The mean and standard deviation of the slope conductance near V_rev built for TM α7 nAChR- (A) and hippocampal CA1 pyramidal NMDAR-mediated responses (B). A significant [Ca²⁺]_o–dependent decrease in the whole-cell conductance of TM α7 nAChR-, but not CA1 NMDAR-mediated responses was observed (97). This decrease was not due to a current rundown because it persisted in experiments where high (i.e., 20 mM) [Ca²⁺]_o was used before low (i.e., 2 mM) [Ca²⁺]_o (97). Examples of TM α7 nAChR-mediated currents obtained by applications of choline at various positive and negative membrane voltages in voltage-clamp in 2 mM [Ca²⁺]_o (C) and 20 mM [Ca²⁺]_o (D). The whole-cell conductance of TM α7 nAChR channels in high [Ca²⁺]_o was always lower than that in low [Ca²⁺]_o, presumably due to a Ca²⁺-dependent block of monovalent ion permeation. E) The current-voltage relationship for responses illustrated in (C) and (D). No considerable current rectification was observed owing to Mg²⁺-free external and internal solutions and the presence of F⁻ ions in the internal solution. The I–V curves were fitted with second-order polynomial equations. Panels C–E illustrate data obtained from the same acutely dissociated TM neuron. Examples of CA1 NMDAR-mediated currents obtained by applications of NMDA plus glycine at various positive and negative membrane voltages in voltage-clamp in 2 mM [Ca²⁺]_o (F) and 20 mM [Ca²⁺]_o (G). H) The current-voltage relationship for responses illustrated in (F) and (G). The whole-cell conductance of NMDAR channels in 20 mM [Ca²⁺]_o was similar to that in 2 mM [Ca²⁺]_o, indicating a lack of significant Ca²⁺-dependent block of monovalent ion permeation. The I–V curves were fitted with second-order polynomial equations. Panels F–H illustrate data obtained from the same acutely dissociated hippocampal CA1 neuron. Note that although the application pipettes were filled with 40 mM choline or 200 µM NMDA + 20 µM glycine, the effective concentrations of choline or NMDA+glycine near the recorded neurons were unknown and considerably lower than the concentrations of agonists in application pipettes. However, in each given experiment these concentrations were very stable evidenced by stable responses (97). Reprinted from (97) with permission from Blackwell Publishing in the format Journal via Copyright Clearance Center.

A) NS-1738, 5-HI, Invermectin and Genistein represent the family of Type-I α7-PAMs. Schematic current traces illustrate the effects of Type-I α7-PAMs on α7 nAChRs: Type-I α7-PAMs increase the peak of α7 nAChR-mediated responses but do not alter the rate of desensitization of α7 nAChRs. B) PNU-120596, TQS, A867744, JNJ-1930942 represent the family of Type-II α7-PAMs. Schematic current traces illustrate the effects of Type-II α7-PAMs on α7 nAChRs: Type-II α7-PAMs increase the peak of α7 nAChR-mediated responses and considerably reduce the desensitization of α7 nAChRs.

The spontaneous firing of TM neurons was native as current injections were not applied (i.e., 0 pA). Horizontal bars indicate −65 mV. In current-clamp, in the absence of PNU-120596 and choline, TM neurons exhibited regular patterns of spontaneous firing (A). In these control experiments, when the membrane voltage was hyperpolarized to −65 mV by injections of a small current, step-like depolarizations were not observed (B). Recordings in A and B were obtained from the same TM neuron 1 min apart. After the sustained repetitive activation of TM nAChRs was observed in voltage-clamp upon administration of 10 µM choline plus 1 µM PNU-120596 (C), current-clamp recordings were conducted using the same TM neuron (D). In current clamp, activation of TM α7 nAChRs resulted in transient repetitive increases in the frequency of spontaneous firing of TM neurons (D, filled arrows). Traces shown in C and D were obtained from the same TM neuron 1 min apart. The framed insert in D) illustrates at a higher time resolution a portion of recording containing one transient excitation. E) The effects of individual step-like depolarizations in current clamp. When a hyperpolarizing current (~ −40 pA) was injected in the recorded TM neuron (the injection time is marked by *) during a prolonged interval of increased frequency (the interval between open and filled triangles), it resulted in cessation of spontaneous firing, allowing detection of the final portion of an underlying step-like depolarization. Therefore, a prolonged depolarization was observed as both an increase in spontaneous firing in the beginning of depolarization (open triangle) and a depolarizing step at the end of depolarization (filled triangle). Subsequent step-like depolarizations are also seen between the two dashed lines in insert. The insert illustrates this transition process at a higher resolution. In these experiments, ACSF contained 20 µM, gabazine, 15 µM DNQX, 50 µM AP-5 and 40 µM picrotoxin. The internal solution was K-gluconate-based. Reprinted from (34) with permission from ASPET.

The left most pathway: ACh esterase inhibitors (e.g., donepezil) increase the CSF level of ACh and promote activation of both nAChRs and mAChRs. Despite cognitive benefits (dashed line), the lack of selectivity may cause considerable side effects (e.g., autonomic). The right most pathway: α7 nAChR agonists. A moderate activation of α7 nAChRs by selective agonists (e.g., DMXBA) protects neurons, benefits cognition and appears to be clinically safe. The middle pathway: positive allosteric modulators (PAMs) of α7 nAChRs. Choline is a low-potency endogenous selective agonist of α7 nAChRs, but its potency can be considerably increased by Type-II α7-PAMs, such as PNU-120596. α7-PAMs do not activate α7 nAChRs in the absence of nicotinic agonists. Instead, α7-PAMs lower the energy barrier, allowing lower concentrations of nicotinic agonists to activate the receptor. In the presence of Type-II α7-PAMs, endogenous choline may become effective in producing moderate persistent activation of native α7 nAChRs. This type of activation of α7 nAChRs may promote neuroprotection and benefit cognition.

A–C) Current deviations were completely and reversibly blocked by 20 nM MLA, confirming the involvement α7 nAChRs. All current traces in A–C) were obtained from the same TM neuron. D–E) Step-like responses were observed in both voltage-(D) and current-clamp (E) recordings. Traces in D) and E) were obtained from the same TM neuron 1 min apart. In these experiments, the frequency of step-like current events appeared to be sensitive and rapidly responsive to changes in the ACSF concentrations of choline and PNU-120596 (34; 35). Activation of α7 nAChRs in current-clamp elicited transient repetitive step-like depolarizations: ~4 mV for individual events and ~25 mV for simultaneous multiple events (E). The bottom trace in D) and the top trace in E) share the same time scale shown between these traces. The vertical scale bar indicates either 20 pA (for traces in D) or 20 mV (for traces in E). In experiments shown in D–E), 0.3 µM TTX was continuously present in ACSF and the internal pipette solution contained CsMeSO₃. In voltage-clamp experiments, the membrane voltage was held at −60 mV. F) To visualize individual step-like depolarizations, a small continuous hyperpolarizing current (−5 pA) was injected into the recorded neuron resulting in cessation of spontaneous firing. Under these silent conditions, transient step-like depolarizations triggered short trains of action potentials (open arrows). However, occasionally, depolarizations did not trigger action potentials or triggered only a single action potential per depolarization (filled arrows). Step-like voltage and current deviations were resistant to 20 µM gabazine, 15 µM DNQX, 50 µM AP-5, 40 µM picrotoxin, and 0.3 µM TTX applied to ACSF. Reprinted from (34) with permission from ASPET.

A) Sequential images from a time series showing two Ca²⁺ blinks separated by 1.1 µm in a single filopodia. Top left image shows the regions used for measurements overlaid on the fluorescence image, subsequent F/F0 images were captured every second during application of nicotine + PNU-120596. B) Time-course of the F/F0 in two regions (#2 and #4) that exhibit repetitive Ca²⁺ elevations lasting ~3 s and in contiguous regions (#1, #3, and #5) that did not display considerable Ca²⁺ activity. C) Intensity profile of the F/F0 signal at t = 1 s in regions #2 and #4, showing the spatial spread of the Ca²⁺ elevations. The cross-section at >20% of the peak fluorescence averaged 0.67 µm and 0.64 µm for regions #1 and #2, respectively. Cell calcium by CHURCHILL LIVINGSTONE. Reproduced from with permission of CHURCHILL LIVINGSTONE in the format Journal via Copyright Clearance Center.