Introduction

Abstract Introduction Methods Results Discussion References

Although the psychological effects of the alkaloid nicotine have long been acknowledged (for a review see Stolerman et al., 1995), the physiological functions of neuronal nAChRs in the mammalian CNS remain unclear. For many years, the lack of specific agonists and antagonists for a given neuronal nAChR subtype severely limited the identification of functional nAChRs in various brain areas. This challenge was further aggravated by the fast kinetics of inactivation of some nAChR subtypes (reviewed in Sargent, 1993; Lindstrom, 1995; Albuquerque et al., 1995, 1997). With the development of systems that allow for agonists to be rapidly applied to and removed from the vicinity of neurons, and with the discovery of new pharmacological tools, it became possible to identify the various subtypes of functional nAChRs present on neurons from different brain areas, including the hippocampus (Alkondon and Albuquerque, 1993, 1994). Electrophysiological studies have shown that rapid exposure of 85% of cultured hippocampal neurons to nicotinic agonists results in activation of fast-decaying currents that are referred to as type IA currents. Type IA currents 1) are sensitive to blockade by -BGT, MLA or -CTX-ImI, 2) show an inward rectification that depends on the intracellular levels of Mg⁺⁺, 3) display a rundown that can be prevented to a great extent by the presence of ATP-regenerating compounds in the intracellular solution and 4) are highly sensitive to modulation by extracellular levels of Ca⁺⁺ (Alkondon et al., 1992, 1994; Alkondon and Albuquerque, 1993; Bonfante-Cabarcas et al., 1996; Pereira et al., 1996). Only 10% of the cultured hippocampal neurons respond to nicotinic agonists with slowly decaying currents that are sensitive to blockade by DHE and are referred to as type II, and no more than 2% of the neurons respond to such agonists with slowly decaying currents that are sensitive to blockade by mecamylamine and are referred to as type III (Alkondon and Albuquerque, 1993; Alkondon et al., 1994).

The pharmacological and kinetic characteristics of type IA, type II and type III currents suggest that they are subserved by -7 nAChRs, 42 nAChRs, and 34 nAChRs, respectively (Alkondon and Albuquerque, 1993; Alkondon et al., 1994). Potentially, activation of each of these receptors can lead to an increase of intracellular levels of Ca⁺⁺ either indirectly via depolarization and consequent activation of voltage-gated Ca⁺⁺ channels or directly because of Ca⁺⁺ permeation through the nAChR channel. In this respect, -7 nAChRs on hippocampal neurons, similar to -7 nAChRs ectopically expressed on Xenopus oocytes, are highly permeable to Ca⁺⁺ (Bertrand et al., 1993; Séguéla et al., 1993; Castro and Albuquerque, 1995).

Many of the biological functions identified to date for neuronal nAChRs have been associated with receptor-mediated changes in intracellular Ca⁺⁺ and include modulation of release of different neurotransmitters (McGehee et al., 1995; Sacaan et al., 1995; Alkondon et al., 1996b; Gray et al., 1996; Sershen et al., 1997), modulation of neurite outgrowth (Pugh and Berg, 1994), control of synthesis and/or release of neurotrophins, regulation of early gene (e.g., c-fos) transcript levels (Greenberg et al., 1986) and activation of second messengers (MacNicol and Schulman, 1992; Vijayaraghavan et al., 1995). In addition, mounting evidence suggests that CNS nAChRs are involved in controlling memory and learning. In fact, it seems that the brain cholinergic system (Aigner and Mishkin, 1986; Durkin, 1989; Dunnett and Fibiger, 1993) and the hippocampus (Squire, 1992; Bunsey and Eichenbaum, 1996) are vital components for memory processing in the mammalian CNS. Not only are nicotinic agonists able to improve cognition in experimental animals (see review by Stolerman et al., 1995), but also the number of neuronal nAChRs is markedly decreased in the cortex and hippocampus of patients with Alzheimer's disease, a pathological condition characterized by progressive neurodegeneration and cognitive impairment (see review by Kellar and Wonnacott, 1990; Schröder et al., 1991; Maelicke and Albuquerque, 1996).

Numerous questions have been raised regarding the influence of neuronal nAChR activation on synaptic function in the hippocampus. Although initial studies have shown that, similar to neurons in the rat olfactory bulb and in the chick interpeduncular nucleus (McGehee et al., 1995; Rocha et al., 1995; Alkondon et al., 1996b), neurons in the CA3 field of the rat hippocampus express an -7 nAChR that modulates the release of glutamate from presynaptic terminals (Gray et al., 1996), the effects of nAChR activation on the synaptic activity in other areas of the hippocampus remain obscure. Thus, the main scope of the present study was to determine whether functional nAChRs are present on CA1 neurons, and if so, to investigate the effects of nicotinic agonists on the ongoing synaptic activity in the CA1 hippocampal field, a region that undergoes synaptic plasticity during learning (Huerta and Lisman, 1993; Bliss and Collingridge, 1993) and is also implicated in epileptogenesis (Lothman et al., 1993).

With the use of infrared-assisted videomicroscopy to visualize individual neurons in rat hippocampal slices and the patch-clamp technique, we have demonstrated that functional nAChRs are present on pyramidal neurons and interneurons of the CA1 field of the hippocampus and that activation of nAChRs present on CA1 interneurons induces the release of GABA.

	Material and Methods

Abstract Introduction Methods Results Discussion References

Preparation of hippocampal slices. Sprague-Dawley rats (8 to 24 days old) were used in this study. The rats were deeply anesthetized in an atmosphere of CO₂ with dry ice, and sacrificed by cervical dislocation. Their brains were removed and kept in ice-cold ACSF. The hippocampi were dissected out and cut into slices of 200- to 250-µm thickness either manually or with a vibratome. The cut slices were placed in a chamber containing ACSF at room temperature (20-22°C). The ACSF was continuously bubbled with 95% O₂ and 5% CO₂. After at least 1 h, a single slice was mounted in a round tissue chamber (1.5-ml capacity) and superfused continuously at a rate of 2 ml/min with oxygenated ACSF. ACSF had the following composition (in mM): NaCl, 125; NaHCO₃, 25; KCl, 2.5; NaH₂PO₄, 1.25; CaCl₂, 2; MgCl₂, 1; and glucose, 25. Neurons present in the top layer within a depth of about 60 µm were successfully cleaned by applying a gentle stream of ACSF through a patch pipette with a tip diameter of about 10 µm. In about 30% of the experiments, the slices were exposed to the enzyme hyaluronidase (10 U/ml) for 30 to 45 min before starting the cleaning procedure. The nicotinic responses obtained in the neurons with and without the use of the enzyme did not differ to any noticeable extent. In many experiments cleaning of the neurons was achieved by a stream of internal solution coming out of the recording pipette to which positive pressure was applied.

Preparation of cultures of hippocampal neurons. According to the procedure described in Alkondon and Albuquerque (1993), neurons dissociated from the hippocampus of Sprague-Dawley rat fetuses (16- to 18-day gestation) were plated onto poly-L-lysine-precoated coverslips placed in 35-mm plastic culture dishes (Nunc A/S, Kamstrupvej 90, Denmark). Neurons were used 18 to 21 days after plating.

IR-assisted videomicroscopy. Neurons in culture and in slices were visualized through a Zeiss upright microscope (Zeiss Axioskop, New York, NY) using a 40× water-immersion objective (0.75 numerical aperture and 1.7 mm working distance) and DIC optics (Stuart et al., 1993; Alkondon et al., 1996a). After adjusting the specimen by the normal illumination light settings, an infrared filter (RG-9, _max = 800 nm, Melles Griot, Rochester, NY) was brought into the light path, and the images were then detected with an infrared-sensitive videocamera (Newvicon C2400-07, Hamamatsu Photonics, Hamamatsu City, Japan) and displayed on a standard video monitor. Background subtraction and contrast enhancement of the images were done with an ARGUS 10 image processor (Hamamatsu City, Japan). The images were then either recorded on videotape or captured online by a Pentium computer with a frame grabber and ADOBE PREMIERE software (Version 1.0, Adobe Systems Inc., Mountain View, CA). Images of the neurons were printed on a Codonics NP-1600 photographic network printer (Codonics, Inc., Middleburg Heights, OH) after adjusting the contrast and brightness by the Micrographics program.

Fluorescence microscopy. A fluorescence-sensitive camera was adapted to the Zeiss Axioskop so that it was possible to visualize the CA1 neurons in situ and to determine their morphology with a Lucifer yellow-containing solution in the patch pipettes. The neuronal morphology (pyramidal versus interneuron) could be identified by adding 0.4% Lucifer yellow to the internal pipette solution before making the whole-cell recordings. At the end of each experiment in which Lucifer yellow-containing internal solution was used, the image of the labeled neuron was captured by a color chilled 3CCD camera (Hamamatsu, Japan) and stored in the computer. The images of the neurons were then analyzed for branching pattern, which helped in confirming the morphology of the neurons under study and visualizing spine-rich regions along the dendritic surface.

Whole-cell current and voltage recording. Whole-cell patch recording was performed at the cell soma of the neurons, and current or voltage changes induced by different agonists were recorded from hippocampal neurons according to the standard patch-clamp technique (Hamill et al., 1981) with an LM-EPC7 patch-clamp system (List Electronic, Darmstadt, FRG). The signals were filtered at 2 kHz and either recorded on video tape for later analysis or directly sampled by a microcomputer with the pCLAMP 6 program (Axon Instruments, Foster City, CA). The external bath solution for the slices was the same as the ACSF, but contained atropine (1 µM) to block the muscarinic receptors. The external bath solution for the culture experiment consisted of (in mM): NaCl, 165; KCl, 5; CaCl₂, 2; glucose, 10; and HEPES, 5 (pH adjusted to 7.3 with NaOH; 330 mOsm). The internal solution used to fill the patch pipettes consisted of (in mM): EGTA, 10; HEPES, 10; and either CsCl, 160, KCl, 160, or the combination of CsCl, 80 and CsF, 80 (pH adjusted to 7.3 with CsOH or KOH, depending on the main cation in the solution; 330 mOsm). When the ATP-regenerating compounds phosphocreatine, creatine phosphokinase and ATP were included in the internal salt solution, the concentration of the main cation was decreased appropriately to adjust the osmolarity to 330 mOsm (Alkondon et al., 1994). Patch pipettes were made from a borosilicate glass capillary with a 1.2-mm outer diameter (World Precision Instruments, Sarasota, FL). When filled with the internal solution, the pipettes had resistances ranging from 2 to 6 M. The access resistance during the whole-cell recordings was within 20 M. All the recordings were performed at room temperature (20-22°C)

Agonist and antagonist application. Agonists were applied to the neurons by one of the following protocols. Because of the limited working distance (1.7 mm between the specimen and the objective), a modified U-tube was fabricated in the laboratory by attaching a 1-cm cylindrical glass tube having an inner diameter of about 70 µm (outer diameter  150 µm) to the pore of a regular U-tube (Alkondon and Albuquerque, 1993), and glued at the joints to prevent any leakage. This extension of the U-tube permitted positioning of the tip within a distance of about 200 µm from the surface of the neurons. The modified U-tube had the same advantage as the regular U-tube such that several agonists could be applied to a single neuron. However, once the agonist was applied to the neurons through the modified U-tube, the solution could not be quickly removed by the same tube. In that case, the high flow rate (2 ml/min) and the small volume of the bath (1.5 ml) facilitated the removal of the agonists. In other experiments, a micromanifold system with a tip having 1-cm length and 100-µm inner diameter (ALA Scientific Instruments, Westbury, NY) was used. This system worked basically in the same way as the home-made U-tube. The small dead space of the micromanifold system facilitated rapid switching from one solution to another. However, agonists applied through this system to the neurons resulted in the activation of currents that had slower rise times than those evoked by application of agonists through the modified U-tube.

Data analysis. The peak amplitude, rise time and decay time constants of the currents were analyzed with the pCLAMP program (Axon Instruments, Foster City, CA). The data are given either as mean ± S.E.M. or as individual values.

Drugs used. ACh chloride, choline chloride, cytisine, ()-nicotine hydrogen tartrate, 1,1-dimethylphenyl piperazinium iodide, GABA, picrotoxin, atropine sulfate, tetrodotoxin, d-TC, and hyaluronidase (type I-S) were obtained from Sigma Chemical Co. (St. Louis, MO). CNQX was obtained from Research Biochemical International (Natick, MA). -BGT was purchased from Biotoxins, Inc. (St. Cloud, FL). (+)-Anatoxin-a fumarate was a gift from Prof. H. Rapoport (Department of Chemistry, University of California, Berkeley, CA). Methyllycaconitine citrate was a gift from Professor M.H. Benn (Department of Chemistry, University of Calgary, Alberta, Canada). (+)-Epibatidine hemioxalate was a gift from Dr. Ray Baker (Merck, Sharp & Dohme Research Laboratories, Harlow, Essex, UK). DHE hydrobromide was a gift from Merck, Sharp & Dohme (Rahway, NJ). All chemicals were dissolved in double-distilled water, and the stock solutions (0.01-1 M) were kept frozen until ready to use. Stock solutions of CNQX (10 mM) were made in NaOH (12.5 mM), and those of picrotoxin (250 mM) were made in dimethyl sulfoxide.

	Results

Abstract Introduction Methods Results Discussion References

In this paper we present data on the properties of nicotinic acetylcholine responses recorded from CA1 neurons that underwent normal ontogenic development in vivo. These neurons were visually identified in hippocampal slices and morphologically characterized with the help of IR-DIC and fluorescence microscopy. The CA1 area of the hippocampus consists of a diverse group of neurons, including pyramidal neurons located in the stratum pyramidale and several types of interneurons present in the stratum oriens and alveus, stratum pyramidale, stratum radiatum and stratum lacunosum moleculare (see Morin et al., 1996). As described under "Material and Methods," agonists were applied to neurons in the slices via a modified U-tube (Alkondon and Albuquerque, 1993), a micromanifold system (which served a similar purpose as the modified U-tube) or a single pipette (which was useful to restrict the neuronal surface covered by the agonist) (Alkondon et al., 1996a).

Images of neurons in the CA1 field of hippocampal slices

Based on the morphological features and on the location, the neurons studied in the CA1 region of hippocampal slices (area inside the rectangular box in fig. 1A) could be divided into two groups. The first group consisted of pyramidal neurons that were located in the stratum pyramidale. The second group consisted of interneurons that were located in the stratum pyramidale and in the stratum radiatum up to 300 µm from the midline of the pyramidal layer. Figure 1 illustrates sample images of individual neurons visualized in different layers of the CA1 field of hippocampal slices. The interneurons located in the stratum pyramidale and in the stratum radiatum had non-pyramidal-shaped soma and were devoid of prominent thick apical dendrites (fig. 1B). Most of the neurons in the stratum pyramidale had a pyramidal-shaped soma and prominent apical dendrites (fig. 1C). In addition, protrusions resembling spines were seen on some of the apical dendrites of pyramidal neurons and on the dendrites of interneurons (fig. 2). Fluorescence microscopy of Lucifer yellow-filled neurons not only confirmed the morphological features of pyramidal and non-pyramidal cells but also enabled identification of spiny dendritic regions (fig. 2).

View larger version (142K): [in this window] [in a new window]   Fig. 1.   Images of CA1 neurons in hippocampal slices. (A) Image of a 250-µm-thick slice which illustrates the different fields in the hippocampus. The slice was obtained from a 16-day-old rat. The CA1 and CA3 regions and the dentate gyrus (DG) are indicated by the arrows. Pyramidal neurons and interneurons were identified in the CA1 field (outlined by the rectangular box) by means of IR-DIC or by a combination of IR-DIC and fluorescence microscopy. (B and C) Infrared and fluorescence images of neurons visualized in the CA1 field of the hippocampus. (B, top frame) Infrared image of an interneuron located in the stratum pyramidale of a hippocampal slice obtained from a 17-day-old rat. The interneurons had a non-pyramidal-shaped cell body and thin dendrites, which could be easily seen in the fluorescence image obtained after the cell was filled with Lucifer yellow via a whole-cell recording pipette (B, bottom frame). (C, left frame) Infrared image of a pyramidal neuron visualized in the CA1 field of a hippocampal slice obtained from a 15-day-old rat. Notice the typical characteristics of pyramidal-shaped cell body and thick, long and prominent apical dendrites, which can be easily visualized in the fluorescence image of the Lucifer yellow-filled pyramidal neuron (C, right frame).

View larger version (83K): [in this window] [in a new window]   Fig. 2.   Images of spine-rich dendrites. Upper frame shows the fluorescence images of two Lucifer yellow-filled interneurons in a single hippocampal slice from a 16-day-old rat. The neuron at the top was located 50 µm and the neuron at the bottom, 125 µm from the midline of the stratum pyramidale. The segment of secondary dendrite depicted between the two arrow heads in this frame is shown enlarged in the bottom frame. Arrows shown in the bottom frame indicate the presence of spines on this secondary dendrite. However, the images of most of the spines could not be captured in this two-dimensional picture.

Characteristics of nicotinic responses evoked in CA1 neurons

All experiments were carried out in the presence of atropine (1 µM) to block muscarinic receptors so that the responses evoked by nonspecific cholinergic agonists reflected exclusively the activation of nAChRs. Moreover, the use of TTX-free external solution kept the neuronal circuitry functional, so that it could be known whether action potentials and any subsequent action potential-dependent synaptic events could occur upon activation of nAChRs on CA1 hippocampal neurons.

Nicotinic responses of pyramidal neurons. Application of physiological recording saline to pyramidal neurons caused neither change in the membrane current nor alterations in the frequency or amplitude of spontaneously occurring MPSCs (fig. 3A). On the other hand, a 1-s pulse application of GABA (20 µM) to such neurons resulted in the activation of a slowly rising inward current that, after reaching the peak, declined to the baseline current level within about 5 s (fig. 3A). GABA-induced response was not accompanied by any change in the frequency of MPSCs.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Samples of nicotinic responses recorded from pyramidal neurons. Responses obtained from two neurons (labeled as A and B) are illustrated. Agonists were applied via the U-tube. The solid line shown above the first trace indicates the duration (1 s) of agonist pulses. The hippocampal slices were obtained from rats that were 15 days old (A) and 16 days old (B). The recording pipette was filled with a F-containing, Cs+-based internal solution. A portion of the trace containing the PSCs is shown in an expanded scale in B. Holding potential, 50 mV.

Nicotinic responses of interneurons. Application of agonist-free physiological solution to interneurons changed neither the baseline noise nor the frequency of MPSCs (fig. 4A). However, exposure of these neurons to GABA (20 µM)-containing external solution resulted in the elicitation of small inward currents (fig. 4A), and application of ACh to these neurons resulted in inward currents that had slow kinetics of activation and inactivation (fig. 4). The ACh-evoked currents had a prolonged decay phase, which lasted for several seconds even after termination of the agonist pulse (fig. 4B). Such slow nicotinic currents were recorded from 24 of 27 interneurons tested, and resembled the slowly decaying type II nicotinic currents that can be recorded from cultured hippocampal neurons (Alkondon and Albuquerque, 1993, 1995). In 9 of 24 ACh-sensitive neurons, the slow nicotinic current was accompanied by bursts of current transients (see fig. 4A). These current transients, hereafter referred to as fast current transients, had the shape of inverted action potentials, and probably represented the active propagation of action potentials in the neurons under study. In 1 of 27 interneurons studied, ACh induced fast current transients without any noticeable slow nicotinic current. The frequency of the fast current transients elicited by ACh in the interneurons was variable from one neuron to another, and also in the same neuron, the frequency tended to decrease during the course of the experiment. In 2 of 27 interneurons tested, ACh induced an inward current that had both a rapidly and a slowly decaying component (see trace in fig. 4C); this current resembled type IB nicotinic currents recorded from cultured hippocampal neurons (Alkondon and Albuquerque, 1993). In all 27 interneurons tested, ACh also elicited PSCs (fig. 4); the frequency of ACh-evoked PSCs was variable among different neurons and was always less intense than that observed in pyramidal neurons.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Samples of nicotinic responses recorded from interneurons. Responses obtained from three neurons (labeled A, B and C) are illustrated. Agonists were applied for 2 s via a U-tube to neuron A, for 25 ms via a single agonist pipette to neuron B and for 3 s via a micromanifold system to neuron C. Agonist pulses are indicated by solid lines above the traces in each section. Hippocampal slices were obtained from rats that were 16 days old (A), 12 days old (B) and 15 days old (C). All three interneurons were present in the stratum radiatum, and were located 200 µm (A), 250 µm (B) and 70 µm (C) from the midline of the stratum pyramidale. The recording pipette was filled with a F-containing, Cs+-based internal solution. Holding potentials: 96 mV in A, 70 mV in B and 60 mV in C.

Characteristics of ACh-evoked PSCs. PSCs evoked by ACh were compared with the spontaneously occurring MPSCs. In contrast to randomly and less frequently occurring MPSCs, the PSCs occurred at high frequencies during the agonist application. In the example illustrated in figure 5A, MPSCs occurred at 0.22 Hz, whereas ACh-elicited PSCs occurred at 1.11 Hz. Further, the amplitude distribution of MPSCs and ACh-evoked PSCs showed that the PSCs were substantially larger than the MPSCs (fig. 5A). The amplitude distribution of the MPSCs could be fitted to a single gaussian function with a mean value of 16.8 pA, whereas that of the agonist-evoked PSCs could be fitted by three gaussian functions with mean values of 17.2, 32.2 and 64.4 pA. Moreover, the MPSCs had a rise time (10-90%) of 2.07 ± 0.12 ms and a decay-time constant of 12.9 ± 0.4 ms, whereas the ACh-evoked PSCs had a rise time and a decay-time constant of 2.72 ± 0.13 ms and 19.9 ± 1.0 ms, respectively. These results suggested that both MPSCs and ACh-elicited PSCs are mediated by the same neurotransmitter, and that the PSCs are the result of the recruitment of more release sites during nAChR activation.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Analysis of MPSCs and ACh-elicited PSCs. (A) Plots show the distribution of the amplitudes and the cumulative distributions of the amplitudes of PSCs evoked by application of ACh to a single pyramidal neuron and of MPSCs. As illustrated, in the presence of ACh, a high frequency of large amplitude events could be seen. (B and C) Traces are representative samples of the PSCs recorded during application of ACh to two pyramidal neurons. ACh was applied to the neurons via the U-tube for 1 s (B) and for 2 s (C). Solid lines above the traces indicate the duration of agonist pulses. ACh-evoked PSCs could still be detected after a 12-min superfusion of the neuron with CNQX (right trace in B). However, after a 5-min superfusion of the neuron with picrotoxin, no PSCs could be detected during the application of ACh (right trace in C). Rats from which the slices were taken were 15 days old (A) and 8 days old (B). The recording pipette was filled with a F-containing, Cs+-based internal solution. Holding potential, 50 mV.

Responsiveness of CA1 neurons to receptor subtype-specific nicotinic agonists. Nicotinic agonists are useful, to a certain extent, in discriminating the subtypes of nAChRs present on CNS neurons. To further authenticate the presence of functional nAChRs in the developing CA1 neurons and to get preliminary insights into the subunit composition of these receptors, we used various nicotinic agonists and studied the responses evoked by them.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Responses of pyramidal neurons to different nicotinic agonists. Responses obtained from three neurons (labeled as A, B and C) are illustrated. Agonists were applied to the neurons via the U-tube for 1 s (A and B) and via the micromanifold system for 3 s (C). Solid lines above the traces indicate the duration of the agonist pulses. The rats from which slices were taken were 15 days old (A and B) and 19 days old (C). The recording pipette was filled with a F-containing, Cs+-based internal solution. Holding potentials: 50 mV (A and B) and 60 mV (C).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Responses of interneurons to different nicotinic agonists. Responses obtained from four interneurons (two in A, one in B and one in C) are illustrated. Agonists were applied to the neurons for the duration indicated by the solid lines on top of traces. The U-tube was used to apply the agonists to the first neuron in A, and the micromanifold system was used to apply the agonists to all other neurons. The rats from which slices were obtained were 16 days old (A), 15 days old (B) and 12 days old (C). All four interneurons were present in the stratum radiatum and were located between 125 and 200 µm from the midline of the stratum pyramidale. The recording pipette was filled with a F-containing, Cs+-based internal solution. Holding potentials: 96 mV (top trace in A) and 60 mV (all the other traces). The traces in the right panel are the same recording as in the left panel but are shown on a compressed time scale.

Responsiveness of CA1 neurons to receptor subtype-specific nicotinic antagonists. The nonselective nicotinic antagonist d-TC (10 µM) blocked the ACh-evoked slow inward currents and PSCs in CA1 pyramidal neurons (n = 3 neurons) (fig. 8). In addition, d-TC blocked ACh-evoked fast current transients recorded from interneurons (fig. 9). The blocking effect of d-TC was reversible within about 10 min after washing the neurons with d-TC-free physiological recording solution (figs. 8 and 9).

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Effects of nicotinic blockers d-TC and MLA on ACh-induced responses recorded from pyramidal neurons. Responses obtained from two pyramidal neurons are shown. ACh was applied to the neurons for 1 s via the U-tube as indicated by the solid lines above the traces. (A) ACh-evoked currents were recorded under control conditions (left trace), 3 min after superfusion with 10 µM d-TC (middle trace) and 10 min after washing the neurons with d-TC-free external solution (right trace). (B) ACh-evoked currents were obtained under control conditions (left trace), 8 min after superfusion with 1 µM MLA (middle trace) and 12 min after washing the neurons with MLA-free external solution (right trace). The rat from which slices were obtained was 15 days old. The recording pipette was filled with a F-containing, Cs+-based internal solution. Holding potential, 50 mV.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   Pharmacological characterization of ACh-induced current transients in interneurons. Fast current transient induced by ACh in a single interneuron is shown. ACh was applied for 50 ms (as indicated by the solid line above the top traces) by pressure ejection from a single pipette. Traces in the first row represent ACh-evoked currents under control conditions (left trace) and 3 min after superfusion of the neuron with d-TC-containing external solution (right trace). The blocking effect of d-TC was reversed within 10 min of washing. Traces in the second row represent the current evoked by application of ACh to the same neuron before (left trace) and after 16-min superfusion with 100 nM -BGT (right trace). Traces in the bottom row represent the current evoked by application of ACh to the same neuron before (left trace) and after 4 min superfusion with TTX-containing external solution (right trace). The rat from which hippocampal slice was obtained was 17 days old. The recording pipette was filled with a F-free, Cs+-based solution containing ATP-regenerating compounds. Holding potential, 60 mV.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Pharmacological characterization of ACh-induced slow currents in interneurons. Slow inward currents induced by application of ACh to a single interneuron are shown. ACh was applied to the neuron for 3 s (as indicated by the bar on the top of the first trace) via a micromanifold system. Traces, from top to bottom, represent the ACh-evoked current recorded in the absence of any blocker, 10 min after superfusion with -BGT (100 nM), 3 min after superfusion with DHE (10 µM) and 12 min after wash. The slice was obtained from a 15-day-old rat. This interneuron was located in the stratum radiatum 150 µm from the midline of the stratum pyramidale. Holding potential, 60 mV.

Mechanism underlying the release of GABA by nAChR activation

To delineate the mechanisms by which activation of nAChRs leads to induction of PSCs and fast current transients, we used the Na⁺-channel blocker, TTX. In the absence of TTX, ACh could induce either PSCs or slow inward currents accompanied by PSCs in pyramidal neurons, whereas in the presence of TTX, ACh-evoked PSCs could not be detected (see fig. 11). Further, in interneurons that responded to ACh with fast current transients and few PSCs, TTX completely abolished both types of responses (see fig. 9). These results clearly indicate the involvement of Na⁺ channels in inducing PSCs and fast current transients that occur on nAChR activation.

View larger version (31K): [in this window] [in a new window]   Fig. 11.   Effect of TTX on ACh-induced responses in pyramidal neurons. Responses obtained from two pyramidal neurons (labeled as A and B) are shown. ACh was applied for 1 s via the U-tube as indicated by the solid lines above the traces. Traces in the first row represent ACh-evoked responses recorded before (left trace) and after 2 min superfusion with TTX (right trace). Traces in the second row represent ACh-evoked responses before (left trace) and after 5 min superfusion with TTX (right trace). After recording the control responses, neurons were superfused with TTX-containing physiological solution and subsequently exposed to an admixture of ACh and TTX. The rats from which slices were obtained were 16 days old (A) and 10 days old (B). The recording pipette was filled with a F-containing, Cs+-based internal solution. Holding potential, 50 mV.

Responses of cultured hippocampal neurons to nicotinic agonists in the absence of TTX

In our studies to date, nicotinic responses of hippocampal neurons in culture have always been recorded in the presence of TTX, thus precluding an investigation of the ability of nicotinic agonists to modulate release of neurotransmitters by acting on preterminal nAChRs. Thus, we investigated the responses evoked by application of nicotinic agonists to cultured hippocampal neurons in the absence of TTX. In TTX-free solutions, application of ACh to voltage-clamped hippocampal neurons in culture induced an inward current (indicated by asterisk in left column, trace 2 of fig. 12) that had the characteristics of type IA currents recorded in the presence of TTX (Alkondon and Albuquerque, 1993). However, this inward current, before decaying completely to the baseline level, was accompanied by a burst of GABA-mediated PSCs. The PSCs occurred as a consequence of nAChR activation because application of physiological saline to such neurons did not induce any membrane current changes or PSCs (left column, top trace in fig. 12). These responses resembled those recorded from some of the CA1 interneurons (see fig. 7A). In some cells, application of ACh evoked only PSCs; the direct nicotinic current component was either too small or could not be discerned clearly from the PSCs (data not shown). Choline (10 mM) induced responses identical to those evoked by ACh (see left column, trace 3 in fig. 12), which indicates that the nAChRs that mediate these events in the cultured neurons contain the -7 subunits.

View larger version (15K): [in this window] [in a new window]   Fig. 12.   Pattern of nicotinic responses recorded from cultured hippocampal neurons in the absence of TTX. Left traces represent sample recordings of currents (first three traces) and voltage (last trace) changes induced by application of nicotinic agonists to a single hippocampal neuron cultured for 19 days. The agonists were applied for 2 s via a micromanifold system. Solid line above the top trace indicates the duration of the agonist pulse. Under the voltage-clamp condition, the neuron was held at 70 mV. The inward currents indicated by the asterisks represent the direct response that originates from activation of nAChRs on the neuron under study, whereas the PSCs that follow the initial current probably arise from GABAA receptors stimulated by GABA released by the activation of nAChRs present on neurons that synapse onto the neuron under study. Under the current-clamp mode, the fast voltage transient represents the action potential triggered by depolarization resulting from activation of nAChRs present on the neuron under study, and the subsequent events represent PSPs mediated by GABAA receptors stimulated by GABA released because of activation of nAChRs present on neurons that synapse onto the neuron under study. The neuron had a resting membrane potential close to 60 mV. The recording pipette was filled with a KCl-containing internal solution. Right traces represent samples of currents (first two traces) and voltage (last trace) recorded from another hippocampal neuron cultured for 21 days. The agonist was applied for 25 ms by pressure ejection from a single pipette as shown by a solid line above the top trace. Under the voltage-clamp condition, the neuron was held at 70 mV. The first trace was sampled without application of agonist. Application of ACh induced a burst of fast current transients (second trace) and a burst of action potentials (third trace) in this neuron. The resting membrane potential was close to 70 mV. Recording pipette was filled with a KCl-based internal solution.

Application of either ACh (left bottom trace in fig. 12) or choline (data not shown) to such neurons under current-clamp condition evoked an initial voltage transient, which resembled an action potential and was followed by several PSPs. It is likely that the initial voltage transient was the result of an actively propagating action potential that was initiated because of the depolarization triggered by nAChR activation.

Pressure ejection of ACh onto other hippocampal neurons in culture initiated a burst of fast current transients without noticeable inward currents or PSCs (right column, middle trace in fig. 12). This response is similar to that elicited by ACh in some of the neurons in the hippocampal slices (see fig. 4). Applying ACh to such cultured neurons under the current-clamp condition resulted in the initiation of a burst of action potentials (right column, bottom trace in fig. 12). These observations supported the notion that fast current transients evoked by ACh in the neurons of hippocampal slices are caused by the active propagation of action potentials that were initiated by the depolarization subsequent to nAChR activation.

The subtype of nAChR that is responsible for the initiation of action potentials in the cultured hippocampal neurons was characterized pharmacologically. In the presence of the GABA_A receptor antagonist picrotoxin, random firing of spontaneous action potentials was recorded from some hippocampal neurons in culture. Application of ACh, choline or ()-nicotine to these neurons resulted in the generation of bursts of action potentials that was insensitive to blockade by DHE. When the same agonists were applied to the neurons after bath-application of MLA (1 nM) for 6 to 8 min, they did not elicit the typical burst of action potentials seen under control conditions (fig. 13). Washing the neurons resulted in the restoration of the effects of the nicotinic agonists (fig. 13). These results support the concept that activation of an nAChR that contains the -7 subunit is responsible for the initiation of action potentials in the cultured hippocampal neurons.

View larger version (24K): [in this window] [in a new window]   Fig. 13.   Pharmacological characterization of the nicotinic agonist-mediated action potentials in cultured neurons. Voltage responses of a single hippocampal neuron to the application of different nicotinic agonists. The agonists were applied for 2 s via a micromanifold system. Solid lines shown at the bottom of each column of traces indicate the duration of agonist pulses. The resting membrane potential was close to 70 mV. The neuron was cultured for 19 days. The first row of traces represent sample recordings obtained during the application of physiological saline, which did not change the firing pattern of the neuron. Application of ACh, choline or ()-nicotine induced bursts of action potentials (rows 2-4, column 1). After superfusion of the neuron for 6 to 8 min with MLA, these agonists did not elicit the burst of action potentials (rows 2-4, column 2). After washing the neuron with MLA-free physiological solution for 10 to 12 min, the responses of the neuron to the agonists were similar to those recorded under the control condition (rows 2-4, column 3).

	Discussion

Abstract Introduction Methods Results Discussion References

In the present study, we demonstrate that functional nAChRs are present in visually identified pyramidal neurons and interneurons of the CA1 field of the hippocampus of developing rats. Application of nicotinic agonists to CA1 neurons evoked action potentials as well as whole-cell currents that were either fast decaying (in interneurons) or slowly decaying (in pyramidal neurons and interneurons). Of major interest, however, was the finding that the predominant response of CA1 neurons to nicotinic agonists is the release of GABA, evidenced by the ability of nicotinic agonists to induce GABA-mediated PSCs in pyramidal neurons and interneurons.

Characterization of the nicotinic responses recorded from CA1 neurons in hippocampal slices. Similarly to hippocampal neurons in culture, neurons in the CA1 field of hippocampal slices showed pharmacologically and kinetically distinct responses upon rapid application of nicotinic agonists. To compare the kinetics of the nicotinic responses recorded from hippocampal neurons in slices with that of the nicotinic currents recorded from hippocampal neurons in culture, it was necessary to take into account that the diffusion barriers in the slices slow down the agonist access to the neurons and that the agonist-delivery systems used to investigate the nicotinic responses of neurons in the slices were much slower than those used to obtain the responses of the neurons in culture. These factors contribute to slower kinetics of receptor activation and inactivation. Thus, the fastest nicotinic response of CA1 neurons in the slice (see fig. 7A) would correspond to the type IA currents recorded from the cultured neurons, and the slower decaying current recorded from the neurons in the slice (see figs. 7 and 11) would be equivalent to the type II currents recorded from hippocampal neurons in culture. The fast-decaying current, which was recorded predominantly from interneurons of the CA1 field of the hippocampal slices, was unveiled only when the -7 nAChR-specific agonist choline was used, which suggests that such response is most likely subserved by an -7 nAChR. Most of the neurons that showed a fast-decaying current in response to choline responded to nonspecific nicotinic agonists with a whole-cell current that had a fast- and a slow-decay component, or with a current that decayed very slowly. This finding could be explained by the ability of such nonspecific agonists to activate both rapidly and slowly desensitizing nAChRs, and by the fact that, depending on its magnitude, a slowly decaying current can easily mask a rapidly decaying current. Moreover, this finding indicates that a single neuron can express more than one subtype of functional nAChR.

TTX-sensitive nicotinic responses recorded from hippocampal neurons in culture and from neurons in the CA1 field of hippocampal slices. In voltage-clamped cultured hippocampal neurons, ACh evoked fast current transients similar to those recorded from CA1 interneurons exposed to nicotinic agonists. When the same cultured hippocampal neurons were current clamped, ACh evoked action potentials. These action potentials could also be induced by the -7-selective agonist choline, and were sensitive to blockade by the -7-selective antagonist MLA, which indicates that in cultured hippocampal neurons, activation of -7 nAChRs can result in depolarization sufficient to trigger action potentials.

The involvement of nAChRs in a local neuronal circuitry in the CA1 field of the hippocampus. Our findings provide direct evidence that functional nAChRs are present on the surface of both CA1 pyramidal (P, fig. 14) and interneurons (I, fig. 14) and that the receptors present in the interneurons modulate GABA release.

View larger version (12K): [in this window] [in a new window]   Fig. 14.   Proposed scheme of nicotinic actions on CA1 hippocampal neurons. Three pathways (A-C) are proposed to explain the nicotinic responses recorded from neurons in the CA1 field of the rat hippocampus. In A, the neuron under study receives an autaptic input from its own axon, and a GABAergic input from another neuron. In B, the neuron under study receives a GABAergic input from another neuron. In C, the neuron under study gives out a glutamatergic output to a GABAergic neuron, which in turn provides an inhibitory connection to the primary neuron. P, pyramidal neurons; I, interneurons; a, axon. Open triangles represent GABAergic and filled triangle represents glutamatergic boutons. Arrows indicate the main axonal pathway for the neuron under study. The tip of a patch pipette is shown on each neuron under study.

Physiological significance of nAChR-mediated modulation of GABA release in the hippocampus. After exposure to nicotinic agonists, the frequency of GABA-mediated PSCs was always much higher in recordings obtained from pyramidal neurons than in those obtained from interneurons, which thus indicates that nicotinic agonists may have a profound inhibitory influence on the activity of CA1 pyramidal neurons. According to our findings, if a neuronal network composed of interneuron  interneuron  pyramidal neuron exists, nAChR activation in the second interneuron should be very effective in increasing the inhibitory tonus to the pyramidal neuron, thereby overriding a possible functional disinhibition of the pyramidal neuron (Tóth et al., 1997). This disinhibition could occur in such a neuronal network only if nicotinic agonist-induced GABA release from the first interneuron in the circuitry were sufficient to silence the interneuron that synapses onto the pyramidal neuron. Thus, the frequency and timing of cholinergic stimuli arriving at the various CA1 interneurons may be essential in determining the magnitude of the output signals of the CA1 pyramidal neurons.