Projection neurons can be distinguished from local inhibitory neurons with transgenic mouse lines
The MVN contains two broad classes of neurons with different physiological, neurochemical, and anatomical properties (Bagnall et al., 2007
; Straka et al., 2005
). These complementary populations can be identified in transgenic mouse lines: glutamatergic and glycinergic neurons are fluorescently labeled in the YFP-16 line, and a subset of GABAergic neurons is fluorescently labeled in the GIN line (Bagnall et al., 2007
; Feng et al., 2000
; Oliva et al., 2000
). To determine whether these distinct neuronal classes correspond to known MVN cell types or mediate different circuit functions, which would constrain the consequences of synaptic plasticity, we evaluated their projections to other brain areas. Fluorescent dextran conjugates were injected in vivo
into previously identified targets of the MVN (Highstein and Holstein, 2006
), including the oculomotor nucleus, thalamus, medullary reticular formation, and cerebellum. In YFP-16 mice, YFP-expressing neurons were retrogradely labeled from all targets (). In contrast, GFP-expressing neurons in GIN mice were never retrogradely labeled from injections targeted outside of the vestibular nuclei. GFP-positive synaptic terminals in GIN mice were, however, observed on the somata and proximal dendrites of neurons in the MVN retrogradely labeled from injections to the cerebellum, thalamus, and reticular formation (). These results indicate that fluorescently labeled MVN neurons in the YFP-16 line are predominantly projection neurons, while those in the GIN line provide local inhibition within the bilateral MVN.
Figure 1 Two transgenic mouse lines define populations of projection and interneurons in the MVN. A-C: Neurons expressing fluorescent protein in the YFP-16 mouse line (green) were retrogradely labeled from stereotaxic dye injections (purple) into the thalamic (more ...)
The vestibular nerve synapse is bidirectionally plastic
Vestibular nerve afferents provide the major excitatory drive to the MVN and synapse directly onto both YFP-16 and GIN neurons (Bagnall et al., 2008
). We sought to determine whether these synapses were capable of activity-dependent plasticity that regulated postsynaptic firing responses and whether modifications of synaptic strength depended on postsynaptic cell type. To investigate whether vestibular nerve synapses were plastic, we devised two protocols based on those used to induce LTD (Zhang and Linden, 2006
) and LTP (Pugh and Raman, 2006
) at the analogous excitatory mossy fiber synapse onto deep cerebellar nucleus neurons. Because MVN neurons receive a tonic barrage of both excitatory and inhibitory synaptic inputs, the protocols mimic epochs of either enhanced excitation or inhibition. The “100 Hz stim” protocol comprised 30 repetitions of 550-msec vestibular nerve stimulation at 100 Hz, which elevated the firing rate of postsynaptic neurons (). The “100 Hz stim plus hyperpolarization” protocol comprised 30 repetitions of 550-msec vestibular nerve stimulation at 100 Hz, paired with hyperpolarization of the postsynaptic neuron by ~15–25 mV for 250 msec to simulate strong, coincident inhibition ().
Figure 2 Bidirectional, non-Hebbian plasticity of vestibular nerve synapses onto YFP-16 neurons in the MVN. (A) Representative response to protocol consisting of 100 Hz synaptic stimulation for 550 msec (left). Mean EPSC peak amplitude before and after protocol, (more ...)
We initially examined synaptic plasticity in projection neurons highlighted in the YFP-16 line. Fluorescent neurons were targeted for whole-cell patch recordings in oblique coronal brainstem slices. Neurons were recorded primarily in current clamp to enable spontaneous firing (11 ± 2 Hz) and physiological baseline Ca2+
dynamics. Electrical stimulation of the vestibular nerve elicited synaptic responses in most MVN neurons, as previously reported (Bagnall et al., 2008
). EPSCs evoked by nerve stimulation were measured during brief voltage clamp epochs every 15 sec, and a stable 10 min baseline was established prior to protocol application. The 100 Hz stim protocol evoked postsynaptic firing that averaged 38 ± 10 Hz and induced a short-lasting post-tetanic depression, followed by a robust LTD of the vestibular nerve synapse that reduced the peak EPSC amplitude to 0.82 ± 0.06 of the baseline 15 mins post-protocol (, n=11, p=0.03 vs. no-protocol control recordings: 0.98 ± 0.01, n=9). Thus, the vestibular nerve synapse displays activity-dependent, long-term strength modifications, and coincident pre- and post-synaptic activity results in LTD.
Given the extensive role of inhibition in the control of sensory processing and plasticity in the vestibular system (Buttner et al., 1992
; Gittis and du Lac, 2006
; Ito et al., 1970
; Kato et al., 2003
; Shimazu and Precht, 1966
), we examined whether long-term synaptic efficacy was influenced by postsynaptic hyperpolarization. Remarkably, hyperpolarization of postsynaptic YFP-16 neurons during high-frequency vestibular nerve stimulation reversed the direction of long-term plasticity and resulted in LTP, which increased the peak EPSC amplitude to 1.31 ± 0.13 of the baseline (, n=21, p=0.05 vs. no-protocol control; average hyperpolarization: −18.2 ± 1.3 mV). The stimulation patterns that induced synaptic plasticity did not induce concomitant changes in the intrinsic properties of postsynaptic neurons (Supplemental Table 1
). These data show that the first central synapse in the vestibular system is capable of robust, bidirectional plasticity and that postsynaptic voltage controls the direction of plasticity.
Plasticity modulates the gain of evoked firing in projection neurons
What are the consequences of vestibular nerve synaptic plasticity for postsynaptic neuronal output? Although synaptic efficacy is typically assessed with either single stimuli or pairs of stimuli, vestibular nerve afferents fire at high baseline rates (~30–50 Hz in vivo
) and rarely fall silent for extended periods (Hullar and Minor, 1999
; Lasker et al., 2008
). Consequently, we assessed the effects of synaptic plasticity by stimulating with trains of pulses at rates across the physiologically relevant operating range of the vestibular nerve.
At the vestibular nerve to MVN neuron synapse, stimulus trains evoke EPSCs that depressed rapidly, over the first ~10 pulses, to a steady-state amplitude that is independent of frequency (Bagnall et al., 2008
). Neither the time course nor the relative magnitude of short-term synaptic dynamics were altered following the induction of LTD (n=9, p= 0.57) or LTP (n= 8, p= 0.08)(). Importantly, a linear relationship between the rate of stimulation (5–100 Hz) and the normalized synaptic charge transfer was also preserved following the induction of LTD (R2
= 0.97 ± 0.01, n=11) and LTP (R2
= 0.99 ± 0.01, n= 8, ). These results demonstrate that LTD and LTP influenced EPSC amplitude uniformly across stimulus rates without affecting the short-term synaptic dynamics. Thus, vestibular nerve plasticity scales evoked postsynaptic currents without altering the rate-independence of synaptic transmission and corresponding linear charge transfer.
Figure 3 Synaptic plasticity uniformly scales synaptic currents evoked by stimulus trains. (A) EPSCs measured during vestibular nerve stimulation at 10 Hz rapidly reach a steady-state plateau of 49 ± 4% (EPSC11–20) of the EPSC amplitude response (more ...)
For synaptic plasticity to have functional consequences, it must ultimately influence firing in postsynaptic neurons. The role of firing is exceptionally well characterized in the vestibular system, where head motion modulates the firing rate of MVN neurons. Applying stimulus trains to the vestibular nerve produced increases in MVN firing rates that were proportional to the stimulation rate (). We quantified the firing response as synaptic gain: the slope of the relationship between postsynaptic firing rate and presynaptic stimulus rate. Firing responses varied considerably across neurons; baseline synaptic gains ranged from 0.11 to 0.87, corresponding to firing rates ranging from 17 to 102 Hz in response to 100 Hz stimulation.
Figure 4 LTD and LTP control the gain of synaptically-evoked postsynaptic firing. (A) Representative responses of a YFP-16 neuron to 1-sec stimulation of the vestibular nerve at 25, 50, and 100 Hz. (B) Example synaptic gain recorded in a YFP-16 neuron before (grey) (more ...)
To assess the effects of plasticity on firing, synaptic gain was measured in response to 25–100 Hz trains before and after induction of synaptic plasticity, while DC hyperpolarizing or depolarizing current was injected to maintain the baseline firing rate at ~ 7 Hz to facilitate comparisons across cells. Following the induction of LTD, firing responses to vestibular nerve stimulation decreased (). Conversely, the induction of LTP resulted in increases in synaptic gain (). Across the population, LTD decreased the synaptic gain to 0.83 ± 0.06 of the baseline gain (n=10, p=0.02), and LTP increased the synaptic gain to 1.49 ± 0.16 (n=10, p=0.004)(). The linear relationship between postsynaptic firing and stimulation rate was preserved following plasticity induction (). The baseline firing rate, from which increases in firing rate were evoked, was constant during LTD (Pre: 6.7 ± 0.9 Hz, Post: 6.7± 0.9 Hz) and LTP experiments (Pre: 7.2 ± 0.9 Hz, Post: 7.6 ± 0.9 Hz), and neither protocol altered the intrinsic excitability of postsynaptic neurons (Supplemental Table 1
), suggesting that changes in postsynaptic firing responses were driven completely by changes in synaptic strength. In control experiments, in which 30 repetitions of 250-msec hyperpolarizing steps were applied in the absence of synaptic stimulation, synaptic gain was unchanged (1.00 ± 0.02 of the baseline, n=5, p=0.42, data not shown).
The magnitude of the changes in firing rates produced by vestibular nerve synaptic plasticity varied across stimulation rates and neurons (). The gain changes evoked by LTP corresponded to an average firing rate increase of 13.2 ± 3.3 Hz in response to 100 Hz stimulation, but an increase of 0.8 ± 1.6 Hz in response to 25 Hz stimulation. Similarly, gain changes following LTD decreased the average firing response by 7.2 ± 3.3 Hz during 100 Hz stimulation and by 1.8 ± 1.5 Hz during 25 Hz stimulation. Synaptic gain was unaffected in 2/10 neurons following LTP induction and in 4/10 neurons following LTD induction, despite accompanying changes in EPSC amplitude. The absolute changes in firing responses at any stimulus rate were not correlated with initial firing rate or baseline synaptic gain. These results indicate that although the relationship between synaptic strength and postsynaptic firing responses is influenced by several factors, plasticity at the vestibular nerve synapse functions as a bidirectional, linear gain control mechanism that regulates the strength of signal throughput.
Vestibular nerve synapses contain NMDA and calcium-permeable AMPA receptors
The induction of synaptic plasticity in many systems requires Ca2+
influx through postsynaptic receptors. Although several studies have indicated that vestibular nerve synapses are glutamatergic and contain both NMDA and AMPA receptors (Babalian et al., 1997
; Doi et al., 1990
; Kinney et al., 1994
; Lewis et al., 1989
; Straka et al., 1996
; Takahashi et al., 1994
), relatively little is known about the Ca2+
permeability or voltage dependence of the postsynaptic receptors. We measured current-voltage (IV) relationships of the AMPA and NMDA receptors in YFP-16 neurons in the presence of specific pharmacological antagonists to identify receptor subtypes (). The AMPA-R IV relationship strongly rectified (Rectification Index, +45 mV/−45 mV: 0.44 ± 0.06; range: 0.18–0.79, n=11) (), suggesting the presence of GluA2-lacking, Ca2+
-permeable AMPA-Rs (CP-AMPA-Rs) (Bochet et al., 1994
; McBain and Dingledine, 1993
). The selective blocker of GluA2-lacking AMPA-Rs, Philanthotoxin-433 (PhTx-433, 10μM) reduced EPSC amplitude by 55.9 ± 10.1% (range: 7.4–86.9%, n=9, data not shown), indicating that vestibular nerve synapses onto projection neurons contain a composite of GluA2-lacking and GluA2-containing AMPA-Rs.
Figure 5 LTD and LTP require postsynaptic calcium and distinct classes of ionotropic glutamate receptors. (A) Evoked vestibular nerve EPSCs measured at membrane potentials from −65 to +45 mV in the presence of 10 μM NBQX (top) or 100 μM (more ...)
The NMDA-R contribution to vestibular nerve synaptic transmission was small relative to that of AMPA-Rs (NMDA +45 mV/AMPA −65 mV: 0.48 ± 0.10; range: 0.18–0.89, n=9) (). The NMDA-R current duration, 104 ± 18 msec, and voltage dependence () suggested that NMDA-Rs predominantly contain the NR2A subunit (Cull-Candy and Leszkiewicz, 2004
). These data show that two complementary Ca2+
-permeable glutamate receptors support vestibular nerve transmission: CP-AMPA-Rs, which pass Ca2+
maximally at relatively hyperpolarized postsynaptic membrane potentials, and NMDA-Rs, which pass Ca2+
maximally at relatively depolarized potentials.
LTD and LTP depend differentially on postsynaptic glutamate receptors
Given the presence of two distinct Ca2+-permeable glutamate receptors at this synapse, we tested whether postsynaptic Ca2+ elevations were required for either type of long-term synaptic plasticity. Indeed, both LTD and LTP were blocked by the inclusion of the Ca2+ chelator BAPTA (5 mM) in the recording pipette (100 Hz stim protocol: 1.00 ± 0.03, n=7, p=0.03; 100 Hz stim plus hyperpolarization protocol: 0.98 ± 0.03, n=6, p=0.02, ).
Does Ca2+ influx through NMDA-Rs or CP-AMPA-Rs contribute to the induction of long-term plasticity at the vestibular nerve synapse? We first examined the role of NMDA-Rs. Blockade of NMDA-Rs via bath application of D-APV (100 μM) or intracellular inclusion of MK-801 (1 mM) abolished 100 Hz stim-induced LTD (1.01 ± 0.03, n=6, p=0.02, ), without blocking LTP (1.41 ± 0.13, n=9, p=0.19, ). Thus, postsynaptic NMDA-Rs are required for LTD, but not LTP, at the vestibular nerve synapse.
To test the role of CP-AMPA-Rs, we bath applied the specific blocker PhTx-433 (10 μM). In the presence of PhTx-433, LTD induced by 100 Hz stimulation was unaltered (0.79 ± 0.07, n=6, p=0.59, ). Interestingly, the 100 Hz stim plus hyperpolarization protocol resulted in synaptic depression (0.81 ± 0.10, n=5, p=0.008, ) in the presence of Phtx-433, rather than potentiation, indicating that CP-AMPA-Rs are required for LTP induction. The unmasked LTD indicates that the 100 Hz stim plus hyperpolarization protocol simultaneously recruits LTP and LTD mechanisms, with the balance in favor of LTP under normal conditions. Consistent with this interpretation, applying the 100 Hz plus hyperpolarization protocol in the presence of D-APV resulted in enhanced LTP (), while applying same protocol in the presence of reduced extracellular Mg2+
(0.3 mM) to augment Ca2+
influx via NMDA-Rs suppressed the induction of LTP (0.95 ± 0.4, n=5, Figure S1
). Thus, LTD and LTP at the vestibular nerve synapse operate in parallel and require distinct Ca2+
-permeable glutamate receptors.
To determine whether CP-AMPA-Rs are additionally expressed presynapically, 10 μM PhTx-433 was washed onto slices and the paired-pulse ratio (PPR) at the 20-msec interval was measured. The presence of PhTx-433 did not alter the PPR (Baseline: 93.1 ± 4.2%, PhTx-433: 96.9 ± 3.9%, p=0.20, data not shown), suggesting that CP-AMPA-Rs predominantly exerted their effects on LTP postsynaptically. We conclude that LTP required postsynaptic Ca2+ passed by CP-AMPA-Rs and that LTD requires postsynaptic Ca2+ passed by NMDA-Rs. This complementary dependence on postsynaptic ionotropic glutamate receptors enables membrane voltage to gate the direction of synaptic plasticity.
Postinhibitory rebound depolarization contributes to LTP induction
What are the essential postsynaptic requirements for the induction of LTP? Postsynaptic hyperpolarization produces several distinct effects: it prevents firing, modulates the amplitude of voltage-dependent currents, and recruits low voltage-activated currents. Many neurons in the MVN exhibit rebound firing, a transient elevation in firing rate following relief from hyperpolarization, and the magnitude of rebound firing varies across individual neurons (du Lac and Lisberger, 1995
; Sekirnjak and du Lac, 2002
; Serafin et al., 1991
). During the 100 Hz stim plus hyperpolarization plasticity protocol, 15 of 21 neurons () exhibited rebound firing (42 ± 8 Hz), quantified as the average firing rate increase over the spontaneous rate during the initial 300 msec post-hyperpolarization. LTP was induced in 11 of these 15 neurons (EPSC post-protocol: 1.45 ± 0.16, n=15, p=0.002 vs. control). In contrast, LTP was not induced in any of the remaining 6 neurons that did not exhibit rebound firing post-hyperpolarization (EPSC post-protocol: 0.95 ± 0.03, n=6, p=0.57 vs. control; Rebound vs. Non-rebound: p=0.004, Fisher Exact Test).
To assess whether rebound currents merely distinguished two subtypes of YFP-16 neurons or, additionally, played a causal role in LTP induction, we manipulated the timing of rebound relative to synaptic stimulation in the 100 Hz stim plus hyperpolarization. We first extended the duration of the hyperpolarization step to 550 msec, such that the rebound occurred immediately after, rather than during, the 550-msec stimulus train. This protocol induced LTP of 1.27 ± 0.12 (, n=7, p=0.05), comparable to LTP induced by hyperpolarization lasting 250 msec (p=0.81). We then extended the hyperpolarization step duration to 1550 msec so that rebound followed synaptic stimulation by 1000 msec. This protocol, which temporally dissociated synaptic input and intrinsic rebound, did not induce LTP (, 0.96 ± 0.09 of baseline, n=7, p=0.61 vs. control). These results suggest that intrinsic rebound is a critical component of LTP induction that must to be temporally linked to synaptic stimulation.
Figure 6 LTP of synapses onto projection neurons depends on the relative timing of synaptic stimulation and hyperpolarization offset. (A) LTP is induced by pairing 550-msec, 100 Hz synaptic stimulation with coincident 550-msec hyperpolarization. (B) Extending (more ...)
To test whether this rebound requirement was mediated by enhanced firing following synaptic stimulation, we applied the 100 Hz stim plus hyperpolarization protocol to neurons lacking intrinsic rebound firing and injected a 300 msec suprathreshold depolarizing current following hyperpolarization (Figure S2
). The addition of this mimicked rebound was inadequate to induce LTP in non-rebounding neurons (0.88 ± 0.07 of baseline, n=7, p=0.005 vs. 100 Hz stim plus hyperpolarization, Figure S2
). Furthermore, the magnitude of EPSC potentiation did not correlate with the rate of rebound firing in YFP-16 neurons displaying intrinsic rebound (R2
=0.08). Thus, while rebound firing distinguishes two populations of YFP-16 neurons and is predictive of LTP induction, hyperpolarization offset and rebound currents contribute to LTP via mechanisms beyond simple increases in postsynaptic firing rate.
Vestibular nerve synapses onto GABAergic interneurons are plastic
Thus far we have focused on projection neurons. Are vestibular nerve synapses onto local inhibitory neurons () also plastic? To investigate this potential additional site of plasticity for head movement-driven behaviors, we applied the stimulus protocols that induced LTD and LTP in YFP-16 neurons to GIN neurons. The 100 Hz stim protocol evoked LTD in GIN neurons (0.74 ± 0.05 of baseline, n=6, p=0.001, ). The induction of LTD in interneurons was blocked by intracellular, postsynaptic BAPTA (5 mM) (0.99 ± 0.08, n=6, p=0.02), as well as by the NMDA-R antagonist D-APV (100 μM) (1.01 ± 0.07, n=6, p=0.02, ). These data demonstrate that LTD in vestibular nucleus interneurons requires Ca2+ influx through NMDA-Rs, as is the case for projection neurons ().
Figure 7 Vestibular nerve synapses onto MVN interneurons exhibit LTD. (A) Representative response evoked by protocol consisting of 100 Hz synaptic stimulation for 550 msec (left). Mean EPSC amplitude before and after protocol (right). (B) Representative response (more ...)
Figure 8 Plasticity in interneurons is dominated by NMDA-R-mediated LTD. (A) Inclusion of 5 mM BAPTA in the recording pipette abolished LTD induced by the 100 Hz stim protocol, as did (B) bath application of 100 μM D-APV. (C) Bath application of 100 μM (more ...)
In striking contrast, the stimulus protocol that induced LTP in projection neurons did not result in potentiation of synaptic strength onto interneurons but instead induced a weak LTD (0.90 ± 0.05 of baseline, n=9, p=0.08, ). Interestingly, blocking NMDA-Rs with bath application of D-APV during the 100 Hz stim plus hyperpolarization protocol unmasked an underlying synaptic potentiation (1.25 ± 0.11 of baseline, n=7, p=0.04, ). This D-APV-unmasked LTP in interneurons was sensitive to PhTx-433 (0.95 ± 0.07, n=8, p=0.04, ). These results indicate that, as in projection neurons, CP-AMPA-R activation paired with postsynaptic hyperpolarization can induce LTP in interneurons, but LTP is typically masked by NMDA-R-mediated LTD.
Several factors might underlie LTD dominance in interneurons, including differential expression of glutamate receptors or post-hyperpolarization rebound currents. Indeed, the AMPA-R IV relationship in GIN neurons was less rectifying than that measured in YFP-16 neurons (Rectification Index GIN: 0.69 ± 0.06, n=13, p=0.01, ), while the NMDA/AMPA ratio in GIN neurons was not significantly different from YFP-16 neurons (GIN: 0.47 ± 0.09, n=13, p=0.67, ). The inclusion of a relatively smaller proportion of CP-AMPA-Rs at synapses onto GIN neurons may limit the recruitment of LTP. All GIN neurons exhibited post-hyperpolarization rebound firing, although its magnitude was less than half that of the YFP-16 neurons that rebounded (GIN: 20 ± 3 Hz, YFP-16: 42 ± 8 Hz, p=0.02). Thus, CP-AMPA-R and rebound currents are differentially expressed according to circuit function in the MVN, such that both are minimized in local inhibitory GIN neurons.