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The Journal of Neuroscience, April 15, 1999, 19(8):2887-2896
Nicotinic Receptor Activation Excites Distinct Subtypes of
Interneurons in the Rat Hippocampus
A. Rory
McQuiston and
Daniel V.
Madison
Department of Molecular and Cellular Physiology, Stanford
University School of Medicine, Stanford, California 94305
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ABSTRACT |
We examined the function of nicotinic acetylcholine receptors
(nAChRs) in interneurons of area CA1 of the rat hippocampus. CA1
interneurons could be classified into three categories based on
nicotinic responses. The first class was depolarized by  7 nAChRs,
found in all layers of CA1 and as a group, had axonal projections to
all neuropil layers of CA1. The second class had both fast 7 and
slow non-7 nAChR depolarizing responses, was localized primarily to
the stratum oriens, and had axonal projections to the stratum
lacunosum-moleculare. The third group had no nicotinic response. This
group was found in or near the stratum pyramidale and had axonal
projections almost exclusively within and around this layer. Low
concentrations (500 nM) of nicotine desensitized fast and
slow nAChR responses. These findings demonstrate that there are
distinct subsets of interneurons with regard to nicotinic receptor
expression and with predictable morphological properties that suggest
potential cellular actions for nicotinic receptor activation in normal
CNS function and during nicotine abuse.
Key words:
hippocampus; CA1; interneuron; subtypes; nicotinic
receptor; 7; non-7; nicotine
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INTRODUCTION |
One important target for the actions
of nicotine in the hippocampus appears to be the inhibitory
interneurons. Anatomically, interneurons of the hippocampus express at
least the 7 subtype of nicotinic receptors, as shown by
-bungarotoxin (-BgTx) binding (Freedman et al., 1993 ). These
receptors are found underlying synapses on interneuronal somata (Hunt
and Schmidt, 1978). Furthermore, cholinergic boutons have been found
adjacent to somata of CA1 interneurons (Frotscher et al., 1989), and
in situ hybridization studies have shown that the
hippocampus expresses 2-4, 7, and  2 nicotinic acetylcholine
receptor (nAChR) mRNA subunits (Wada et al., 1989; Séguéla
et al., 1993). 7 nicotinic receptor activity has also been shown to
directly excite morphologically unidentified interneurons (Alkondon and
Albuquerque, 1991; Reece and Schwartzkroin, 1991; Albuquerque et
al., 1995; Alkondon et al., 1997; Jones and Yakel, 1997; Frazier et
al., 1998a), and cultured hippocampal neurons appear to express three
different nAChR subtypes (Alkondon and Albuquerque, 1993). Pyramidal
cells, on the other hand, may not have nicotinic receptors (Frazier et
al., 1998a, but see Alkondon et al., 1997).
Although they comprise perhaps only 10-15% of the neurons in
hippocampus, interneurons are extremely influential in the control of
hippocampal circuitry because their divergent connections modulate virtually all activity of the more numerous principal neurons. Interneurons comprise a very diverse group, with a wide variety of
specialized dendritic and axonal arbors. Different types of interneurons would appear to perform specific and varying functions in
the hippocampus (for review, see Freund and Buzsaki, 1996). For
example, some interneurons potently inhibit pyramidal cells by acting
directly on their cell bodies or axon hillocks (Gulyas et al., 1993a;
Buhl et al., 1994; McBain et al., 1994; Sik et al., 1995; Miles et al.,
1996), whereas others inhibit pyramidal cell activity at their
dendrites (Han et al., 1993; Gulyas et al., 1993a,b; Sik et al., 1995;
Miles et al., 1996). Another group of interneurons appears to
specifically inhibit other interneurons (Acsady et al., 1996a,b; Gulyas
and Freund, 1996; Hajos et al., 1996). Information on how nicotine
affects these different types of interneurons will be valuable in
assessing and understanding the role of acetylcholine (ACh) in
influencing hippocampal and brain circuitry.
In this study we have explored the actions of nAChR activity on
subtypes of hippocampal interneurons and pyramidal cells. By recording
the membrane currents and potentials that result from rapid application
of ACh in whole-cell mode and comparing those responses to the
morphology of reconstructed interneurons, we have determined that the
distribution of nAChRs varies with interneuronal morphology.
Specifically, the type of nicotinic response that an interneuron has is
strongly correlated with the target of its axonal arbors. In our study,
we have found pyramidal cells rarely show nicotinic responses, and when
present these responses are much smaller than in interneurons. In all
cases, low doses of nicotine are sufficient to significantly reduce
responses to rapidly applied ACh, presumably by desensitizing nicotinic receptors. These nicotinic actions in conjunction with the correlated anatomical characteristics suggest specific roles for the nicotinic synapses on interneurons in CA1.
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MATERIALS AND METHODS |
Young rats (16- to 54-d-old) were killed under halothane
anesthesia by decapitation, and their brains were rapidly removed and
placed in cold (4°C) saline (in mM: NaCl 119, KCl 2.5, CaCl2 1.0, MgCl2 3, NaHPO4 1, NaHCO3 26.2, glucose 11, and kynurenic acid 1) bubbled with
95% O2 and 5% CO2, pH 7.4. The brain
was then hemisected, and single-hemisphere coronal slices (300- to 400-µm-thick) were cut on a Vibratome (Lancer) and placed submerged in an incubation chamber at 30°C for 30 min. The slices were then allowed to cool down to room temperature (~23°C) and recorded from
over the next 2-4 hr.
Whole-cell patch-clamp recordings were made from visualized
interneurons in all layers of area CA1 in the hippocampus (MacVicar, 1984; Dodt and Zieglgansberger, 1990). Slices were constantly superfused with room temperature saline (in mM: NaCl 119, KCl 2.5, CaCl2 2.5, MgSO4 1.3, NaHPO4 1, NaHCO3 26.2, glucose 11, atropine
sulfate 1-5, pH 7.4, bubbled with 95% O2 and 5%
CO2) (22-24°C) in a recording chamber mounted on
the stage of a modified Nikon Optiphot 2 microscope. Near infrared
light illuminated the brain slice placed on a cover glass bottom of the
recording chamber and collected by a 40× water immersion objective,
magnified an additional 1.2× before the image was collected by an
intensified CCD camera (Hamamatsu) with contrast enhancement. The image
of the cells in the slice was displayed on a video monitor, and glass patch pipettes were visually advanced through the slice to the surface
of the cell from which we recorded.
Patch pipettes were fabricated from borosilicate glass (KG33; 1.5 mm
outer diameter, 1.0 mM inner diameter; Garner Glass
Company) and filled with one of two intracellular solutions, either (in mM:) KCH3SO4 130, NaCl 8, HEPES 10, MgATP 2, Na3GTP 0.3, and BAPTAK4 0.1, pH 7.25 or a similar solution substituting Cs gluconate for KCH3SO4. When neurobiotin (0.5%) was included
in the internal solution to label cells,
KCH3SO4 was reduced to 120 mM to
maintain similar osmolarity.
Membrane potentials and currents from interneurons were monitored with
an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA), acquired
through a MIO-16-E2 A/D interface (National Instruments) onto a Pentium
personal computer using software written in Labview by members of the
lab (Eric Schaible and Paul Pavlidis). Data were analyzed using another
program written in Labview and in Axum (Mathsoft Inc.), a commercially
available program. Statistical significance was determined by a z-score
or a two-tailed unpaired Student's t test, as indicated.
Mean values are reported as mean ± SEM. Membrane potentials were
corrected for junction potentials by 12 mV, as empirically determined
by Neher (1992).
Nicotinic currents were evoked by ejecting ACh through a small tipped
(<1 µm) pipette that was moved under visual control to within ~30
µm of the soma of the neuron under study. The solution was
pressure-ejected (100 msec, 10 psi) directly onto the cell body and
proximal dendrites using a Picospritzer II (General Valve, Fairfield,
NJ). The pipette solution contained 3 mM ACh dissolved in
extracellular saline, pH 7.4, as described above, except that HEPES was
substituted for NaHCO3. Other drugs and chemicals were applied by bath perfusion.
Standard histological processing for biotin was performed to visualize
the interneurons filled with neurobiotin (Bolam, 1992). Briefly, slices
were fixed overnight in a 0.1 M phosphate buffer solution containing 4% paraformaldehyde, 0.05% glutaraldehyde, and
0.2% picric acid. Slices were then embedded in gelatin and resectioned
to 100 µm. Sections were permeabilized with 0.5% Triton X-100,
treated with 0.3% hydrogen peroxide to reduce background peroxidase
activity, and incubated overnight in avidin-biotin-peroxidase complex. The next day, the sections were stained with diaminobenzidine, intensified with nickel, mounted on slides, cleared, and then coverslipped. Neurons were reconstructed using a Neurolucida.
All chemicals were purchased from Fluka except for the following:
-bungarotoxin, mecamylamine (MEC), dihydro--erythroidine (DHE), methyllycaconitine (MLA),
6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX),
D( )-2-amino-5-phosphonopentanoic acid
(D-APV), bicuculline methiodide (BIC; Research
Biochemicals, Natick, MA), tetrodotoxin (TTX; Calbiochem, La Jolla,
CA), KCH3SO4 (ICN Biochemicals, Costa Mesa,
CA), and paraformaldehyde (Electron Microscope Sciences). -Conotoxin ImI (-CTX ImI) and -conotoxin MII (-CTX MII)
were generous gifts from Dr. B. Olivera (University of Utah, Salt
Lake City, UT).
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RESULTS |
We used the whole-cell patch-clamp technique on area CA1
interneurons visualized in hippocampal slices with Nomarski optics under infrared illumination (IR-DIC). This study includes the data from
recordings of 159 interneurons and 15 pyramidal cells in all layers of
area CA1. ACh (3 mM) was applied in brief pulses directly
to neurons by pressure ejection from a nearby patch electrode. All
responses were recorded in the presence of 1-5 µM
atropine to prevent muscarinic responses.
Interneurons with fast 7 nAChR-mediated responses
The most common nicotinic response observed is illustrated
in Figure 1. Application of a brief (100 msec) puff of 3 mM ACh caused a rapid depolarization of the
membrane potential and a burst of action potentials (Fig.
1A1, right). When the membrane
potential was voltage-clamped near resting membrane potential (VH = 70 mV), 3 mM ACh produced a
rapidly activating and deinactivating inward current (mean amplitude,
680 ± 705 pA; n = 99) (Fig.
1A2,3). Application of the 7
nAChR-selective partial antagonist -CTX ImI (McIntosh et al., 1994;
Johnson et al., 1995; Pereira et al., 1996) inhibited the nicotinic
inward current in a reversible manner (Fig.
1A2,B; 55.3 ± 7.3% of
control; n = 3; p = 0.00015, z-score).
Low concentrations of the selective 7 antagonists MLA (1-10
nM) (Fig. 1A3,B;
0.1 ± 0.1% of control; n = 16; p = 0.000015, z-score) and -BgTx (100 nM) (Fig.
1B; 0 ± 0% of control; n = 9;
p = 0.000015, z-score) both completely inhibited this
fast nicotinic current in a reversible and irreversible manner, respectively. However, the less selective inhibitor MEC (1 µM) had only a small but significant effect (Fig.
1B; 90.2 ± 3.3% of control; n = 5; p = 0.037, z-score), whereas DHE (Fig.
1B; 100 nM; 100.6 ± 3.2%;
n = 4; p = 0.26, z-score) had no
effect. These pharmacological observations suggest that this fast
nicotinic response is mediated by 7 nAChRs.
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Figure 1.
7 NAChR activation excites most interneurons.
A, Whole-cell recordings made from an interneuron
located in SR at the border of SLM. A1,
Left, Electrophysiological responses to hyperpolarizing
and depolarizing current injection (120 pA). Right,
Application of 3 mM ACh by pressure application (100 msec,
10 psi at the arrow, in the presence of 5 µM atropine) caused a depolarization and evoked action
potentials. A2,3, In the same cell, under
voltage clamp, the same pulse of ACh (arrow) produced a
fast inward current that was reversibly inhibited in a dose-dependent
manner by the 7 nicotinic antagonists -CTX ImI
(A2, 500 nM) and MLA
(A3, 2 nM).
A4, A plot of the peak currents versus time for
the experiments in panels A2 and
A3. B, Summary plot from a number
of interneurons showing the effect of various antagonists on the peak
inward currents induced by ACh (3 mM). C, In
another interneuron, a current-voltage relationship was constructed
for the ACh response. This current showed mild inward rectification and
reversed between 0 and +10 mV. Raw data traces are shown on the
left with the holding potential for each current trace
shown. ACh was applied as before, at the arrow. The peak
currents are plotted against the membrane holding potential on the
right. Calibration: A1,
Vertical, 20 mV, horizontal,
left, 200 msec, right, 500 msec;
A2,3, vertical, 40 pA,
horizontal, 500 msec; B,
vertical, 100 pA, horizontal, 1 sec;
C, vertical, 1000 pA,
horizontal, 500 msec. All arrows in all
traces indicate the point at which 3 mM ACh was
pressure-applied (100 msec, 10 psi) to the soma of the respective
interneuron.
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Previous studies have shown that nAChRs can cause the release of
neurotransmitters in the hippocampus (Alkondon et al., 1997; Wonnacott,
1997) through the activation of nAChRs on the presynaptic terminals
(Gray et al., 1996; Wonnacott, 1997). We sought to determine whether
the nicotinic responses we observed were caused by the direct
activation of postsynaptic nAChRs or indirectly through the release of
neurotransmitter onto the interneuron via the activation of nAChRs on
the presynaptic terminals. Inhibiting ionotropic glutamate receptors
(NBQX, 30 µM; D-APV, 50 µM)
(Fig. 1B) and GABAA receptors
(bicuculline, 10 µM) (Fig. 1B;
99.7 ± 2.7% of control; p = 0.24;
n = 4) did not inhibit nicotine responses nor did
inhibiting voltage-dependent calcium channels (VDCC) with cadmium
(Cd2+; 200 µM) (Fig.
1B) and voltage-dependent sodium
(Na2+) channels (VDSC) with TTX (1 µM)
(Fig. 1B; 98.0 ± 1.2% of control; p = 0.075; n = 4). Therefore, the
nicotinic responses are likely caused by direct activation of
postsynaptic nAChRs consistent with receptor localization studies in
slices (Hunt and Schmidt, 1978; Freedman et al., 1993).
Neuronal nAChRs have previously been shown to have inwardly rectifying
current-voltage (I-V) relationships,
passing little outward current and having zero current between 0 and 10 mV (for review, see Role, 1992). To examine the
I-V relationship of these fast nicotinic
currents, we recorded the nicotinic currents with an intracellular
solution containing Cs+ instead of
K+ to block K+ currents,
extracellular TTX (1 µM) to block VDSCs, and
Cd2+ (200 µM) to block VDCCs. This
allowed for better control of the membrane potential at more
depolarized potentials and prevented action potentials and
calcium-dependent neurotransmitter release. Consistent with previous
findings, fast nicotinic responses, when measured at varying membrane
potentials, produced an inwardly rectifying I-V
relationship, passing reduced (Fig. 1C) outward current at
positive membrane potentials. As expected for the magnesium concentration in our internal solution, this rectification was not
complete (cf. Albuquerque et al., 1996), but on average, near zero
current was seen between membrane potentials of 0-10 mV
(+5.6 ± 4.4 pA; n = 14). Thus, the
I-V relationship of the fast nicotinic response
is consistent with the activation of neuronal nAChRs.
A subset of interneurons express two types of NAChRs
A subset of interneurons in the stratum oriens (SO) showing
horizontally oriented spindle-shaped cell bodies, when viewed in live
slices with IR-DIC, responded to puffs of 3 mM ACh with the
fast response and an additional slower response (Fig.
2A2). The slow
component was always seen in combination with a fast component. The
fast response was identical to that seen in other interneurons. As in
interneurons showing only the fast response, the fast phase of the
nicotinic response was completely blocked by application of -BgTx
(Fig. 2A2; 100 nM) or MLA (1-10 nM), but the slow response was unaffected. Recordings in
current clamp (Fig. 2A1, right)
showed that the isolated slow response could cause the interneuron to
fire a barrage of action potentials in response to a brief puff of 3 mM ACh. The presence of an -BgTx-insensitive slow
response suggests that these neurons express an additional nAChR that
is not of the 7 type. Depolarizing current injection into one of
these interneurons produced a nonaccommodating train of action
potentials (Fig. 2A1), however, the presence or absence of accommodating trains of action potentials was not indicative of the presence or absence of a nicotinic slow component in
an interneuron. In the illustrated neuron, a hyperpolarizing current
injection resulted in a hyperpolarization with a depolarizing sag and
after the termination of the pulse an overshoot of the resting membrane
potential (Fig. 2A1). Again, the presence of
a depolarizing sag was not indicative of a cell displaying a slow
nicotinic response.
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Figure 2.
A subset of interneurons in SO are excited by two
kinetically distinct nicotinic responses. A1,
Left, Cell membrane responses to hyperpolarizing and
depolarizing current injection (120 pA). Right, Slow
membrane depolarization and action potentials of a cell in response to
brief pressure application of ACh (arrow) onto the soma.
The cell was treated with atropine (1 µM) to block
muscarinic receptors and 100 nM -BgTx to block the fast
7 nAChRs. A2, Left, In another
SO interneuron, pressure application of ACh to the cell soma induced a
fast and a slow inward current response voltage-clamped at 70 mV. The
7 antagonist a-BgTx (100 nM) completely blocked the fast
inward current but did not affect the slow inward current.
A3, The 32 nAChR antagonist -CTX MII
(200 nM) did not affect the slow nicotinic current in
another SO interneuron. A4, The nonselective
nicotinic antagonist MEC (1 µM) reversibly inhibited the
slow nicotinic current in another SO interneuron.
A5, The nonselective nicotinic antagonist DHE
inhibited the slow nicotinic current in another SO interneuron.
A6, Left, A plot of the peak
inward current of a slow nicotinic response and its inhibition by MEC
over time. Data from the same cell in A4.
Right, A plot of the peak inward current of the slow
nicotinic response and its concentration-dependent inhibition by DHE
from the cell in A5. B, Summary
plot of the effects of different antagonists on the slow nicotinic
current expressed as a percentage of control amplitude.
C, Left, Raw data traces of the slow
nicotinic current response held at varying membrane potentials.
Right, The peak amplitudes of the nicotinic currents
plotted against the membrane potentials at which they were recorded.
Calibration: A1, Vertical, 20 mV,
horizontal, left, 200 msec,
right, 1 sec; A2,
vertical, 100 pA, horizontal, 1 sec;
A3, vertical, 20 pA,
horizontal, 1 sec; A4,5,
vertical, 20 pA, horizontal, 1 sec;
C, vertical, 100 pA,
horizontal, 1 sec.
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In voltage clamp, the fast current was completely inhibited by the 7
nAChR antagonists -BgTx (100 nM; Fig.
2A2) or 10 nM MLA (data not
shown), but the slow component remained. To investigate the properties
of this slower current in isolation, we performed all subsequent
experiments in either -BgTx (100 nM) or MLA (10 nM) to inhibit the fast current. The nicotinic slow current
(mean amplitude, 97.2 ± 65.4 pA; n = 52;
VH = 70 mV) was not inhibited by the 32
nicotinic-selective antagonist -CTX MII (200 nM;
95.3 ± 3.4% of control; p = 0.35; z-score, n = 3) (Fig.
2A3,B) (Cartier et al.,
1996). However, the slow current was reversibly inhibited by the
nonselective nicotinic antagonist MEC (1 µM) (Fig.
2A4,B; 25.8 ± 4.5% of control; p = 0.00001; z-score, n = 6) and dose dependently by DHE (100 nM) (Fig.
2A5,B; 74.6 ± 7.5%
of control; p = 0.032; n = 10; 1 µM; 42.7 ± 5.7% of control; p = 0.005; z-score, n = 20). These data suggest that in
addition to expressing 7 receptors, this subset of interneurons also
expresses another unidentified kinetically slower nAChR. As was the
case with the fast response, glutamate antagonists (NBQX, 30 µM; D-APV, 50 µM) and
GABAA antagonists (BIC; 10 µM; Fig.
2B; 96.4 ± 2.7% of control; p = 0.3; z-score, n = 6) and/or TTX (1 µM)
and Cd2+ (200 µM) (96.4 ± 2.7%
of control; p = 0.36; z-score, n = 6)
did not inhibit the slow nicotinic response. Therefore, it is likely that ACh acts directly on nAChRs on the cell bodies of the interneurons.
Under the same conditions used to measure the fast nicotinic current,
the measurement of the slow I-V relationship
showed a strong inward rectification (Fig. 2C). Little or no
current was observed between 0 and +10 mV (+4.4 ± 5.5 pA;
n = 9). These observations are consistent with the
currents being the result of the activation of a neuronal nicotinic
current. A few interneurons having a slow response were also found in
the stratum lacunosum/moleculare (4 of 20 interneurons). These
responses were considerably smaller than those recorded in interneurons
of the SO. In the overall population of interneurons, ~50% showed
only the fast response, whereas ~36% showed both the fast and slow
response, although most of these were found in the SO. The percentage
of interneurons with both fast and slow responses is somewhat
over-represented in the SO because late in the study we found we were
able to identify them before recording, on the basis of their
morphological characteristics. The breakdown of response type by
hippocampal layer is shown in Table 1.
Some interneurons and most pyramidal cells do not have
nicotinic responses
Although virtually all interneurons located in the
neuropil layers showed a nicotinic response, many interneurons located primarily in or near the pyramidal cell layer did not respond at all to
brief applications of ACh. (Fig. 3, Table
1). An example of such an interneuron is
illustrated in Figure 3. This cell displayed nonaccommodating rapidly
firing action potentials in response to depolarizing current injection
and a small depolarizing sag to hyperpolarizing current injection that
overshot the resting membrane potential after termination of the
current pulse (Fig. 3A). However, ACh-nonresponsive
interneurons showed a variety of firing behaviors. Some showed slower
firing rates and significant accommodation to depolarizing current
pulses. In this cell, ACh did not cause a membrane depolarization in
current clamp (Fig. 3B) or an inward current in voltage
clamp (VH = 70 mV; Fig. 3C). However, ACh (Fig. 3C, arrow) did sometimes
induce a barrage of spontaneous EPSCs (observed in 3 of 22 nonresponsive interneurons). These presumed presynaptic actions were
not subsequently investigated, although they were also observed with
similar frequency in ACh-responsive interneurons.
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Figure 3.
Some interneurons do not respond to nicotinic
agonist. All recordings in this figure are from an interneuron with its
cell body in the pyramidal cell layer. A, Membrane
responses to depolarizing and hyperpolarizing current injection (120 pA). B, Membrane potential response to pressure
application of 3 mM ACh. C, Voltage-clamp
recording (VH = 70 mV) measuring the
current response to 3 mM ACh. Calibration:
A, B, Vertical, 20 mV,
horizontal, 200 msec (A) 500 msec
(B); C, vertical 20 pA, horizontal 500 msec.
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Table 1.
Distribution of nicotinic receptor actions among the
different layers in the CA1 region of the hippocampus
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Like nonresponsive interneurons, pyramidal cells usually displayed no
response to brief applications of ACh (Fig.
4A). Application of ACh
to the soma of pyramidal cells usually did not cause a depolarization
(Fig. 4A2) or an inward current under voltage
clamp (Fig. 4A3). However, 2 of 15 pyramidal cells did respond to ACh with small depolarizations and inward currents
(10 and 20 pA peak amplitudes; Fig. 4B2,3)
that were much smaller than those typically seen in responsive
interneurons. These depolarizations and inward currents were inhibited
by MLA (10 nM, Fig. 4B4).
Pyramidal cells recorded in this study were easily recognized by their
familiar electrophysiological properties. They showed spike frequency
accommodation to depolarizing current injection, and their action
potentials were of longer duration than in interneurons and were
followed by small brief afterdepolarizations (Fig.
4A1,B1). Spike
trains were followed by a slow afterhyperpolarizing potential.
Hyperpolarizations resulting from current injection showed a
depolarizing sag and an overshoot of the resting membrane potential
after the termination of the current pulse (Fig.
4A1,B1).
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Figure 4.
Most pyramidal cells do not respond to
nicotinic agonist, but some show a small response. A, A
recording from a pyramidal cell that was unresponsive to nicotinic
agonist, representing the majority of pyramidal cells recording.
A1, Membrane potential responses to depolarizing
and hyperpolarizing current injections (300 pA).
A2, The lack of effect of pressure application
of 3 mM ACh (applied at the mark) on the
membrane potential or the membrane current (A3;
voltage clamp, VH = 70 mV) of the
pyramidal cell. B, Recordings from a pyramidal cell
showing a small response to nicotinic agonist, representing the
minority of pyramidal cells. B1, Membrane
response to depolarizing and hyperpolarizing current injection.
B2, Response of membrane potential to pressure
application of 3 mM ACh to the soma.
B3, Response in membrane current to identical
application of ACh. B4, This response was
blocked by 10 nM MLA. Calibration: A, 10 mV,
100 pA, 200 msec, 500 msec; B1,2, 10 mV, 200 msec, 500 msec; B3,4, 20 pA, 500 msec.
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Low concentrations of nicotine inhibit nicotinic responses
in interneurons
Low concentrations of nicotine, similar to the plasma
concentrations reached in smokers after a single cigarette (Benowitz et
al., 1989), have been shown to inhibit the activation of different types of nAChR subunits expressed in Xenopus oocytes,
presumably because of the desensitization of the receptor (Hsu et al.,
1996; Fenster et al., 1997). Figure
5A shows an example of the
effect of bath application of low concentrations of nicotine on an
interneuron expressing only a fast 7 nicotinic response. A 500 nM concentration of nicotine in the bath reduced the
fast nicotinic response in 10 of 14 trials. On average, the fast
nicotinic response was inhibited ~31% (69.5% of control
amplitude ± 6.1%, p = 0.04; z-score,
n = 14) from a mean control amplitude of 950 ± 693 pA by 500 nM nicotine, a concentration reached in
plasma after a cigarette. Higher concentrations of nicotine were more
effective, reducing the nicotinic response in all trials. A 1.5 µM concentration of nicotine reduced the fast nicotinic
current by an average of ~70% (30.2% of control amplitude ± 7.5%; n = 5), and 5 µM nicotine reduced
it by ~92% (7.9% of control ± 3.0%; n = 7).
These effects were reversible (Fig.
5A,B).
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Figure 5.
Low concentrations of nicotine inhibit
both the fast 7 nicotinic and the slow non-7 nicotinic response.
A1, Reversible inhibition of the fast nicotinic
current by bath-applied nicotine (5 µM).
A2, A plot of the peak fast inward currents
(same cell as A1) throughout the entire
experiment showing the reversible inhibition by the varying
concentrations of nicotine on the nicotinic currents.
B1, Reversible inhibition of the slow nicotinic
currents by bath-applied nicotine (5 µM).
B2, A plot of the peak slow inward currents
(same cell as B1) throughout the experiment show
the reversible inhibition of the slow nicotinic currents by
concentrations of nicotine. C, Summary showing that 500 nM to 5 µM nicotine significantly inhibits
both the slow and fast nicotinic current responses across all tested
cells. Calibration: A, Vertical, 50 pA,
horizontal, 500 msec; B,
vertical, 40 pA, horizontal, 1 sec.
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Low concentrations of nicotine also desensitized the slow nicotinic
response. Slow nicotinic currents were reduced by bath application of
500 nM nicotine in the bath in 12 of 16 trials. The
nicotinic currents (mean amplitude, 97 ± 34 pA; n = 16) were reversibly inhibited in a dose-dependent manner (500 nM, 1.5 µM, and 5 µM), as seen
in Figure 5B. On average, slow nicotinic currents were
inhibited ~37% by 500 nM nicotine in the bath (63.4% of
control ± 6.2%; p = 0.03; z-score,
n = 16), ~57% by 1.5 µM nicotine
(42.7 ± 5.4%; n = 6), and by ~87% in 5 µM nicotine (13.3 ± 2.5%; n = 8;
p = 0.00001, z-score, all concentrations, fast and
slow) (Fig. 5C).
Distribution and morphology of the different subtypes
of interneurons
Based on nicotinic responsiveness, there appears to be three
"response types" of interneurons in area CA1 of the hippocampus. The majority of interneurons have only the fast 7-mediated response. A second response type shows both the fast and the slow
non-7-mediated response. Third, are interneurons that respond not at
all to nicotinic agonist. Although at least the first two of these
types are found at least to a minor extent in all layers of CA1, they
are not uniformly distributed across these layers. Table 1 shows the distribution of interneurons of different response types across the
layers of the hippocampus. All interneurons in the stratum radiatum
(SR) and stratum lacunosum moleculare (SLM) responded to ACh. Of these
interneurons, almost all exhibited only the 7-mediated fast
responses (37 of 41). A few of these interneurons (4 of 41) also showed
a very small slow non-7 nicotinic response in addition to the fast
response. These were all found within the SLM. The slow responses that
were recorded in SLM were considerably smaller in amplitude than those
recorded in the SO (an average of 18 ± 19 pA, n = 4 in SLM, compared with 97 ± 34 pA, n = 16 in a
representative sample of SO interneurons; VH = 70 mV). When recording membrane potential, the slow response in SLM
interneurons averaged <2 mV.
The SO contained interneurons that fell into all three categories of
nicotinic responses. However, unlike the SR and SLM, a large proportion
of interneurons recorded here expressed both the fast 7 nicotinic
response and the slow non-7 nicotinic response (63% of cells
recorded showed both responses). In our recordings, the large majority
of interneurons having slow responses are found in SO (>90%).
However, as mentioned before, the proportion of slow-responding cells
within SO is somewhat overestimated, because late in this study it
became possible to identify these interneurons by their characteristic
morphology. This ability to select for slow responding cells makes it
impossible to give an accurate numerical indication of the proportion
of the whole SO population having both fast and slow responses.
However, what is clear is that these slow nicotinic responses are
almost absent from other CA1 layers, and in the SO, are confined
primarily to neurons having spindle-shaped dendritic fields and somata
that are horizontally oriented relative to the alveus (Fig.
6B). Besides
interneurons having a slow response, there were also many that had only
the fast response (32%), and some did not respond to ACh at all (5%), although these nonresponders were always found near the border with
SP.
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Figure 6.
Morphology of different subsets of interneurons
that express different compliments of nAChRs. The lighter
line in each tracing is the axon, and the darker
line is the dendrite of each illustrated neuron.
A, An example of an interneuron with its soma located at
the SR/SLM border that responded to nicotinic agonist with only a fast
7 nicotinic response. Its dendrite ramified mostly within SR, but
also in SLM. Its axon was contained almost entirely within SLM with a
small projection to SR. B, An example of an OLM-type
interneuron that responded with both the fast 7 and slow non-7
nicotinic response. Its dendrite was located entirely within SO and was
oriented parallel to the alveus. Its axon had a small projection to SO,
but ramified widely in SLM. C, An example of an
interneuron that did not respond to nicotinic agonist. Its dendritic
tree was found across all layers of CA1, but its axon was confined to
an area in and bordering SP. D, A pyramidal cell that
does not respond to nicotinic agonist. We detected no difference in the
morphologies of pyramidal cell that did not respond to agonist and
those that responded with a small response.
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Stratum pyramidale (SP) also contained interneurons of all response
types, including nicotinic-insensitive neurons. The fast 7 nicotinic
response was observed in 44% of the interneurons of the SP. However,
nicotinic responses in interneurons in SP were typically smaller than
those seen in other layers (peak current amplitude, 299.3 ± 101.0 pA; n = 14 for fast responses in SP vs 730.2 ± 80.4 pA; n = 80 for fast responses in other layers;
p = 0.0021, unpaired t test). These smaller
responses were often insufficient to evoke action potentials. Only 6%
of the interneurons in SP showed both fast and slow nicotinic
responses, many fewer than in SO. The interneuron population differed
greatly from the other layers in that 50% of interneurons in SP showed
no response to ACh at all. Eighty-two percent of nonresponsive
interneurons were localized to the SP layer or its boundary. The
remaining nicotinic-unresponsive interneurons were located in SO.
Although some interneurons with similar anatomical morphologies and
roles in the hippocampus are concentrated in one layer of CA1, other
morphological types are found in several layers of CA1 (Freund and
Buzsaki, 1996). Therefore, the location of the cell body of the
interneuron does not necessarily reflect the role of that interneuron
in the hippocampus. To examine directly whether nicotinic response
types could be correlated with certain morphological characteristics,
we filled a distributed sample of interneurons (and also pyramidal
cells) with neurobiotin for later anatomical identification. Seventeen
interneurons and three pyramidal cells were examined in this way. Both
their axons and dendrites could largely be reconstructed and localized.
An interneuron typical of those that exclusively displayed a fast 7
nicotinic response is shown in Figure 6A. This
interneuron had its cell body located at the border of SR and SLM, its
dendrites were confined to SR and SLM, and its axon projected to SLM
exclusively. Four other reconstructed interneurons from SR and SLM,
which only exhibited 7 responses, showed varying morphology, but
their axons always ramified in hippocampal layers containing the
pyramidal cell dendrites (SLM, SR, and/or SO) while avoiding the
pyramidal cell body layer. Similarly, an SO interneuron responding with only an 7 response also had an axonal projection to SO and SR, avoiding the cell body layer except to pass through to the SR.
A representative tracing of an SO interneuron that showed both the fast
and the slow nicotinic response is shown in Figure 6B. This type of interneuron, previously identified
and termed an oriens lacunosum moleculare, or OLM cell (Freund
and Buzsaki, 1996), had its cell body localized to the SO and
horizontally oriented dendrites confined to the SO. Its axon showed a
small amount of local ramification, but most of the axon projected to the SLM where it profusely ramified within that layer. Similar morphology was seen in the other four reconstructed cells displaying both slow and fast nicotinic responses.
Figure 6C shows an interneuron typical of those with no
response to ACh. Its cell body was found in at the border of the SP and
SO, and its dendrites were found in all layers, whereas its axon was
confined primarily to the pyramidal cell body layer. Four other
nonresponding interneurons showed similar morphology. Two other
nonresponding cells, one with its cell body in SO and one at the border
of SR and SP, both had axons projecting selectively to the pyramidal
cell body layer. Therefore, it would appear that interneurons that do
not express functional somatic nAChRs exert their inhibition at the
cell bodies of neurons within the pyramidal layer, whereas nicotine
responsive interneurons exert their influence outside this layer. This
appears to be a strong principal of CA1 interneuronal organization, but
not completely absolute, because we did identify one interneuron with
its soma in SO that both responded to ACh and had axon projecting to
the pyramidal cell body layer.
Finally, a pyramidal cell that did not produce a nicotinic response is
shown in Figure 6D. One other reconstructed pyramidal cell that did respond to ACh and one that did not respond to ACh showed
similar morphology.
| |
DISCUSSION |
Interneurons in hippocampal area CA1 fall into three classes with
regard to their nicotinic response to rapid ACh application: (1)
interneurons with a nicotinic fast inward current mediated by 7
nAChR activation, (2) interneurons with a fast response and an
additional slow non-7-mediated inward current, and (3) interneurons
that do not respond. This classification correlates strongly with
morphological characteristics of interneurons. Interneurons with only a
fast response project their axonal arbors to neuropil layers in area
CA1. An individual interneuron of this type may have its arbors
confined to a particular layer, but as a group, they project to all
layers, except for being almost entirely excluded from the pyramidal
cell layer. Most of the interneurons having an additional slow
response, OLM cells as described by Freund and Buzsaki (1996), are
found in SO, although a few having a much smaller slow nicotinic
response were also found in SLM. Unresponsive interneurons are found
almost exclusively in or on the border of SP and appear to be basket
cells or axo-axonic cells.
The fast nicotinic response is clearly mediated by an 7 nicotinic
receptor. It is completely blocked by low concentrations of
-bungarotoxin and MLA. It shows the inward rectification
characteristic of 7 responses, although we find this rectification
to be more variable than sometimes previously reported, ranging from
mild as seen in Figure 1, to nearly complete. However, the amount of rectification observed is not outside the normal range seen for these
receptors, particularly for the internal magnesium concentration used
(cf. Albuquerque et al., 1996). The slow response, on the other hand,
is mediated by a nicotinic receptor of an unidentified type. It is not
blocked by the -bungarotoxin or MLA, but is reduced by MEC and high
concentrations of DHE. This response was always seen in combination
with the fast response, never alone.
Three different types of functional nAChRs in interneurons have been
described (Alkondon and Albuquerque, 1993). They found that >90% of
nicotinic responses were caused by the activation of 7 receptors
(type 1a). Two other slower responses were described, one inhibited by
low concentrations of DHE (10 nM) (type II) and another
inhibited by MEC (1 µM) (type III) (Alkondon and
Albuquerque, 1993). Cells with type II responses expressed 4 nAChR
mRNA (Albuquerque et al., 1995). Although the hippocampus contains
small amounts of mRNA for 2-4 (Wada et al., 1989), it is unlikely
that the slow nicotinic response we observed is the result of the
activation of the 4-containing receptor observed by Albuquerque et
al. (1995), because the slow response we observed was not sensitive to
low concentrations of DHE. However, it cannot be ruled out that the slow response we observed includes an 4-containing receptor, because
the subunits can dramatically influence nAChR pharmacology. nAChRs
in culture and nAChRs we observe may contain different compliments of
subunits while still containing the 4 subunit. However, the
pharmacology and kinetics of our slow response is more consistent with
the MEC-sensitive type III nicotinic response observed in culture
(Alkondon and Albuquerque, 1993). The slow nicotinic response is not
likely caused by the activation of an 32-containing nAChR,
because it was not inhibited by -CTX MII (Cartier et al., 1996).
Another candidate for the slow nicotinic response may be an nAChR that
includes the 2 subunit (Luetje and Patrick, 1991).
We also made several recordings from pyramidal neurons and tested their
responsiveness to nicotine. Reports from studies in cultured neurons
(Alkondon et al., 1996) reported that both "pyramidal-type" cells
and presumed interneurons in primary culture were sensitive to
nicotine. Pyramidal cells in hippocampal slices were also reported respond to nicotine with a slow response (Alkondon et al., 1997). However, Frazier et al. (1998a) found pyramidal cells to be completely unresponsive to nicotinic receptor activation. Our results agree to the
largest extent with Frazier et al. (1998a). Although we did find
pyramidal cells that responded to nicotine with a fast 7-mediated
inward current, this was rare, and when they did appear, the responses
were very small, averaging ~ th the size of those seen on
average in interneurons. We never recorded a slow nicotinic response in
pyramidal cells. The earlier reports of nicotinic responsiveness of
cells in primary culture could have arisen from two sources. First,
pyramidal type neurons may not actually be pyramidal neurons,
but rather interneurons with pyramidal shape. Second, it is possible
that pyramidal cells in culture may express a different compliment of
nicotinic receptors than mature neurons in brain. The conclusion we
reach from our own data are that pyramidal cells are largely
nonresponsive to nicotine, although perhaps not entirely in all cases.
Potential functional consequences of nonuniform nicotinic
responsiveness in interneurons
Interneurons falling into different morphological categories have
varying targets of their axonal arbors and thus, presumably have
different roles in the hippocampus (Freund and Buzsaki, 1996). Functionally, these morphological subclasses of interneurons are believed to include those that inhibit pyramidal cell dendrites or
somata (Harris et al., 1985; Nunzi et al., 1985; Kawaguchi and Hama,
1988; Gulyas et al., 1991, 1993a; Buhl et al., 1994; Sik et al.,
1995; Acsady et al., 1996a,b) to project out of CA1 (Sik et al.,
1994, 1995) or to innervate other interneurons (Acsady et al., 1996a,b;
Gulyas and Freund, 1996; Hajos et al., 1996).
Although nicotinic fast-only responders comprise a diverse group of
morphological types, it is clear that virtually all of these
interneurons share the characteristic of having their axonal projections in the neuropil layers of CA1 and excluded from the pyramidal cell layer. These interneurons may mediate feed-forward inhibition (Lacaille and Schwartzkroin, 1988) because they are in a
position to receive input from fibers entering the CA1 and to inhibit
the dendritic areas of pyramidal cells. In addition to being activated
by glutamatergic inputs, these interneurons may be excited by
cholinergic inputs acting on their 7 receptors. It has been reported
that at least some hippocampal interneurons (in SR) receive functional
cholinergic inputs that generate 7-mediated synaptic responses
(Frazier et al., 1998b). These synaptic responses are incompletely
inhibited by concentrations of the 7 antagonist MLA (50-75
nM), higher than needed to give complete block of exogenous ACh responses (1-10 nM). Despite considerable effort, we
were unable to detect any fast excitatory synaptic potentials that were
not blocked by elevated concentrations of the glutamate receptor antagonist NBQX (40-50 µM; data not shown).
Interneurons that show both fast and slow nicotinic responses appear to
be mostly the OLM cells described by Freund and Buzsaki (1996). These
cells may monitor activity of CA1 pyramidal cells and in turn, potently
inhibit excitatory input coming into the hippocampus from the perforant
path or other dendritic inputs (Gulyas et al., 1993a,b; McBain et al.,
1994; Sik et al., 1995; Yanovsky et al., 1997). In this sense, these
are more in the character of classical feedback inhibitory
interneurons, except that they may be inhibiting the inputs to
pyramidal cells (Yanovsky et al., 1997), rather than directly exerting
this inhibition on the somata. Because these cells have 7 nicotinic
responses, they could be directly excited by cholinergic input in the
classical sense of nicotinic synapses. If a slower nicotinic response
is also synaptically evoked, then these interneurons might also exhibit
a prolonged activity after this activation. Such non-7-mediated
nicotinic prolonged synaptic potentials (DHE-sensitive) have been
recorded in ferret visual cortex (Roerig et al., 1997).
The axonal arborization patterns of nicotine-responsive interneurons
suggest that as a group, these interneurons exert their synaptic
inhibition primarily on the dendrites of CA1 pyramidal neurons or on
the inputs to those dendrites. Nonresponsive interneurons, on the other
hand, appear to be mostly basket type or axo-axonic interneurons having
both axons and dendrites that ramify almost exclusively around the
pyramidal cell layer. As such, they must exert their inhibition mostly
on the pyramidal cell somata. From this, one could hypothesize that as
a group, nicotine-responsive interneurons may be involved primarily
with influencing the input to area CA1, whereas unresponsive
interneurons may have their influence primarily on the output. Thus,
the influence of cholinergic inputs from outside the hippocampus would,
at least in CA1, be to suppress input activity by stimulating
interneurons that suppress this input.
This hypothesis is particularly interesting when considered in light of
the data from this study and others (Frazier et al., 1998a) on the
effects on tonic nicotine application on nicotinic responses in
interneurons. The blockade of ACh responses by bath-applied nicotine
suggests that the action of self-administered nicotine is not to
activate the nicotinic receptors of the brain, but rather to
functionally inhibit them. Second, they suggest that ingestion of
nicotine would hamper the ability of cholinergic nuclei outside the
hippocampus to influence the inhibition in area CA1, without a direct
effect on the output from CA1 pyramidal cells. In the particular case
of OLM cells, self-administered nicotine might hamper the ability of
pyramidal cell output to feedback-regulate the inputs to the distal
dendrites of the same or other pyramidal cells.
| |
FOOTNOTES |
Received Nov. 19, 1998; revised Jan. 26, 1999; accepted Jan. 28, 1999.
This work was supported by National Institute of Mental Health Grants
MH48874 and MH56454 to D.V.M. We would like to thank Eric Schaible and
Paul Pavlidis for writing the acquisition and analysis software, Isabel
Parada-Riquelme for advice on histological procedures, David Prince and
John Huguenard for allowing us to use of their Neurolucida for neuronal
reconstructions, and Bill Colmers, Van Doze, Brie Linkenhoker, and
Bruce MacIver for helpful discussions and careful reading of this
manuscript. We are grateful to B. Olivera for the gifts of
-conotoxin ImI and -conotoxin MII.
Correspondence should be addressed to Daniel V. Madison, Department of
Molecular and Cellular Physiology, Beckman Center, Room 111, Stanford
University School of Medicine, Stanford, CA 94305-5345.
| |
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TOP
ABSTRACT
INTRODUCTION
