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Muscarinic Modulation of Glutamatergic Transmission at the Mossy Fiber
to Mossy Cell Synapse in the Dentate Gyrus
Nathan Jay Weinstock
Mossy cells are glutamatergic local circuit neurons in the dentate hilus of the hippocampus. Here I
investigate cholinergic modulation by a certain class of receptors, muscarinic acetylcholine receptors, in the
hilar region of the dentate gyrus. Electrophysiological measurements were used to study spontaneous
excitatory postsynaptic currents (EPSCs) in slices of the rat hippocampus. Application of 10 pM carbachol (CCh) in
the presence of 1 pM tetrodotoxin (TTX) reliably produced a reduction in mini-EPSC frequency without changing
the amplitude. This effect was not blocked by pre-treatment with 5 pM atropine, a mAChR antagonist. Due to
the apparent atropine insensitivity, additional pharmacological experiments will be necessary to fully characterize
The hippocampus is a region of the brain within the temporal lobe that is essential for the formation of
new memories.1 There are two primary afferent projections to the hippocampal formation, one from the
entorhinal cortex, and one from the medial septum.2,3 Inputs from the entorhinal cortex innervate granule cells
of the dentate gyrus in a targeted manner as part of the classic hippocampal trisynaptic circuit.4,5 Septo-
hippocampal cholinergic activation operates in a diffuse manner across a wide variety of targets and has
been demonstrated to play a key role in the modulation of hippocampal excitability.6 Cholinergic afferentation to
the hippocampus from the medial septum has been implicated in memory formation and retrieval.7,8 Degradation
of this afferent pathway may play a role in the pathology of Alzheimer's disease and geriatric memory dysfunction.9,10
Cholinergic activity is modulated by two distinct classes of receptors for acetylcholine: muscarinic
receptors (mAChRs) which are G-protein coupled receptors and nicotinic receptors (nAChRs) which are non-
selective ligand-gated cation channels.11,12 Muscarinic receptors have been reported to show presynaptic
inhibitory actions and postsynaptic excitatory effects.13 Activation of postsynaptic nicotinic receptors results
in excitation by evoking inward currents.14 However, presynaptic nAChRs also exist on some terminals, where
they are thought to enhance transmitter release by increasing calcium influx into the terminal.15
In the current study, I investigate whether presynaptic mAChRs modulate transmitter release at the mossy fiber
to mossy cell synapse in the rat dentate gyrus. Mossy fibers are axons of the principle output neurons of the
dentate gyrus, the granule cells,16 while mossy cells are unique excitatory local circuit neurons that participate
in recurrent excitatory connections with granule cells.17 The question of direct muscarinic modulation of this
synapse is of particular interest due to a few distinctive properties of mossy cells. In epilepsy, mossy cells die at
a more rapid rate than any other cell body in the hippocampus, and they are particularly sensitive
to excitotoxicity.18,19 Additionally, there has been a recent inconsistency in the literature regarding a different
target, the MF to CA3 pyramidal cell synapse. When this synapse was examined by Williams and Johnston,20
they found that 10 pM muscarine produced a reliable depression of both excitatory postsynaptic potentials
(EPSPs) and excitatory postsynaptic currents (EPSCs). Williams and Johnston concluded that a presynaptic site
of action for muscarine seemed to be the most plausible explanation. However, a separate study conducted by
Vogt and Regehr21 found direct synaptic modulation mediated by mAChRs only in the associational-commisural (A/
C) fiber system, a network of excitatory connections between CA3 pyramidal cells. Here they found that
synaptic inhibition primarily reflects presynaptic calcium channel inhibition mediated by mAChR activation.
However, at the mossy fiber terminal, they found modulation by muscarine through indirect means. Specifically,
they concluded that muscarine increases the firing frequency of GABAergic interneurons in area CA3 of
the hippocampus, which increases GABA release and ultimately leads to inhibition of MF synapses by
activating presynaptic GABAB receptors.21 By determining the specific manner in which acetylcholine acts at
the mossy fiber to mossy cell synapse (a collateral synapse of the same pathway studied by Williams and
Johnston20 and Vogt and Regehr21) cholinergic modulation of hippocampal function will be further illuminated.
I used a combination of electrophysiological measurements of spontaneous EPSCs and optical imaging to study
the mossy fiber to mossy cell synapse in slices isolated from the rat brain. I found a statistically significant
reduction in mEPSC frequency (in the presence of TTX) with bath application of 10 pM carbachol, a mAChR
agonist. Surprisingly, this effect was not blocked by pre-treatment with 5 pM atropine, a mAChR antagonist.
This study illustrates that there is some evidence for direct modulation of neurotransmitter release at the mossy
fiber to mossy cell synapse by presynaptic mAChRs, however further experiments will be necessary before
that conclusion can be reached with confidence.
MATERIALS AND METHODS
Sprague-Dawley rats post-natal 18- to 25- days old were used to obtain 300 pm horizontal slices. Slices were
cut using a vibratome (Pelco, Redding, CA) and transferred to a holding chamber filled with dissecting solution at
320C for 30 minutes. After 30 min at 320C, slices were allowed to equilibrate to room temperature, where
they remained until transferred to the recording chamber. The artificial cerebral spinal fluid (ACSF) used for
cutting and incubating contained (in mM) 124 NaCI, 2.5 KCI, 1.2 NaH2PO4, 2.5 MgSO4, 10 A-glucose, 1 CaCl2,
and 25.9 NaHCO3, saturated with 95% 02-5% CO2. After transfer to the recording chamber, slices were
visualized using a differential interference contrast microscope ( BX51WI, Olympus, Melville, NY) and superfused at
a rate of 2 ml/min with ACSF that was heated to 300C and contained (in mM) 126 NaCI, 3 KCI, 1.2 NaH2PO4,
1.5 MgSO4, 11 A-glucose, 2.4 CaCl2, and 25.9 NaHCO3, saturated with 95% 02-5% CO2. A Sutter Instrument
Co. (Novato, CA) Flaming/Brown Micropipette puller was used to make pipettes for the whole-cell patch
clamp recordings of mossy cells. Pipette resistance was 3-5 MQ after being filled with an internal solution
that contained (in mM) 125 K-gluconate, 5 NaCI, 0.1 CaCI2, 2 MgCI2, 1 EGTA, 4 MgATP, 0.3 Na3GTP, 10 K-
HEPES, and 10 QX-314. Access resistances ranged from 10 - 40 MQ. The cells were voltage clamped at -70mV. As
an additional visual measure to ensure recordings were actually being taken from mossy cells, sulforhodamine
101 was added to the internal solution and after recording was completed, neurons were visualized using light from
a xenon lamp filtered at 510-560 nm. In order to amplify the voltage and current signals an Axon Multi-clamp
700A amplifier (Axon Instruments, Union City, CA) was used. The data was sampled at 20 kHz, filtered at 2
kHz, using a Digidata 1320A (Axon Instruments, Union City, CA), and then recorded on a personal computer
using Clampex version 9 (Axon Instruments, Union City, CA). In order to analyze the data Clampfit 9.2
(Axon Instruments, City, CA), OriginPro v. 7.5 (OriginLab, Boston, MA), MiniAnalysis (Synaptosoft, Decatur, GA)
and Excel 2000 (Microsoft, Seattle, WA) were used. Drugs were bath applied from an automated syringe pump at
40 pL/min. All chemicals used in these experiments including 5 pM muscarine, 10 pM carbachol, 1 pM TTX, and 5
pM atropine were obtained from Sigma (St. Louis, MO).
Although the principle goal of this project was to test the hypothesis that presynaptic mAChRs are expressed
on mossy fiber terminals to mossy cells, it quickly became apparent that mossy cells exhibit robust expression
of postsynaptic mAChRs. Specifically, bath application of 5 mM muscarine induced a steady state depolarization
of mossy cells in current clamp mode that was clearly suprathreshold for induction of action potentials (Figure
1A). This effect was readily blocked by bath application of 5 mM atropine, a mAChR antagonist. Further, when
this steady state current was eliminated by a holding current of equal amplitude but opposite polarity, it was
clear that muscarine had also dramatically altered the response of mossy cells to brief depolarizing pulses. Figure
1B shows the response of a typical mossy cell to two hyperpolarizing pulses and one depolarizing pulse in
the absence of carbachol. After bath application of carbachol the response to equivalent hyperpolarizing pulses
is unaltered, but the response to brief depolarization is dramatically increased (Figure 1C). This effect is known as
an after depolarization (ADP). Both this ADP and the previously described steady state current resemble effects
of postsynaptic mAChR activation noted in other cell types.22,23
Mosy cell C
A S~nmcaine B C
Cell _ ' -__ J _
Figure 1. Steady-state responses recorded from current-clamped hilar mossy cells using K-
gluconate internal. A) Postsynaptic effect of 5 pM atropine in the presence of 5 pM muscarine,
results consistent with previous reports. B) Representative trace showing hilar mossy cell activity.
C) ADP in the presence of 5 pM muscarine and -60 pA holding current.
In order to test the hypothesis that presynaptic mAChRs are expressed on mossy fiber terminals to mossy cells,
it was necessary to control for these postsynaptic events. Fortunately the steady state depolarization was
effectively eliminated by running the experiment in voltage clamp mode (where current is injected into the cell
as needed to keep the voltage across the membrane constant). Because the ADP was voltage sensitive (data
not shown), this also prevented induction of a muscarinic ADP. Finally, I added 1 mM TTX to the bath for all
future experiments, to block voltage gated Na+ channels and effectively eliminate action potentials.
Under those conditions, we recorded from mossy cells and observed miniature excitatory postsynaptic
currents (mEPSCs) that were likely mediated by spontaneous and action potential independent release of
glutamate from mossy fiber terminals. Because these events are likely mediated by release of a single vesicle (i.
e., because they are likely to be quantal in nature), any carbachol-induced change in frequency may be
interpreted as a presynaptic effect, while changes in amplitude would indicate a postsynaptic phenomenon.
Our results demonstrated that administration of 10 mM CCh produced a statistically significant reduction in
mEPSC frequency (Figure 2A). A second experiment was conducted after pre-treatment with 5 pM atropine
(Figure 2B) that did not block the CCh mediated reduction of mEPSC frequency. Previous work has shown
that atropine is a nonselective mAChR antagonist.24-26 Figure 2C summarizes the % decrease in mEPSC
frequency, highlighting the negligible effect of atropine on blocking the CCh induced reduction of mEPSC frequency.
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Figure 2. Hilar mossy cells were voltage-clamped at -70mV in the presence of 1pM TTX with K-
gluconate internal. A) Reduction of mEPSC frequency with application of 10pM carbachol for 10
minutes, n = 7. B) Slight block of the reduction of mEPSC frequency with the application of
10pM carbachol for 10 minutes in the presence of 5pM atropine, n = 12. C) Summary plot illustrating
%/o decrease in mEPSC frequency. Error bars indicate the SE.
My results indicated that activation of postsynaptic mAChRs expressed by hilar mossy cells has two clear
effects. First, it produces a steady state current that is suprathreshold for induction of action potentials. Second,
it will reliably produce an ADP in current-clamped hilar mossy cells. Both of these effects are consistent with
those observed subsequent to activation of postsynaptic mAChRs on other cell types.27-29
In order to test the hypothesis that mossy fiber inputs to hilar mossy cells express presynaptic mAChRs, hilar
mossy cells were voltage-clamped at -70 mV, and action potentials were blocked by bath application of 1 mM
TTX. Under these conditions bath application of 10 mM CCh significantly reduced mEPSC frequency without
changing mEPSC amplitude. This result points toward the presence of mAChRs on the presynaptic terminal. Now
that the existence of mAChRs is suspected, this synapse can be better characterized by testing the
atropine sensitivity of the muscarinic ADP. If the effect is mAChR dependent treatment of atropine should block
the reduction of mEPSC frequency. Surprisingly, when CCh was administered with 5 pM atropine on board
the reduction in TTX-insensitive mEPSC frequency was not blocked.
How can mEPSC frequency be reduced by CCh without being blocked by atropine? One explanation is that
the atropine was contaminated in some way, rendering it inactive. To control for this possibility atropine
was regularly tested against a carbachol induced ADP recorded in current clamp. These experiments suggested
that the atropine was in fact effective, and yet still failed to completely block the carbachol mediated decrease
in mEPSC frequency. Nevertheless, to my knowledge a carbachol mediated atropine insensitive effect would
be without precedent in the CNS. Therefore it is vital that this hypothesis be subjected to more
rigorous pharmacological analysis. For example, the argument that the current effect is indeed mediated by
mAChRs would be strengthened by experiments that identified another muscarinic agonist capable of
reducing mEPSC frequency. Similarly, a selective mAChR antagonist capable of blocking the effects of
carbachol would strengthen the argument that our current effect is indeed muscarinic.
Another potential strategy that could clarify our current results is to examine the effects of carbachol on
some measure of presynaptic function other than mEPSC frequency. Paired pulse facilitation of TTX sensitive
evoked EPSCs may be an appropriate measure in this regard. In paired pulse experiments, two stimuli are
delivered every 30 seconds with an inter-stimulus interval of ~40ms. Paired pulse facilitation (PPF) is described as
an increase in release probability of the second stimulus in the protocol. Due to residual calcium in the
presynaptic terminals after the first stimulus the second response is more robust.30 Increases in PPF
are characterized by changes in release probability and suggest a presynaptic site of action.31,32 Thus, if the effect
of carbachol on mEPSC frequency described here is in fact mediated by activation of mAChRs, we would
hypothesize that application of muscarinic agonists should also increase PPF at these synapses.
Overall, I observed an effect of carbachol on mEPSC frequency that is consistent with the hypothesis that
presynaptic mAChRs are expressed at the mossy fiber to mossy cell synapse. However, the inability of atropine
to completely block this effect was quite surprising, and it is clear that further experiments will be necessary to
fully characterize the effects of muscarinic compounds on glutamate release at this synapse.
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