Jerrel L. Yakel

Nicotinic receptors in the brain: correlating physiology with function

Nicotinic ACh receptors (nAChRs) have been implicated in a variety of brain functions, including neuronal development, learning and memory formation, and reward. Although there are substantial data indicating that nAChR subunits are found in many brain regions, the precise cellular roles of these subunits in neuronal functions have remained elusive. Until recently, nAChRs were thought primarily to serve a modulatory role in the brain by regulating neurotransmitter release from nerve terminals. However, new evidence has revealed that nAChRs also function in a postsynaptic role by mediating fast ACh-mediated synaptic transmission in the hippocampus and in the sensory cortex, and are found at somatodendritic as well as nerve terminal sites in the reward system. It is possible that presynaptic and postsynaptic nAChRs mediate changes in the efficacy of synaptic transmission in these brain regions. These changes could underlie the proposed functions of nAChRs in cognitive functions of the hippocampus and cerebral cortex, in neuronal development in the sensory cortex, and in reward.

Desensitization of nicotinic ACh receptors: shaping cholinergic signaling

Nicotinic ACh receptors (nAChRs) can undergo desensitization, a reversible reduction in response during sustained agonist application. Although the mechanism of desensitization remains incompletely understood, recent investigations have elucidated new properties underlying desensitization, indicating that it might be important to control synaptic efficacy, responses to cholinergic agents, and certain nAChR-related disease states. Thus, studying how different nAChR subunits contribute to desensitization might help to explain variations in responsiveness to drugs, and might thus improve their therapeutic applications. Agonist-specific desensitization, desensitization arising from resting receptors, natural mutations dramatically altering desensitization, and the possibility that recovery from desensitization is an important process for modulating receptor function, together provide a new framework for considering desensitization as a target to shape cholinergic signaling.

Functional nicotinic ACh receptors on interneurones in the rat hippocampus

1 Neuronal nicotinic ACh receptors (nAChRs) were studied in the rat hippocampal slice preparation using whole‐cell patch‐clamp recording techniques. 2 Responses to ACh (100 μm) were detected on inhibitory interneurones in the CA1 field of the hippocampus proper and in the dentate gyrus, but not on principal excitatory neurones in either region. The different neuronal types were identified based on their morphology and location. 3 ACh excited interneurones in the hippocampus and dentate gyrus in current‐clamp recordings. In voltage‐clamp recordings, ACh‐activated inward currents were recorded from interneurones in the presence of blockers of synaptic transmission and the muscarinic ACh receptor antagonist atropine. The zero current potential for this response to ACh was near 0 mV. 4 The effect of ACh was mimicked by the nAChR‐selective agonists nicotine (100 μm) and 1,1‐dimethyl‐4‐phenyl‐piperazinium iodide (DMPP, 100 μm). The response to ACh was reversibly antagonized by the neuronal nAChR antagonist mecamylamine (10 μm). The nAChR α7 subunit‐selective antagonists α‐bungarotoxin (100 nm) and methyllycaconitine (10 nm) also inhibited the response to ACh. 5 These observations demonstrate the presence of functional nAChRs on inhibitory interneurones in the rat hippocampus. Thus, a novel mechanism by which ACh can regulate neuronal activity in the hippocampus is revealed.

CALCINEURIN REGULATION OF SYNAPTIC FUNCTION : FROM ION CHANNELS TO TRANSMITTER RELEASE AND GENE TRANSCRIPTION

Calcineurin is a calcium (Ca2+)/calmodulin (CaM)-dependent protein phosphatase that has been shown to regulate the activity of ion channels, neurotransmitter and hormone release, synaptic plasticity and gene transcription. At glutamatergic synapses, the inhibition of calcineurin with immunosuppressant drugs has been reported to enhance both the presynaptic release of glutamate and postsynaptic responsiveness. Several other ligand- and voltage-gated ion channels are negatively regulated by calcineurin. Hormone release in insulin-secreting pancreatic beta cells and pituitary corticotrope tumour (AtT20) cells is also negatively regulated by calcineurin. In this article, Jerrel Yakel discusses the evidence that calcineurin plays a vital role in regulating neuronal excitability and hormone release.

Rat nicotinic ACh receptor α7 and β2 subunits co‐assemble to form functional heteromeric nicotinic receptor channels

Rat hippocampal interneurons express diverse subtypes of functional nicotinic acetylcholine receptors (nAChRs), including α7‐containing receptors that have properties unlike those expected for homomeric α7 nAChRs. We previously reported a strong correlation between expression of the α7 and of the β2 subunits in individual neurons. To explore whether co‐assembly of the α7 and β2 subunits might occur, these subunits were co‐expressed in Xenopus oocytes and the functional properties of heterologously expressed nAChRs were characterized by two‐electrode voltage clamp. Co‐expression of the β2 subunit, both wild‐type and mutant forms, with the α7 subunit significantly slowed the rate of nAChR desensitization and altered the pharmacological properties. Whereas ACh, carbachol and choline were full or near‐full agonists for homomeric α7 receptor channels, both carbachol and choline were only partial agonists in oocytes expressing both α7 and β2 subunits. In addition the EC50 values for all three agonists significantly increased when the β2 subunit was co‐expressed with the α7 subunit. Co‐expression with the β2 subunit did not result in any significant change in the current‐voltage curve. Biochemical evidence for the co‐assembly of the α7 and β2 subunits was obtained by co‐immunoprecipitation of these subunits from transiently transfected human embryonic kidney (TSA201) cells. These data provide direct biophysical and molecular evidence that the nAChR α7 and β2 subunits co‐assemble to form a functional heteromeric nAChR with functional and pharmacological properties different from those of homomeric α7 channels. This co‐assembly may help to explain nAChR channel diversity in rat hippocampal interneurons, and perhaps in other areas of the nervous system.

Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons

1 The molecular and functional properties of neuronal nicotinic acetylcholine receptors (nAChRs) were characterized from CA1 neurons in rat hippocampal slices using single‐cell reverse‐transcription polymerase chain reaction (RT‐PCR) in conjunction with whole‐cell patch‐clamp recordings. 2 We analysed the presence of the neuronal nAChR subunit mRNAs α2–7 and β2–4, along with the mRNA for the GABAergic markers GAD (glutamic acid decarboxylase) 65 and 67 isoforms, and VGAT (vesicular GABA transporter) in interneurons from the stratum radiatum and stratum oriens, and in CA1 pyramidal neurons. Functional nAChR‐mediated currents were detected in both interneuron populations, but not in pyramidal neurons. 3 The neuronal nAChR subunit mRNAs detected in > 20 % of the populations examined were α4, α5, α7 and β2–4 in stratum radiatum interneurons; α2, α3, α4, α7, β2 and β3 subunits in stratum oriens interneurons; and β2–4 in pyramidal neurons. High levels of GABAergic marker mRNAs were detected in both interneuron populations, but not in pyramidal neurons. 4 Significant co‐expression of nAChR subunits within individual neurons was detected for α3 +α5, α4 +β2, α4 +β3, α7 +β2, β2 +β3 and β3 +β4. 5 The kinetics of the nAChR‐mediated currents in response to the application of 100 μm ACh were significantly correlated with the expression of particular nAChR subunits. The α3, α7 and β2 nAChR subunits were individually correlated with a fast rise time, the α2 nAChR subunit was correlated with a medium rise time, and the α4 nAChR subunit was correlated with a slow rise time. The α2 and α4 nAChR subunits were also significantly correlated with slow desensitization kinetics.

Timing-Dependent Septal Cholinergic Induction of Dynamic Hippocampal Synaptic Plasticity

Cholinergic modulation of hippocampal synaptic plasticity has been studied extensively by applying receptor agonists or blockers; however, the effect of rapid physiological cholinergic stimuli on plasticity is largely unknown. Here, we report that septal cholinergic input, activated either by electrical stimulation or via an optogenetic approach, induced different types of hippocampal Schaffer collateral (SC) to CA1 synaptic plasticity, depending on the timing of cholinergic input relative to the SC input. When the cholinergic input was activated 100 or 10 ms prior to SC stimulation, it resulted in α7 nAChR-dependent long-term potentiation (LTP) or short-term depression, respectively. When the cholinergic stimulation was delayed until 10 ms after the SC stimulation, a muscarinic AChR-dependent LTP was induced. Moreover, these various forms of plasticity were disrupted by Aβ exposure. These results have revealed the remarkable temporal precision of cholinergic functions, providing a novel mechanism for information processing in cholinergic-dependent higher cognitive functions.

Nicotinic ACh receptors as therapeutic targets in CNS disorders.

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor (nAChR) channels. These receptors are widely distributed throughout the central nervous system (CNS), being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however, the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimers disease, AD), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT3R.

Inhibition of neuronal nicotinic acetylcholine receptor channels expressed in Xenopus oocytes by β-amyloid1–42 peptide

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a variety of physiological processes, including cognition and development. Dysfunctions in nAChRs have been linked to Alzheimer’s disease (AD), a human neurological disorder that is the leading cause of dementia. AD is characterized by an increasing loss of cognitive function, nAChRs, cholinergic neurons, and choline acetyltransferase activity. A major hallmark of AD is the presence of extracellular neuritic plaques composed of the β-amyloid (Aβ1–42) peptide; however, the link between Aβ1–42 and the loss of cognitive function has not been established. Many groups have shown direct interactions between Aβ1–42 and nAChR function, however, with differing results. For example, in rat hippocampal CA1 interneurons in slices, we found that Aβ1–42 inhibits nAChR channels directly, and non-α7 receptors were more sensitive to block than α7 receptors. However, some groups have found that α7 subtypes were potently blocked by Aβ1–42, whereas other groups reported that Aβ1–42 can activate nAChRs (i.e., both α7 and non-α7 subtypes). To further investigate the link between nAChR function and Aβ1–42, we expressed various subtypes of nAChRs in Xenopus oocytes (e.g., α4β2, α2β2, α4α5β2, and α7) and found that Aβ1–42 blocked these various non-α7 nAChRs, without any effect on α7 nAChRs. Furthermore, none of these channels was activated by Aβ1–42. The relative block by Aβ1–42 was dependent on the subunit makeup and apparent stoichiometry of these receptors. These data further support our previous findings that Aβ1–42 directly and preferentially inhibits non-α7 nAChRs.