Nicotine depolarizes deep layers of the EC and Sb through the activation of non-α7 nAChRs in Wistar Rats
Voltage sensitive dye imaging (VSDI) provides high temporal and spatial resolution in monitoring membrane potential changes in neuronal populations in brain slices or under the surface of intact brains (Airan et al., 2007
; Ebner and Chen, 1995
). Di-4-ANEPPS, a styryl dye that linearly reflects membrane potential changes at a rate of 1% per 10 mV under physiological conditions (Loew et al., 1992
), has been found to reliably detect neuronal membrane potential changes within milliseconds (Airan et al., 2007
; Mann et al., 2005
). The voltage sensitivity of Di-4-ANEPPS was verified in our experimental setup () by fluorescent responses to Schaffer collateral stimulation in rat CA1 hippocampal slices stained with this dye, as well as the absence of fluorescent responses in slices stained with the voltage-insensitive
dye FM 1–43 (which was used as a control, ). As previously reported (Tominaga et al., 2000
), we also observed significant fluorescence drift (likely due to photobleaching and other factors) in our slice recordings during 30 min of imaging, which was accounted for and removed (to obtain a stable VSDI signal baseline) by using a 2-dimensional drift-removal function in the BV-Analysis software ().
After adding tetrodotoxin (TTX; 1 μM) to remove action potential-dependent synaptic transmission between neurons, we found that bath-applied nicotine (10 μM) significantly depolarized neuronal populations in Layer VI of the EC (ECVI) and stratum oriens of the Sb (SbSO), whereas other regions were depolarized to a lesser extent ().
Nicotine depolarization of hippocampal formation was most evident in deep layers of EC through non-α7 nAChRs
The two major subgroups of nAChRs in hippocampal neurons are the α7-containing nAChRs (α7 nAChRs) and a diverse subgroup of non-α7-containing nAChRs (non-α7 nAChRs), the most prevalent of which is the α4β2 subtype in the hippocampus and cortex (Alkondon and Albuquerque, 1993
; Alkondon et al., 1994
; Jones and Yakel, 1997
; Sudweeks and Yakel, 2000
). We found that the non-α7 nAChR antagonist dihydro-ß-erythroidine (DHßE, 1 μM) blocked nicotine-induced VSDI responses, while the α7 nAChR-selective antagonist methyllycaconitine (MLA, 10 nM) did not (), which demonstrates that the nicotine-induced neuronal depolarizations were mediated by non-α7 nAChRs. The viability of neurons in slices was verified by the ability of 4 mM KCl to induce significant depolarization (, ), or in some cases by Schaffer collateral stimulation-evoked responses recorded through field electrodes (). Brain slices stained with the voltage-insensitive dye FM 1–43 did not show any fluorescent responses to nicotine (10 μM, ), which suggests that nicotine-induced fluorescent responses using Di-4-ANEPPS were not due to some interaction between nicotine and the fluorescent dye (independent of membrane potential changes), the influence of nicotine on slice optical characteristics, nor any potential membrane surface change as a result of synaptic vesicle release triggered by nicotine.
Layer ECVI neurons are the most responsive to nicotine
We used whole-cell patch-clamp techniques to record from individual neurons within various regions and layers of the hippocampal formation (HF, ) to directly measure the ability of nicotine to induce electrical responses, and how this pattern of responsiveness compared to the VSDI results. Under current-clamp we found that all ECVI neurons that we recorded from (n = 26) were depolarized by bath-applied nicotine (10 μM; , ), and that the amount of depolarization was larger when compared to neurons in other regions of the HF ().
Patch-clamp recordings confirm ECVI neurons as the most nicotine-sensitive neuronal population in the HF
Comparing nAChR-mediated responses in ECVI neurons to those in other regions.
To compare the kinetics of the nicotine-induced depolarizations in different regions of the HF, we averaged the membrane potential recordings in response to nicotine (). We found that the depolarizations induced by the bath-application of nicotine for 5 min reached peak within 1 min and then slowly repolarized back to baseline. The repolarizations were caused by the decay of nicotine responses (most likely due to desensitization) and not as a result of membrane potential drift since the nicotine responses were completely blocked (i.e. complete recovery of membrane potential to the value prior to nicotine application) by DHßE (either 1 μM or 100 μM for complete inhibition). The decay rate (i.e. the % of decay after 5 min of nicotine application) in the CA1 SR neurons (17 ± 6%, n = 4) was similar to that in ECVI neurons (15 ± 4%, n = 16), both of which were significantly slower than that in the SbSO neurons (57 ± 6%, n = 9) or ECV neurons (43 ± 8%, n = 8) (p<0.01, 1-way ANOVA followed by Newman-Keuls multiple comparison test). When DHßE was replaced by the ionotropic glutamatergic receptor blockers NBQX (20 μM) and APV (50 μM), the nicotine-induced responses in ECVI neurons were not affected (n = 3, data not shown), precluding the contribution of glutamate release to the nicotine-induced depolarization.
The concentration of nicotine required to produce half-maximal depolarizations (i.e. the EC50
value) was 0.6 μM for ECVI neurons (), which was significantly lower than for either SbSO (1.3 μM) or ECV (5.5 μM) neurons (p
<0.01, 2-way ANOVA using concentration and region as variables followed by Bonferroni posttests). The bath application of a low concentration of nicotine (0.1 μM), which is within the range found in the blood of average smokers (i.e. 0.06–0.31 μM) (Hukkanen et al., 2005
; Moriya et al., 2006
; Moriya and Hashimoto, 2004
), still significantly depolarized ECVI neurons, unlike either SbSO or ECV neurons ().
Functional distribution of nAChR-mediated current responses in the HF
We recorded nAChR-mediated current responses under voltage-clamp during the rapid pressure application of ACh to the soma of neurons in different regions of the HF in the presence of TTX (1 μM) and atropine (10 μM). Under these conditions, there will be little to no desensitization of nAChR-mediated responses, which is particularly important for α7-containing nAChRs that are characterized by rapid desensitization (Alkondon and Albuquerque, 2002
; Jones and Yakel, 1997
; Khiroug et al., 2003
; McQuiston and Madison, 1999
). To isolate the non-α7 nAChR-mediated current responses, we activated responses with ACh in the presence of MLA (10 nM), and to isolate the α7 nAChR component, we activated responses with the full α7 nAChR-selective agonist choline (). Although MLA has previously been reported to block α6-containing nAChRs (Klink et al., 2001
), the α6 subunit is not expressed in rat hippocampus and entorhinal cortex (Mugnaini et al., 2002
; Sudweeks and Yakel, 2000
), and thus this should not affect the interpretation of our results. We found that ECVI and SbSO neurons had the largest non-α7 nAChR-mediated responses ( and ), similar to what we observed for nicotine-induced depolarizations. These data indicate that ECVI and SbSO neurons had the highest levels of functionally expressed non-α7 nAChRs, which is consistent with the previous VSDI results that ECVI and SbSO were the most nicotine-responsive regions in the HF.
Functional distribution of α7 and non-α7 nAChRs in the HF
After blocking α7 nAChRs with MLA (10 nM), the bath application of DHßE (1μM) blocked over 95% of the nicotine-induced depolarizations in neurons of the ECVI (median, 96.6%; inter-quartile range, 83.3–99.3%; n = 15), the SbSO (98.3%; 96.0–98.9%; n = 5), and the CA1SR (98.3%; 94.3–99.1%; n = 11). The α4β2 nAChR subtype, besides being the most prevalent subtype in the hippocampus and cortex, has the highest affinity for nicotine and DHβE (Harvey et al., 1996
; Marks et al., 1999
). The pharmacological data for our non-α7 nAChR-mediated responses are most consistent with their being comprised mostly of the α4β2 subtype, and not consistent with the other common subtypes of non-α7 nAChRs expressed in the brain; e.g. the α3β4 subtype (Jones and Yakel, 1997
; Sudweeks and Yakel, 2000
). Neither the α3 nor the α2 nAChR subunits were previously detected in the deep layers of the EC whereas the α4 and β2 subunits were (Wada et al., 1989
), as well as the lack of presence of the α6 nAChR subunit.
The functional distribution of α7 nAChR-mediated current responses (i.e. those responses activated by choline) is shown in and . Interestingly, most ECVI neurons (89%) had functional α7 responses with a median amplitude not significantly different from CA1 interneurons (p>0.05, Kruskal-Wallis test and post hoc Dunn's multiple comparison test). Since α7 nAChRs desensitize rapidly, the most likely reason why we did not observe an α7 receptor-mediated depolarizing component in response to bath-applied nicotine in ECVI neurons was due to desensitization. To address this point directly, we utilized the α7-selective allosteric potentiator PNU-120596 (PNU), which dramatically increases α7 response amplitudes in part by reducing desensitization (Hurst et al., 2005
; Poisik et al., 2008
). Bath application of nicotine (under current-clamp; n = 3) to CA1 SR interneurons which functionally expressed only α7 nAChRs (and not non-α7 nAChRs) failed to induce any membrane depolarization (). However after the application of PNU (10 μM; to other α7-only CA1 SR interneurons), the bath application of nicotine was now able to induce significant membrane depolarizations (; n = 3). These results demonstrate that desensitization prevented α7 nAChRs from contributing to the neuronal depolarization induced by the bath application of nicotine under physiological conditions.
Desensitization prevents α7 nAChRs from contributing to membrane depolarizations induced by the bath application of nicotine
The α7-only nature of these responses was verified by similar (defined as less than 5% difference) rise and decay kinetics between responses to ACh and the full and selective α7 nAChR agonist choline, and the significantly slower kinetics of non-α7 nAChRs. The absence of non-α7 nAChR-mediated currents in these α7-only neurons was further confirmed by the lack of changes in the kinetics of responses to ACh after the bath application of DHßE (1 μM). As a final confirmation, 21 α7-only neurons were selected from 68 CA1 SR neurons using these criteria, and ACh-evoked currents in all of these neurons were completely (>95%) blocked by 10 nM MLA.
Characteristics of ECVI neurons
Morphological properties of ECVI neurons
As previously reported (Dugladze et al., 2001
), the shapes of ECVI neurons may be either pyramidal or multipolar, while ECV neurons are primarily pyramidal shaped. When observed under IR-DIC (infrared differential interference contrast) optics, the soma diameters of ECVI neurons were not significantly different from CA1 interneurons, but were significantly larger than the diameters of either ECV or CA1 pyramidal neurons (). The measurements of neuronal size under IR-DIC are consistent with a previous report (Ruan et al., 2007
). The membrane capacitance (an indicator of membrane surface area) of ECVI neurons was significantly larger than CA1 neurons, suggesting that ECVI neurons had more dendritic arboration than CA1 neurons since their soma sizes were similar.
Size and electrophysiological properties of ECVI neurons.
Electrical properties of ECVI neurons
The resting membrane potentials (RMPs) of ECVI neurons were more negative than those of either CA1, Sb, ECII, or ECVIII neurons (). In addition the ECVI neurons had the lowest level of h-current (Ih
) among all regions of neurons studied. Ih
is a current mediated by a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel (Rosenbaum and Gordon, 2004
; Siu et al., 2006
). Although HCN channels are weakly selective for K+
with permeability ratios of about 4:1, under physiological conditions, Ih
is mostly carried by Na+
due to a greater driving force (Siu et al., 2006
has a significant impact on the RMP since inhibition of Ih
hyperpolarizes neurons (Aponte et al., 2006
; Lupica et al., 2001
). Therefore a low Ih
amplitude, such as that found in ECVI neurons, can contribute to the more hyperpolarized RMP of these neurons. The ECVI neurons also had smaller input resistances than CA1 interneurons, spiked at a frequency slower than CA1 interneurons but faster than CA1 pyramidal neurons when depolarized (i.e., medium frequency spiking), and had similar action potential thresholds ().
ECVI neurons provide both excitatory and inhibitory inputs to ECV neurons
Both ECVI and ECV neurons project mainly to superficial layers of the EC (Dolorfo and Amaral, 1998
; Hamam et al., 2000
; Kohler, 1986
), and a wide range of cortical areas including the temporal pole, medial frontal and orbitofrontal cortices, and the rostral part of the polysensory area of the superior temporal sulcus cortices (Deller et al., 1996
; 2001; Hamam et al., 2000
; Munoz and Insausti, 2005
; Wyss and Van Groen, 1992
). Through these projections, ECVI and ECV neurons may impact indirectly on hippocampal functions since the neurons in superficial layers of the EC (ECII or ECIII) are the major sources of inputs received by the dentate and the hippocampal proper. Projections from ECVI or ECV were also identified targeting neurons in the subiculum and the dentate gyrus directly (Dugladze et al., 2001
), suggesting that direct influence on hippocampal function by deep EC neurons is also possible. We found that ECVI neurons, which are mainly excitatory neurons (Hamam et al., 2000
), were the most responsive to nicotine (based on the VSDI and patch-clamp experiments described above). Thus far there are no reports demonstrating the functional synaptic influence of the neuronal activation of ECVI on neurons in other regions. Because the ECV neurons are the closest neuronal population to the ECVI region, any connection between ECV and ECVI neurons may be well-preserved in the hippocampal slices that we have studied. In addition, ECV neurons are known to mediate interactions between the hippocampal proper and a wide range of neocortical areas (Insausti et al., 1997
; Naber et al., 2001
). Therefore if ECV neurons were influenced directly by ECVI neurons, nicotine may influence the function of various hippocampal regions through the activation of neurons in the ECV, which will further influence ECII/III neurons, the major source of inputs for the dentate gyrus.
To investigate whether ECV neurons are postsynaptically activated by ECVI neurons, we recorded spontaneous glutamatergic () or GABAergic () postsynaptic currents (sEPSCs and sIPSCs) in ECV neurons with whole-cell voltage-clamp recordings while bath-perfusing the slices with nicotine (10μM), or by locally activating non-α7 nAChRs in ECVI neurons and other neighboring regions. Bath-applied nicotine (10μM) significantly increased the frequency of sEPSCs (n = 5, see and ) but not that of sIPSCs (n = 5, p>0.05, KS test, see for an example) recorded in ECV pyramidal neurons, demonstrating that bath-applied nicotine enhanced excitatory but not inhibitory inputs on to ECV neurons through the activation of non-α7 nAChRs. The brief (350 ms duration) local application of ACh (4 mM; ~200 μm away from recorded neurons) to ECVI neurons in the presence of antagonists of mAChRs (atropine, 10μM) and α7 nAChRs (MLA, 10 nM) also significantly increased the frequency of sEPSCs onto ECV neurons (n = 5, 0.29 ± 0.08 Hz in control condition as compared to 7.3 ± 2.0 Hz within 10 s following brief local activation of non-α7 nAChRs in ECVI neurons, p<0.05, paired t test, see for an example). This increase did not occur when ACh was locally applied to ECIII or neighboring ECV neurons that were similar distances away from the recorded ECV neuron (n = 5, p>0.05, paired t test), indicating that ECVI neurons act as a major source of excitatory inputs for ECV.
Nicotine-sensitive ECVI neurons provide glutamatergic inputs to pyramidal neurons in ECV
Nicotine-sensitive ECVI neurons also influenced GABAergic inputs to pyramidal neurons in ECV
When we increased the duration of local application of ACh to the ECVI region to 1 min, we could now observe a significant increase in the frequency of sIPSC events in ECV neurons (), demonstrating that inhibitory synaptic connections from ECVI to ECV neurons exist, but they are relatively harder to activate. Increases in the frequency of sIPSCs did not occur when ACh was locally applied to the ECV or ECIII regions similar distances away from the recorded ECV neuron (), demonstrating that non-α7 nAChR-containing inhibitory neurons that target ECV neurons exist mainly in the ECVI region. This increase in sIPSC frequency recorded from ECV neurons was also blocked by DHßE (1 μM, n = 3, ), further confirming that these inhibitory ECVI neurons innervating ECV neurons were activated by ACh through the activation of non-α7 nAChRs. The increase in sIPSC frequency in ECV neurons was also prevented by pretreatment of TTX (1 μM, n = 3, ), indicating that the increase in sIPSC frequency was a result of increased action potential frequency, not a consequence of higher synaptic vesicle release probability (often due to increased intracellular calcium in presynaptic terminals). Statistical analysis revealed a significant decrease in sIPSC interval by the local application of ACh to ECVI neurons (n = 5; ), further suggesting that ECVI GABAergic neurons may provide a major source of inhibitory synaptic influence during systematic non-α7 nAChR activation.
Low concentration of nicotine enhances both amplitude and plasticity in Sb-ECVI synapses
Since we found that both the Sb and ECVI contain nicotine-sensitive neurons, and it is known that Sb neurons project to the ECVI (van Strien et al., 2009
), we studied the effect of nicotine on Sb-ECVI synapses by recording evoked EPSCs (eEPSCs) from ECVI neurons while stimulating the Sb near the CA1 region. The bath application of a low concentration of nicotine (0.1 μM) increased the eEPSC amplitude by 40 ± 2% (n = 5; ). The increase in eEPSC amplitude was also observed when nicotine was applied locally to the recorded ECVI neuron (33 ± 3%; n = 6, ), and this effect was blocked by either DHßE (0.1 μM, ) or MLA (10 nM, , comparing the filled symbols), demonstrating that the enhancement of Sb-ECVI synaptic transmission by nicotine is mediated by both α7 and non-α7 nAChRs at a nicotine concentration related to smoking. The paired-pulse ratio (PPR), which is inversely related to the presynaptic neurotransmitter release probability (Dobrunz and Stevens, 1997
; Lagostena et al., 2008
), was 1.70 ± 0.05 (n = 6) under control conditions (), and decreased significantly to 1.22 ± 0.08 (n = 6) after the local application of nicotine (), suggesting a presynaptic mechanism of action.
Nicotine selectively enhances synaptic transmission and plasticity in Sb-EC synapses
Next we examined if nicotine had any affect on synaptic plasticity in ECVI neurons. A tetanus stimulation (100 Hz, 100ms) delivered to the Sb region elicited short-term (<20 min) potentiation (STP) in the Sb-ECVI synapses (). When the tetanus was preceded by the local perfusion of nicotine (0.1 μM) for 10 min, the STP was boosted to long-term (>50 min) potentiation (LTP, see ), an effect that was blocked by the non-α7 nAChR antagonist DHßE (1 μM, see ). Interestingly the PPR during STP and LTP (1.72 ± 0.11 and 1.58 ± 0.07, respectively, n = 3) was not significantly different than control (1.69 ± 0.15 and 1.65 ± 0.1, respectively, n = 3), suggesting that this enhancement is due to a postsynaptic mechanism of action.
The bath application of the α7-selective antagonist MLA (10 nM) appeared to prevent the presynaptic action of nicotine since under these conditions, nicotine failed to enhance the amplitude of the eEPSC and alter the PPR in the presence of MLA ( and ). However, STP was still boosted to LTP (). These data suggest that nicotine (at a concentration similar to the blood level in cigarette smokers) is acting presynaptically to enhance glutamate release via the activation of α7 nAChRs on presynaptic terminals, and in addition acting postsynaptically via non-α7 nAChRs to convert STP to LTP. A further indication of a postsynaptic mechanism of action for LTP is that dialyzing ECVI neurons with the calcium chelator BAPTA (10 mM) reduced the extent of LTP (30 min after the tetanus) by 70 % (from 82 to 25%; ).