF modulates epilepsy.

Neurotensinergic Excitation of Dentate Gyrus Granule Cells via Gαq-Coupled Inhibition of TASK-3 Channels

Neurotensin (NT) is a 13-amino acid peptide and serves as a neuromodulator in the brain. Whereas NT has been implicated in learning and memory, the underlying cellular and molecular mechanisms are ill-defined. Because the dentate gyrus receives profound innervation of fibers containing NT and expresses high density of NT receptors, we examined the effects of NT on the excitability of dentate gyrus granule cells (GCs). Our results showed that NT concentration dependently increased action potential (AP) firing frequency of the GCs by the activation of NTS1 receptors resulting in the depolarization of the GCs. NT-induced enhancement of AP firing frequency was not caused indirectly by releasing glutamate, GABA, acetylcholine, or dopamine, but due to the inhibition of TASK-3 K(+) channels. NT-mediated excitation of the GCs was G protein dependent, but independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Immunoprecipitation experiment demonstrates that the activation of NTS1 receptors induced the association of Gαq/11 and TASK-3 channels suggesting a direct coupling of Gαq/11 to TASK-3 channels. Endogenously released NT facilitated the excitability of the GCs contributing to the induction of long-term potentiation at the perforant path-GC synapses. Our results provide a cellular mechanism that helps to explain the roles of NT in learning and memory.

Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.