Slice preparation

Sprague-Dawley rats (18- to 54-day-old; Harlan, Indianapolis, IN, USA) were anesthetized with urethane (1.25 g/kg) and killed by decapitation. Transverse hippocampal slices (375 μm) were prepared and maintained at 30–32°C in artificial cerebrospinal fluid containing (in mM): NaCl 124, KCl 5, NaH₂PO₄ 1.25, MgSO₄ 2, CaCl₂ 2.5, NaHCO₃ 22, and glucose 10 and oxygenated with 95% O₂/5% CO₂, for at least 1 hour before recordings.

Electrophysiological recordings

Current- and voltage-clamp recordings were made from the somatic region of pyramidal cells and interneurons as described previously (Yamazaki et al., 2005, 2006). Slices were placed in a recording chamber, submerged, and continuously perfused at 2–3 ml/min with oxygenated artificial cerebrospinal fluid at 30°C. Neurons were visualized using a 40x water-immersion objective, infrared differential interference contrast (IR-DIC) system (Axioskop, Zeiss, Germany), and a charge-coupled device camera C2400 (Hamamatsu Corporation, Hamamatsu, Japan). Patch electrodes were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) using a micropipette puller (P-97, Sutter Instrument, Novato, CA, USA). The pipettes had a resistance of 3–8 MΩ after being filled with pipette solution. For voltage-clamp recordings, pipette solution contained (in mM) 117 Cs-methanesulfonate, 10 HEPES, 0.5 EGTA, 2.8 NaCl, 5 TEA-Cl, 5 QX-314, 2.5 Mg-ATP, and 0.3 Na-GTP, adjusted to pH 7.3 with CsOH. For current clamp recordings, pipette solution contained (in mM) 140 K-gluconate, 10 HEPES, 0.5 EGTA, 10 NaCl, 1 MgCl₂, 2 Mg-ATP, 0.2 Na-GTP, and QX-314, adjusted to pH 7.3 with KOH. The series resistance, normally about 15–20 MΩ, was monitored throughout the experiment; if it changed more than 20%, the recording was discarded. Responses of currents and potentials were recorded using Axopatch-200B or Axoclamp-2B (Axon Instruments, Union City, CA, USA), and were filtered (1–2 kHz), digitized at 1–5 kHz, and stored on a computer. In LTP experiments, excitatory postsynaptic currents (EPSCs) were evoked in oriens/alveus interneurons using a fine tip bipolar tungsten stimulating electrode (TT201-029, Unique Medical, Tokyo, Japan), which was positioned approximately 100–150 μm from the recorded cell. Because there was a possibility that EPSCs contained not only monosynaptic EPSCs but also polysynaptic responses, we measured the amplitude of the first negative peak to evaluate changes in monosynaptic EPSCs. When the amplitude of monosynaptic EPSCs could not be measured accurately due to overlap with a second EPSC peak or an unusual long EPSC peak latency, the recording was discarded. We also applied the NMDAR antagonist 2-amino-5-phosphopentanoate (AP5; 50 μM) throughout recordings to prevent a possible passively propagated form of LTP in interneurons.

Calcium imaging

For simultaneous Ca²⁺ imaging and electrophysiological recordings, interneurons were first loaded with the calcium indicator calcium green 1 (100 μM; Molecular Probes, Eugene, OR, U.S.A.) via the recording pipette. It was allowed to diffuse for a minimum of 20 min before starting optical and electrophysiological recordings. Fluorescence images were obtained by using MetaMorph Imaging System (Universal Imaging Corporation, West Chester PA, USA) and a fluorescent microscope (Axioskop, Zeiss) equipped with a mercury lamp, 488-nm excitation and 520-nm emission filters, and a charge-coupled device camera M4314 (Hamamatsu Corporation). Fluorescence intensities (F_post) over the region of interest in the soma at a given time point were normalized to baseline values [expressed as %ΔF/F = (F_post − F_rest)/(F_rest −F_back) × 100, where F_rest is the baseline fluorescence and F_back is the background fluorescence measured outside the cell] and plotted against time. To monitor the responsiveness of interneurons at the beginning and end of experiments, they were depolarized by 15mV by a current injection to generate calcium responses via activation of voltage-dependent Ca²⁺ channels.

Biocytin staining

During whole-cell recording, interneurons were passively filled with biocytin (0.5%) through the recording pipette for subsequent morphological identification (dendrite orientation, somata shape, and axon distribution) of interneurons. Following recordings, slices were incubated for 30 min and fixed overnight in 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde and 0.05% glutaraldehyde. Biocytin staining was processed using an avidin-peroxidase reaction (Vectastain ABC Standard Kit, Vector, Burlingame,CA, USA) with 3,3′-diaminobenzidine. The slice was then mounted on gelatin-coated glass slides for light microscopic examination.

Double-fluorescence immunohistochemistry

Hippocampal slices containing a biocytin-loaded interneuron were fixed by immersionin 4% paraformaldehyde in 0.1 M PB, pH 7.4, for several hours. After washing in 0.1 M PB, they were incubated with Alexa 546-conjugated streptavidin (Molecular Probes) diluted 1:500 with 0.1 M PB containing 0.1% Triton X-100 overnight at room temperature. Following washing in 0.1 M PB, the slices were incubated with an antibody against somatostatin-28 (IHC8004; Peninsula Laboratories, San Carlos, CA, USA; diluted 1:200) in 0.1 M PB containing 0.1% Triton X-100 for 10–12 h at room temperature. After washing, the slices were allowed to react with Alexa 488-conjugated goat anti-rabbit IgGs (Molecular Probes; diluted 1:500) in 0.1 M PB for 2 h at room temperature. The slices were then washed and coverslippedin Vectashield (Vector). Slices were observed under a confocal fluorescence microscope (LSM510 META, Zeiss).

In situ hybridization

The localization of α2 mRNA-positive cells by non-radioactive in situ hybridization was carried out as previously described (Ishii et al., 2005).

Single-cell reverse transcription (RT)-multiplex polymerase chain reaction (PCR)

To monitor the expression of molecular markers (calbindin D28k, parvalbumin, calretinin, neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin) in recorded cells, single-cell RT-multiplex PCR was performed by a previously described procedure (Cauli et al., 1997). After recordings were completed, the cytoplasm of individual neurons was harvested by applying suction on a patch pipette filled with sterile intracellular pipette solution. The contents of the pipette were expelled into a PCR tube by the application of positive pressure, quickly frozen on dry ice, stored at −80°C overnight, and subjected to RT-multiplex PCR using the Titan One Tube RT-PCR System (Roche Molecular Biochemicals, Indianapolis, IN, USA) and different pairs of PCR primers for simultaneous amplification of different molecular markers. This was followed by a second round of PCR using 1 μl of the first PCR product as a template. In this second round, each target cDNA was amplified individually using its specific primer pair. Each individual PCR reaction was run on an agarose gel and visualized with ethidium bromide for identification of target cDNAs.

Nicotine-sensitive interneurons are oriens-lacunosum moleculare cells

There are different subtypes of horizontally oriented interneurons in the stratum oriens/alveus, including oriens-lacunosum moleculare cells, basket cells, and oriens-bistratified cells (Freund & Buzsaki, 1996; Maccaferri, et al., 2000; Maccaferri, 2005). Basket cells are parvalbumin and cholecystokinin positive (Freund & Buzsaki, 1996; Maccaferri, et al., 2000). Both oriens-lacunosum moleculare cells and oriens-bistratified cells are somatostatin immunoreactive (Maccaferri, et al., 2000), and oriens-lacunosum moleculare cells also contain neuropeptide Y (Freund & Buzsaki, 1996). To gain insight into a nicotine-sensitive interneuron subtype, we combined patch-clamp recording and single-cell RT-multiplex PCR to monitor the differential expression of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin) that define interneuron types with partial overlaps (Freund & Buzsaki, 1996; Parra et al., 1998). To detect the mRNAs of these neurochemical markers simultaneously in a single cell, two steps of multiplex PCR were performed (Cauli et al., 1997; Jia et al., 2009). Initially, the efficiency and specificity of this protocol was tested on total RNA isolated from rat hippocampi. As Fig. 2A1 (top) shows, the seven specific mRNAs were detected, each generating a PCR product of the size predicted by its mRNA sequence. Subsequently, the differential expression of the seven markers was examined at the single cell level. We found that nicotine-sensitive interneurons were consistently positive for somatostatin and neuropeptide Y mRNAs (in 5 of 5 interneurons), and one of these neurons was positive for both calretinin and vasoactive intestinal polypeptide mRNAs (Fig. 2A1, bottom, and Fig. 2A2).

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Figure 2

A subtype of nicotine-sensitive oriens/alveus interneurons and its molecular markers

(A1, A2) Nicotine-sensitive interneurons contain somatostatin and neuropeptide Y mRNAs. (A1, top) RT-multiplex PCR amplification of different marker sequences. Hippocampal RNA (500 pg) was subjected to a RT-multiplex PCR protocol to detect the expression of calbindin D28k (CB), parvalbumin (PV), calretinin (CR), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SS), and cholecystokinin (CCK). (A1, bottom) RT-multiplex PCR applied on a single nicotine-sensitive interneuron after electrophysiological recording to detect molecular markers. An example of single-cell RT-multiplex PCR, showing the presence of CR, NPY, VIP, and SS in a recorded cell. RT-PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide. The amplified fragments had the sizes (in bp) predicted by the mRNA sequences: 432 (CB), 388 (PV), 309 (CR), 359 (NPY), 287 (VIP), 209 (SS), 216 (CCK). A 100 bp DNA ladder was used as a molecular weight marker. (A2) Summary data of single-cell RT-multiplex PCR. (B) One biocytin-labeled nicotine-sensitive interneuron with characteristics resembling oriens-lacunosum moleculare cells. (C1, C2) Double-labeling of a nicotine-sensitive interneuron with biocytine and immunohistochemistry for somatostatin. Immunohistochemical localization of somatostatin-containing interneurons (C1) and co-localization of somatostatin (green) and biocytin (red) in a nicotine-sensitive interneuron (C2).

In a further effort to identify a nicotine-sensitive interneuron subtype, we passively filled these interneurons with biocytin through recording pipettes for subsequent morphological identification. All biocytin-filled cells had horizontally oriented dendrites. When axonal arborizations were visible (n = 10), they showed morphological characteristics of oriens-lacunosum moleculare cells (Fig. 2B), which project to the most distal dendrites of pyramidal cells (Freund & Buzsaki, 1996; Maccaferri et al., 2000; Maccaferri, 2005). To confirm the presence of somatostatin protein in nicotine-sensitive interneurons, some nicotine-responding interneurons were passively filled with biocytin for subsequent double-fluorescence immunohistochemistry with a somatostatin antibody. We found that biocytin-labeled cells were somatostatin-positive (Fig. 2C1, C2). These results demonstrate that nicotine-sensitive interneurons are oriens-lacunosum moleculare cells. However, because we were unable to follow the complete axonal arborization of some biocytin-filled interneurons, we cannot exclude the possibility that nicotine-responding interneurons include other subtypes of horizontally oriented interneurons, which express both somatostatin and neuropeptide Y.

Nicotine increases intracellular Ca²⁺ concentrations in oriens/alveus interneurons

Different subtypes of nAChRs are known to be differentially permeable to Ca²⁺ (Role & Berg, 1996; Fucile, 2004). Thus, activation of non-α7 nAChRs in oriens/alveus interneurons could elevate intracellular Ca²⁺ levels directly by being permeable to Ca²⁺ themselves, and indirectly by generating sufficient depolarizing current to activate voltage-dependent Ca²⁺ channels. Because increases in intracellular Ca²⁺ play a critical role in LTP induction (Topolnik et al., 2005, 2006), we next examined whether nicotine causes an increase in intracellular Ca²⁺ levels. To measure changes in intracellular Ca²⁺ levels in the soma, we used a fluorescent assay with calcium green-1. Current-clamped cells were loaded with calcium green-1 via the patch pipette (Fig. 3A1, A2).

Subsequent bath application of nicotine (10 μM) in the presence of DNQX (20 μM) and AP5 (40 μM) produced detectable increases in fluorescent intensity at the soma, as shown in pseudocolor images (Fig. 3A2, A3). To determine whether the increased fluorescent intensity was due to the activation of non-α7 nAChRs, we simultaneously recorded nicotine-induced changes in membrane potential and fluorescent intensity in current-clamped cells. Bath application of nicotine (10 μM) induced a depolarization in interneurons (10.4 ± 1.2 mV, n = 11) and generated action potentials, which were completely blocked in the presence of DHβE (2 μM; Fig. 3B, C1; 0.2 ± 0.5 mV, n = 9, t₁₈ = −7.01, P < 0.001). At the same time, bath application of nicotine (10 μM) increased Ca²⁺ responses (17.2 ± 3.1% increase over basal levels, n = 11), which were well maintained during nicotine-induced membrane depolarization, and this effect was almost completely inhibited in the presence of DHβE (2 μM; Fig. 3B, C2; 2.3 ± 0.6% increase over basal levels, n = 9, t₁₈ = −4.32, P < 0.001). Reapplication of nicotine after approximately 10-min drug washout reproducibly induced interneuronal depolarization and intracellular Ca²⁺ rises (Fig. 3B, right). These results suggest that activation of non-α7 nAChRs causes increases in intracellular Ca²⁺ concentrations in these interneurons.

It has been shown that bath application of kainate caused depolarization and repetitive action potential firing in oriens-lacunosum moleculare cells (Cossart et al., 1998), which resembles the effects of nicotine on oriens/alveus interneurons (Jia et al., 2009). Thus, we next examined whether activation of kainate receptors induces Ca²⁺ rises in nicotine-sensitive oriens/alveus interneurons. Bath application of kainate (1 μM) induced a depolarization in interneurons (8.3 ± 1.7 mV, n = 5) and generated action potentials (Fig. 3D1,D2), and simultaneously increased Ca²⁺ responses (Fig. 3D1,D2; 14. 5 ± 4.4% increase over basal levels, n = 5). Changes in membrane potential and Ca²⁺ responses caused by application of kainite were very similar to those induced by application of nicotine (Fig. 3D2). Depolarizing the cells with 30 mM K⁺ to activate voltage-dependent Ca²⁺ channels induced a membrane depolarization (Fig. 3D1,D2; 29.9 ± 3.2 mV, n = 6) that was significantly larger (Fig. 3D2; ANOVA, F_(2,19) = 31.9, P < 0.001) than that caused by nicotine (P < 0.001) or kainite (P < 0.001), and caused increases in Ca²⁺ responses (Fig. 3D1,D2; 28.3 ± 3.3% increase over basal levels, n = 6) that were significantly higher (Fig. 3D2; ANOVA, F_(2,19) = 3.54, P = 0.049) than the increases induced by nicotine (P = 0.034) or kainite (P = 0.028). Thus, nicotine-induced intracellular Ca²⁺ increases were much less than the Ca²⁺ increases that were produced by the activation of voltage-dependent Ca²⁺ channels. Ca²⁺ permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) are expressed in oriens/alveus interneurons (Topolnik et al., 2005, 2006; Kullmann, & Lamsa, 2007). In our experiments, these receptors, which could be activated by kainate, were not blocked. Thus, although kainate receptors can be Ca²⁺ permeable depending on the degree of post-transcriptional RNA editing of their subunits (Bernard et al. 1999; Lerma et al. 2001), the observed kainate-induced Ca²⁺ rises might be at least in part due to the activation of Ca²⁺ permeable AMPARs.

Non-α7 nAChRs on oriens/alveus interneurons are Ca²⁺ permeable

Our results described above demonstrate that activation of non-α7 nAChRs caused intracellular Ca²⁺ rises in oriens/alveus interneurons. However, it was unclear whether this was due to direct entry of Ca²⁺ through the receptor channel, depolarization-induced activation of voltage-dependent Ca²⁺ channels, or both. Initially, we attempted to pharmacologically dissect out the relative contribution of voltage-dependent Ca²⁺ channels using Cd²⁺ as a voltage-gated Ca²⁺ channel blocker. We compared the amplitude of nicotine-induced Ca²⁺ elevations in current-clamped interneurons before and after addition of 100 μM Cd²⁺, which almost completely blocked K⁺ (30 mM) induced Ca²⁺ elevations (data not shown). However, we found that the inhibitory effect of Cd²⁺ on nicotine-induced Ca²⁺ rises was quite variable among different cells, perhaps due to differences in the relative contribution of voltage-dependent Ca²⁺ channels to a total Ca²⁺ elevation. Nevertheless, these experiments suggest the involvement of voltage-dependent Ca²⁺ channels in nicotine’s effects. Subsequently, we decided to simply determine whether nicotine increases intracellular Ca²⁺ concentrations without the contribution of voltage-dependent Ca²⁺ channels. Thus, we measured nicotine-induced Ca²⁺ responses in interneurons, which were voltage-clamped below the threshold for activation of voltage-dependent Ca²⁺ channels. Interneurons were loaded with calcium green-1 via the patch pipette and the cells were voltage-clamped at −70 mV in the presence of DNQX (20 μM), AP5 (40 μM). Simultaneous measurements of currents and Ca²⁺ responses were carried out. As reported previously (Jia et al., 2009), bath application of nicotine (0.5–10 μM) produced long-lasting inward currents that were inhibited by DHβE, but not MLA (data not shown). On average, the application of 10 μM nicotine caused a maximum response (Fig. 4A, top, 87.9 ± 2.0 pA, n = 6). Bath application of nicotine (10 μM) also produced a fluorescence response (Fig. 4A, bottom, B; 9.9 ± 1.3% increase over basal levels, n = 6), suggesting that activation of non-α7 nAChRs can produce increases in intracellular Ca²⁺ levels by direct passage of Ca²⁺ through the receptor channel. The response kinetics were different between current-clamped and voltage-clamped conditions (Fig. 3B and Fig. 4A). Furthermore, the increase of intracellular Ca²⁺ concentrations in voltage-clamped cells was smaller than that observed in current-clamped cells, although the difference did not reach statistical significance (Fig. 4B; t₁₅ = 1.69, P = 0.11). These observations further support the idea that depolarization-induced activation of voltage-dependent Ca²⁺ channels via non-α7 nAChRs contributes to nicotine-induced intracellular Ca²⁺ rises in current-clamped cells.

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Figure 4

Nicotine elevates intracellular Ca²⁺ levels in oriens/alveus interneurons via Ca²⁺ entry through non-α7 nAChRs

(A) Simultaneous recordings of nicotine-induced currents (top) and changes in Ca²⁺ fluorescence signal (bottom) in a voltage-clamped oriens/alveus interneuron (at −70 mV). Recordings were carried out in the presence of DNQX (20 μM) and AP5 (40 μM). Nicotine (10 μM) was bath-applied as indicated by the horizontal bar. (B) Summary graph showing Ca²⁺ fluorescence signal (mean ± SEM) elicited in current-clamped (CC) and voltage-clamped (VC) oriens/alveus interneurons by bath application of nicotine (10 μM).

Nicotine facilitates LTP induction at excitatory synapses on oriens/alveus interneurons

Many interneurons in the stratum oriens/alveus, including oriens-lacunosum moleculare cells, show NMDAR-independent LTP (Perez et al., 2001; Lapointe et al., 2004; Topolnik et al., 2006; Lamsa et al., 2007; Kullmann & Lamsa, 2008; Oren et al., 2009), which can be induced by different LTP induction protocols. To examine whether nicotine facilitates the induction of LTP in oriens/alveus interneurons, we recorded EPSCs in voltage-clamped cells (at −70 mV). Most of the excitatory input to oriens-lacunosum moleculare cells arises from recurrent collaterals of CA1 pyramidal cells. Thus, we evoked EPSCs by stimulation in the stratum oriens, close to the patched interneurons (Fig. 5A). Tetanic stimulation (100 Hz, 100 pulses) was used to induce LTP under voltage-clamp conditions. Although NMDA receptor-dependent LTP does not occur in oriens/alveus interneurons (Perez et al. 2001; Lamsa et al. 2007), NMDAR-dependent LTP occurring at the Schaffer collateral-pyramidal cell synapses can passively propagate to these interneurons (Perez et al. 2001). Thus, to prevent a possible passively propagated form of LTP in interneurons, all recordings were performed in the presence of the NMDAR antagonist AP5 (50 μM). In addition, bicuculline (10 μM), MLA (50 nM), and atropine (1μM) were added to the bath perfusate to block GABA_A receptors, α7 nAChRs, and muscarinic receptors, respectively. Under these conditions, tetanic stimulation failed to induce LTP (Fig. 5B,F; 104.3 ± 8.3% 50–55 min after tetanus, n = 4) in voltage-clamped interneurons. However, when tetanic stimulation was applied in the presence of nicotine (10 μM), LTP was induced (Fig. 5C,F; 153.3 ± 14.8% 50–55 min after tetanus, n = 4; tetanus alone vs. tetanus + 10 μM nicotine, t₆ = 3.15, P = 0.020). We have previously reported that bath application of nicotine (0.5–1 μM) produced long-lasting inward currents (10–47 pA) in oriens/alveus interneurons (Jia et al., 2009). Thus, we next examined whether a lower concentration (1 μM) of nicotine facilitates LTP induction in oriens/alveus interneurons, and found that LTP was induced in nicotine-responding interneurons (Fig. 5D,F; 139.4 ± 14.8% 50–55 min after tetanus, n = 5; tetanus alone vs. tetanus + 1 μM nicotine, t₇ = 2.63, P = 0.033). Bath application of nicotine for 1.5 min caused decreases in the amplitude of EPSCs and increases in holding current, indicating the presence of nicotine-induced inward currents (Fig. 5C,D). Increases in holding current reached its peak value in approximately 3 min after nicotine application, at which time the tetanus was delivered, and returned to a baseline level following washout of nicotine. The time course of changes in EPSC amplitude following nicotine application mirrored that of changes in holding current that occurred after nicotine application. This suggests that nicotine-induced inward currents suppresses EPSCs, perhaps due to reduced driving force for Na⁺ and decreased membrane resistance, which causes a shunting effect. To exclude the possibility that the observed effect of nicotine on LTP induction was due to a long-lasting effect of nicotine, we next examined the effect of nicotine alone (i.e., without tetanic stimulation) on EPSCs. Bath application of nicotine (10 μM) for 1.5 min caused decreases in the amplitude of EPSCs, reaching its minimum in approximately 3 min after nicotine application and returning to basal levels following washout of nicotine (Fig. 5E,F; 105.5 ± 6.1% 53–58 min after nicotine application, n = 4; tetanus + nicotine vs. nicotine alone, t₆ = 3.45, P = 0.017). Thus, nicotine alone had no long-lasting effect on EPSCs. These results suggest that nicotine facilitates the induction of LTP in oriens/alveus interneurons via activation of non-α7 nAChRs.

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Figure 5

Nicotine promotes the induction of LTP at excitatory synapses onto oriens/alveus interneurons

(A) Scheme of recording setup showing the position of stimulating and recording electrodes. Oriens/alveus interneurons were voltage-clamped at −70 mV, and evoked EPSCs were recorded in the presence of AP5 (50 μM), bicuculline (10 μM), MLA (50 nM), and atropine (1 μM). (B–E) Changes in the amplitude of EPSCs were plotted as the percent change of initial baseline responses against time. Each trace above the graph in (B–E) was recorded at the time indicated. (B) A tetanus (100 pulses at 100Hz) alone failed to induce LTP. (C) A tetanus applied in the presence of nicotine (10 μM) induced LTP. (D) A tetanus applied in the presence of nicotine (1 μM) induced LTP. (E) Application of nicotine (10 μM) alone had no long-lasting effect on the amplitude of EPSCs. (C-E) Bath application of nicotine caused decreases in the amplitude of EPSCs (top) and increases in holding current (bottom). Administration of nicotine is indicated by the bar and delivery of tetanic stimulation is indicated by the arrow. (F) Histograms show the percent change (mean ± SEM) in the amplitude of EPSCs measured 50–60 min after delivery of tetanic stimulation in the absence and presence of nicotine, or after application of nicotine alone. *P < 0.05.

A non-α7 nAChR subtype expressed in nicotine-sensitive oriens/alveus interneurons

It was previously reported that horizontally oriented interneurons in the stratum oriens/alveus, including oriens-lacunosum moleculare cells, contained both α7 and non-α7 nAChRs, which were activated by ACh puffs but were desensitized in the presence of nicotine (Frazier et al., 1998a; McQuiston & Madison, 1999; Sudweeks & Yakel, 2000). Because it was not tested whether bath application of nicotine elicited non-α7 nAChR-mediated responses in these interneurons, whether these interneurons expressed slowly or non-desensitizing nAChR subtype was unclear. Furthermore, single-cell RT-PCR analysis showed that these interneurons were consistently positive for all α subunit subtypes (α2-α7 mRNAs), making it difficult to identify a non-α7 nAChR subtype that mediated the responses (Sudweeks & Yakel, 2000). Our previous study demonstrated that bath application of nicotine produced slow inward currents in a subset of horizontally oriented interneurons in the stratum oriens/alveus, which were well maintained and inhibited by the non-α7 antagonist DHβE, but not the α7 antagonist MLA (Jia et al., 2009). In situ hybridization demonstrate that the α2 mRNA is uniquely expressed in a subset of interneurons in the stratum oriens/alveus (Ishii et al., 2005; Jia et al., 2009). Furthermore, single-cell RT-PCR analysis demonstrated that these nicotine-responding interneurons were consistently positive for the α2 subunit mRNA (in 8 of 8 interneurons) and two of these cells were positive for the α3 subunit mRNA, but not other α subunit mRNAs (Jia et al., 2009). Thus, the nicotine-induced facilitation of LTP induction is most likely mediated via activation of α2* nAChRs. However, the absence of α3 and α4 subunit mRNAs in many or all nicotine-sensitive interneurons, respectively, can easily be due to a lack of the sensitivity needed to detect less-abundant nAChR subunit mRNAs than α2 subunit mRNAs in these interneurons. It remains to be determined whether these cells contain functional α2* nAChRs that mediate the effects of nicotine.

An interneuron subtype containing non-desensitizing non-α7 nAChRs

The present study demonstrated that biocytin-filled nicotine-sensitive cells showed morphological characteristics of oriens-lacunosum moleculare cells, when axonal arborizations were visible. Furthermore, all non-α7 nAChR-containing interneurons tested in the present study were positive for somatostatin and neuropeptide Y, both of which are known to be expressed in oriens-lacunosum moleculare cells (Freund & Buzsaki, 1996; Maccaferri et al., 2000).

Nicotine-sensitive horizontally oriented interneurons in the stratum oriens/alveus are connected to pyramidal cells (Jia et al., 2009). In the literature (Freund & Buzsaki, 1996; Maccaferri et al., 2000; Maccaferri, 2005), at least three different subtypes of horizontally oriented interneurons (oriens-lacunosum moleculare cells, basket cells, and oriens-bistratified cells) that innervate pyramidal cells have been identified and characterized. Both oriens-lacunosum moleculare cells and oriens-bistratified cells are somatostatin immunoreactive, but basket cells, which innervate the soma, are not (Freund & Buzsaki, 1996; Maccaferri et al., 2000). Thus, although rigid classification of the subtypes of interneurons may be difficult, it is most likely that interneurons activated by nicotine are not basket cells. Because a previous study demonstrated that non-α7 nAChR-mediated responses in oriens-lacunosum moleculare cells desensitized in the presence of nicotine (McQuiston & Madison, 1999), our results suggest the presence of a separate population of oriens-lacunosum moleculare cells, expressing non-desensitizing non-α7 nAChRs.

Oriens-lacunosum moleculare cells project to the most distal dendrites of pyramidal cells where the temporoammonic path terminates (Freund & Buzsaki, 1996; Maccaferri et al., 2000; Maccaferri, 2005). Bath application of nicotine inhibits field excitatory postsynaptic potentials at the temporoammonic pathway, which requires GABAergic inhibition (Nakauchi et al., 2007a,b). This effect of nicotine is prevented, if a knife cut is made along the pyramidal layer in the stratum oriens to sever the axons of interneurons projecting to the apical dendrites of pyramidal cells (Nakauchi et al., 2007b). These observations suggest that the effect of nicotine is attributed to GABA release from the terminals of oriens-lacunosum moleculare cells. The axons of oriens-bistratified cells primarily terminate in the stratum radiatum and overlap the dendritic trees of pyramidal cells (Maccaferri et al., 2000). Bath application of nicotine has no significant effect on field excitatory postsynaptic potentials at the Schaffer collateral pathway (Fujii et al., 1999; Nakauchi et al., 2007a), suggesting that there is no significant GABA release from the terminals of oriens-bistratified cells during nicotine application. These observations also support the idea that oriens-lacunosum moleculare cells are a major subtype of nicotine-sensitive interneurons in the stratum oriens/alveus, which express α2 mRNAs. Identification of a nicotine-sensitive interneuron subtype as an oriens-lacunosum moleculare cell is significant, because nicotine-induced tonic inhibition at the most distal dendrites would depress direct information flow into CA1 pyramidal cells from entorhinal input (the temporoammonic path) and might preferentially direct information flow to CA1 pyramidal cells through the trisynaptic circuitry in the presence of nicotine.

Roles of Ca²⁺ entering through nAChR channels in circuit activity and plasticity

The α7 nAChR is expressed at high levels by interneurons in the hippocampal CA1 region (Alkondon & Albuquerque, 2001; Alkondon et al., 2000; McQuiston & Madison, 1999; Sudweeks & Yakel, 2000; Ji et al., 2001; Buhler & Dunwiddie, 2001, 2002; Frazier et al., 1998a, 1998b, 2003), in which activation of α7 nAChRs causes an increase in intracellular Ca²⁺ concentrations without involving CICR or activation of voltage-dependent Ca²⁺ channels (Khiroug et al., 2003; Fayuk & Yakel., 2005, 2007). This source of Ca²⁺ triggers signaling, leading to the suppression of GABA_A receptor-mediated responses in the interneurons (Wanaverbecq et al., 2007; Zhang & Berg, 2007).

Oriens/alveus interneurons in the hippocampal CA1 region express non-α7 nAChRs, which desensitize in the presence of nicotine (McQuiston & Madison, 1999). The activation of these non-α7 nAChRs via 5 sec pressure pulses of ACh (2 mM) in the presence of MLA does not significantly change intracellular Ca²⁺ concentrations (Fayuk & Yakel., 2005). However, in the present study, we found that bath application of nicotine increases Ca²⁺ levels in nicotine-sensitive interneurons at the stratum oriens/alveus border. This increase is at least in part attributed to Ca²⁺ entry through non-α7 nAChRs, which are continuously activated in the presence of nicotine. The change in intracellular Ca²⁺ concentrations caused by a brief activation of non-α7 nAChRs in oriens/alveus interneurons may be too small to be detected, and only becomes detectable when they are continuously activated in the presence of nicotine. In this case, Ca²⁺ signals leading to LTP induction can be only elicited in interneurons expressing non-desensitizing non-α7 nAChRs, because they are continuously activated in the presence of nicotine.

This research was supported by the National Institute on Drug Abuse (DA014542, DA025269, DA026458).