Introduction
Understanding how the brain processes emotions holds major potential for fundamental
and medical research. Precisely timed neuronal activity across brain regions is crucial
for cognitive processing (Singer, 1999). Studies in humans (Richardson et al., 2004)
and rodents (Maren and Fanselow, 1995) indicate that cooperation between amygdala
and hippocampus is critical for emotional memory formation. This communication involves
the synchronization of neuronal activity at theta (θ) frequencies (4–10 Hz) across
the basolateral amygdala complex (BLA) and the CA1 hippocampal field. In fear conditioning,
a model of emotional memory, animals learn to associate a negative emotional valence
to an initially neutral stimulus (e.g., a tone) after its repetitive pairing with
an aversive stimulus (e.g., an electrical footshock) (LeDoux, 2000). Unconditioned
animals show hippocampus-related θ oscillations in BLA at the levels of individual
principal cells and neuron populations (as reflected in local field potentials, LFPs)
(Paré and Gaudreau, 1996). Amplitude and power of this rhythm increase after auditory,
contextual or social fear learning (Jeon et al., 2010; Paré and Collins, 2000; Seidenbecher
et al., 2003). Moreover, the degree of θ synchrony between BLA and CA1 after fear
conditioning predicts memory performance (Popa et al., 2010). Precise timing of activity
in the BLA is likely important not only for oscillations. It may also be critical
for memory encoding, by selectively assigning emotional valence to incoming sensory
stimuli. However, how BLA network activities are coordinated remains unknown.
Several lines of evidence suggest that GABAergic neurons may be instrumental in controlling
θ oscillations and integrating salient sensory stimuli in the BLA. The BLA is a cortical-like
area; in cortex, GABAergic interneurons can synchronize the activity of large cell
assemblies (Bonifazi et al., 2009; Cobb et al., 1995). Persistent BLA θ oscillations
are accompanied by fear extinction deficits in GAD65 knockout mice (Sangha et al.,
2009). Furthermore, electrical footshocks evoke synchronous GABAergic currents in
BLA principal neurons (Windels et al., 2010).
GABAergic cells in the BLA are comprised of several groups (McDonald, 1982; Sosulina
et al., 2010), with diverse neurochemical expression profiles (Jasnow et al., 2009;
Mascagni and McDonald, 2003; Rainnie et al., 2006; Smith et al., 2000). These might
play specific physiological roles. However, GABAergic cell types of the BLA have not
been fully characterized, and there is a pressing need to define the nature and function
of such cellular diversity (Ehrlich et al., 2009). A division of labor between GABAergic
cell types in controlling local network activities is exemplified in hippocampus,
where cells innervating distinct neuronal compartments fire at specific oscillation
phases (Klausberger et al., 2003; Tukker et al., 2007). We hypothesized that BLA GABAergic
cells contribute in a type-specific manner to the coordination of θ oscillatory interactions
with the hippocampus and local responses to salient sensory stimuli. We investigated
this by recording the spontaneous and noxious stimulus-driven firing of anatomically-identified
BLA interneurons in vivo. Our findings demonstrate that distinct types of BLA GABAergic
cell fulfill specialized and complementary roles in regulating behaviorally relevant
network activities.
Results
We simultaneously recorded spontaneous single-neuron activity in BLA (comprised of
the lateral and basal nuclei) and hippocampal θ oscillations in dorsal CA1 (dCA1)
LFPs of urethane-anesthetized rats. Prominent θ oscillations (4.15 ± 0.23 Hz, mean ±
SD) occurred during cortical activated states in dCA1 (Klausberger et al., 2003),
but not in BLA LFPs. Gamma (γ) oscillations were also detected in dCA1 LFPs (42.1 ±
1.60 Hz, mean ± s.d.).
We recorded interneuron responses to noxious stimuli by delivering electrical shocks
and pinches to the hindpaw controlateral to the recording sites. We also examined
the firing of BLA glutamatergic principal neurons in relation to dCA1 θ. After recordings,
neurons were juxtacellularly filled with Neurobiotin, allowing for their unambiguous
identification.
Interneurons with somata in the BLA were recorded and labeled (Figure S1, available
online, shows cell locations). These were GABAergic, as all tested cells expressed
the vesicular GABA transporter (VGAT) and/or glutamate decarboxylase (GAD; Figures
3F and 4I), and all synapses examined with electron microscopy were symmetric. Interneuron
types were distinguished according to the combination of their postsynaptic targets,
neurochemical markers and axo-dendritic patterns. Twenty eight GABAergic cells could
be classified in four types: axo-axonic, parvalbumin-expressing basket, calbindin-expressing
dendrite-targeting, and “AStria-projecting” cells.
Axo-Axonic Cells Increase Their Firing in Response to Noxious Stimuli
Axo-axonic cells (n = 6) were recorded and anatomically indentified. During dCA1 θ,
they spontaneously fired action potentials at a mean frequency of 12.4 Hz (range 6.5–15.9 Hz;
Table 1; Figure 1A). The firing of 4 of 6 cells was significantly modulated in phase
with dCA1 θ (p < 0.005, Rayleigh test), albeit weakly (mean modulation depth (r) =
0.05, see Experimental Procedures). Two cells fired independently from the hippocampal
θ rhythm (Figure 1A). The four θ-modulated cells fired preferentially between the
peak and the descending phase of dCA1 θ (range 187.0–283.7°, where 0° and 360° represent
θ troughs; θ phase histograms of single neurons are illustrated in Figure S2). However,
statistical analysis showed that these four cells did not form a synchronized population
in relation to dCA1 θ (R′ = 1.03, R0.05,4 = 1.09, Moore test). Furthermore, the firing
of axo-axonic cells did not show statistically significant modulation in phase with
dCA1 γ oscillations (p > 0.1, Rayleigh test, n = 6; Figure S3; Table S3).
Axo-axonic cells displayed dramatic short-latency excitations in response to noxious
stimuli. All axo-axonic cells increased their firing rates upon hindpaw pinches (+377%
of baseline, latency 267 ms, peak 377 ms, n = 6; ranges: 133%–606%, latency 200–400 ms,
peak 400–600 ms, respectively; Table 2; individual histograms are shown in Figure S4).
This excitation rapidly adapted, and was curtailed at stimulus offset (Figure 5D).
Responses to electrical footshocks were similarly pronounced (mean 226% of baseline,
latency 50 ms, peak 225 ms, n = 4/4; ranges 133%–606%, 20–100 ms, 20–420 ms, respectively;
Figure 1C; Table 2; individual histograms, Figure S5).
These neurons exhibited typical axo-dendritic patterns. Their axons formed cartridges.
Almost all of large-axon varicosities were in close apposition with ankyrin G-expressing
axon initial segments, (n = 6/6 cells), as seen with immunofluorescence (Figure 1D).
We analyzed randomly-sampled synapses from two of these cells using electron microscopy.
The vast majority of postsynaptic targets were axon initial segments (95.4%, n = 43
synapses; Figure 1E; Table S1), confirming that these cells were of the axo-axonic
type. All axo-axonic cells expressed parvalbumin (PV), sometimes weakly (Figure 1F),
but were never calbindin (CB)-positive. Two of 6 neurons densely expressed the GABAAR-α1
subunit on their dendrites (immunohistochemical results are summarized in Table S2).
Axo-axonic cells were bitufted. Their dendrites did not branch immediately, were tortuous
and sparsely spiny (Figure 1G). Axonal arborizations of all 6 cells were very dense
and mostly contained within the dendritic field. Axons were always restricted to the
BLA, but could be distributed between lateral and basal nuclei.
These results show that the firing of axo-axonic cells of the BLA dramatically increases
in response to salient sensory stimuli. However, their spontaneous population activity
is not tightly synchronized with hippocampal θ (Figure 5).
Parvalbumin-Expressing Basket Cell Assemblies Tonically Inhibit Principal Cells
Next, we studied the firing of parvalbumin-expressing (PV+) basket cells (n = 15).
During dCA1 θ oscillations, PV+ basket cells fired at a mean frequency of 11.0 Hz
(range 1.8–27.2 Hz; Table 1), some tonically (coefficient of variation (CV) < 1, n =
8/15), others irregularly (CV > 1, n = 7/15). It has been frequently speculated that
PV+ basket cells pace θ rhythms in the BLA (reviewed in Ehrlich et al., 2009). Instead,
we found that most cells were only weakly modulated with dCA1 θ (mean r = 0.06; Figure 2A),
and at dispersed phases (Table 1; Figures 5B and S2). In keeping with this, the firing
of PV+ basket cells as a population was not synchronized with this rhythm (R' = 0.73,
R0.05,12 = 1.042, Moore test; Figure 5A). The firing of PV+ basket cells was not modulated
with dCA1 γ oscillations (p > 0.04, Rayleigh test, n = 15; Figure S3; Table S3).
As with θ modulation, PV+ basket cells displayed heterogeneous and generally moderate
responses to noxious stimuli (Figure 2B; Table 2). Half of the cells tested (6/12)
were excited by hindpaw pinches, three were inhibited, two showed an excitation-inhibition
sequence, and one cell did not respond significantly (Figure S4). Several cells tested
(5/11) were inhibited by electrical footshocks, three cells were excited, and three
other cells did not change their firing rates (Figure S5). Cells that were excited
in response to one type of noxious stimulus could be inhibited by the other stimulus
(Table 2). This further shows that the firing of PV+ basket cells is not selectively
tuned by noxious stimuli. Importantly, heterogeneous firing among PV+ basket cells
does not reflect spatial segregation of activity patterns in the BLA (see Figure S1A
and Table 1).
Axon varicosities of these cells were large and clustered. Light microscopic analysis
(n = 12 cells) revealed that they mostly made close appositions with somata and large
dendrites of BLA neurons expressing the calcium/calmodulin-dependent kinase II alpha
subunit (CaMKIIα; Figure 2C), a marker of principal cells (Supplemental Experimental
Procedures). Electron microscopic analysis confirmed that the main postsynaptic targets
were somata (55%; n = 40 synapses, 2 cells; Figures 2D and S6C) and proximal dendrites
(45%; diameter 1.29 ± 0.1 μm; Figures S6A and S6B; Table S1). For 72.5% of these synapses,
the postsynaptic target was unambiguously identified as a CaMKIIα+ principal neuron
(Figures S6A and S6C, Table S1). Thus, our results established that these interneurons
were basket cells.
In addition to PV, these cells always expressed CB and an accumulation of the GABAAR-α1
subunit along their somatodendritic plasma membranes (n = 12/12 cells; Figures 2E
and 2F; Table S2). This neurochemical pattern is distinct from those of the other
cell types studied here. Three PV+ neurons were classified as basket cells based on
these features, although their axons could not be analyzed. In addition, PV+ basket
cells displayed characteristic axonal and dendritic fields. They were multipolar.
Their dendrites were varicose, typically aspiny, straight, and branched rarely (Figure 2G).
Axonal arborizations were dense within the dendritic field and extended beyond it in radial
branches, sometimes over long ranges (Figure S7A). This suggests that some PV+ basket
cells influence neuronal activities in large parts of the BLA. Overall, PV+ basket
cells show distinct postsynaptic targets and neurochemical contents, demonstrating
they are different cell types in the BLA.
As a group, PV+ basket cells do not appear to fire tuned to dCA1 θ or noxious stimuli
(Figure 5). Thus, assemblies of them may tonically inhibit principal neurons. The
finding that axo-axonic and PV+ basket cell groups do not fire in synchrony with hippocampal
θ rhythm raises the question of which interneurons might fulfill this role.
Calbindin-Expressing Dendrite-Targeting Cells Fire Synchronously with Hippocampal
Theta Oscillations
Dendrite-targeting CB+ cells spontaneously fired at a mean frequency of 3.5 Hz (range
3.0–4.3 Hz, n = 3; Table 1). Their firing was consistently and strongly modulated
with the late ascending phase of dCA1 θ (Figure 3A; mean angle 144.9°, mean r = 0.13;
Figures 5B and S2; Table 1). Thus, as a population, CB+ dendrite-targeting cells did
fire tightly synchronized with hippocampal θ (R′ = 1.15, R0.05,3 = 1.095, p < 0.05,
Moore test; Figure 5A). In contrast, none of these cells fired in phase with dCA1
γ (p > 0.1, Rayleigh test, n = 3; Figure S3; Table S3).
Responses to hindpaw pinches could be tested in two cells. One cell did not significantly
change its firing (Figure 3B); the other was inhibited (latency 4.2 s, peak 4.4 s;
Table 2; Figure S4). Electrical footshocks were applied during recording of the third
cell. In this experiment, only 53 shocks were applied and no change in firing was
observed. Such a sample size is a limitation of the juxtacellular recording/labeling
technique used. It cannot be ruled out that more heterogeneous activity relationships
with θ oscillations or sensory stimuli would emerge if a larger sample of CB+ cells
were available.
When examined with light microscopy, axons of the three cells were distributed in
the BLA neuropil. Some axon varicosities made close appositions with dendrites of
CaMKIIα+, principal neurons. A substantial proportion was not in apposition with identifiable
CaMKIIα+ structures (Figure 3C) and likely contacted small dendritic processes that
could not be resolved with light microscopy. Electron microscopic analysis demonstrated
that postsynaptic targets were exclusively dendrites of small to medium diameter (0.59 ±
0.05 μm, n = 41 synapses, 2 cells; Figure 3D; Table S1). Notably, this diameter value
was the smallest among the neuron types studied (p < 0.05, Kruskal-Wallis test with
Dunn's multiple comparison; Figure S6E). In 24% of these synapses, targets were confirmed
to be CaMKIIα+ dendrites of principal neurons (Figure 3D).
In addition to strongly expressing CB (Figure 3E), two neurons tested contained very
low levels of PV in their somata (but no detectable PV in their dendrites). One cell
was GABAAR-α1+. The cells were immunonegative for other molecules tested, including
somatostatin (Table S2). Dendrites emerged in bipolar arrangement from the soma. They
were tortuous, rough, and sometimes spiny. Axons and dendrites were restricted to
the BLA, but could span lateral and basal nuclei (Figure 3G).
These results show that CB+ dendrite-targeting cells represent a specific cell type,
whose firing is synchronized with CA1 θ (Figure 5A).
Amygdalo-striatal Transition Area-Projecting Neurons Are Inhibited by Noxious Stimuli
We discovered a GABAergic cell type that projects to the amygdalo-striatal transition
area (AStria, hence its name), as well as innervating the BLA (Figures 4C and S7B).
The firing of most AStria-projecting cells (mean frequency 4.01 Hz, range 3.4–6.0 Hz,
n = 4; Table 1) was related to dCA1 θ (n = 3/4, mean r = 0.12). Two of these cells
preferentially fired before the peak (Figure 4A) and one fired most during the descending
phase of the θ rhythm (Figures 5B and S2; Table 1). As a result, this cell population
was not statistically phase-locked to hippocampal θ (R′ = 0.86, R0.05,3 = 1.095, Moore
test). The firing of AStria-projecting neurons was not modulated with dCA1 γ oscillations
(p > 0.04, Rayleigh test, n = 4; Figure S3; Table S3).
In contrast to the previous three cell types, AStria-projecting cells were robustly
inhibited by noxious stimuli. Hindpaw pinches suppressed the firing of 3/4 cells tested
(Figure 4B; mean latency 2,133 ms, peak 2,200 ms; ranges, 1,000–3,800 ms for peak
and latency; Table 2; Figure S4). In two cells, this inhibition persisted for several
seconds after the pinch offset (Figure 5D). Electrical footshocks also elicited strong
inhibitory responses in AStria-projecting cells (−85% of baseline, latency 33 ms,
peak 380 ms, n = 3; ranges: 75%–100%, 20–60 ms, 20–740 ms, respectively; Figures S5
and 5C).
The axon projecting to the AStria innervated somata and dendrites of DARPP-32+ cells,
likely medium-sized spiny neurons (Anderson and Reiner, 1991), which also expressed
CaMKIIα (Figures 4D, 4E, and S6D). Most of the axons were distributed in the BLA,
where they made dense ramifications (Figures 4C and S7B). Studied with light microscopy,
a proportion of the large axon varicosities made multiple perisomatic contacts with
CaMKIIα+ BLA principal neurons; the others possibly contacted small dendrites (Figure 4G).
Electron microscopic analysis confirmed that postsynaptic targets in the lateral nucleus
were dendrites (Figure 4F) and somata (35% and 65%, respectively, n = 40 synapses,
2 cells; Table S1). Of these, 35% were confirmed CaMKIIα+ neurons (Figure 4F, Table
S1). Dendrites targeted by AStria-projecting neurons were smaller than those postsynaptic
to PV+ basket cells but larger than those targeted by CB+ dendrite-targeting cells
(diameter 0.79 ± 0.06 μm, p < 0.05; Figure S6E).
All AStria-projecting neurons expressed PV (Figure 4H), and half also expressed CB.
GABAAR-α1 was moderately enriched in the plasma membrane of one cell but was never
strongly expressed, in contrast to PV+ basket cells (Table S2). Dendrites were multipolar
and branched profusely. They were short, smooth, and very tortuous (Figures 4C and
S7B).
The distinct dendritic and axonal patterns and postsynaptic targets demonstrate that
AStria-projecting cells may constitute a specific cell type. The present data indicate
that they do not form a synchronous cell population with respect to dCA1 θ but dramatically
decrease their firing in response to noxious stimuli (Figure 5).
Overall, various BLA interneuron types appear to fire differently in relation to network
activities. However, they could not be separated on the basis of their spike shapes
and durations (Figure S8; Table S5).
BLA Principal Neurons Fire Heterogeneously in Relation to Hippocampal Theta Oscillations
Next, we assessed the firing modulation of glutamatergic principal neurons in phase
with hippocampal θ, because they are a major target of the interneurons defined above
and represent the main output of the BLA (n = 23 cells; see Figure S1B for somata
locations). Principal cells fired at very low rates during hippocampal θ (mean 0.29 Hz,
range: 0.03–1.34 Hz; n = 23; Table S4). Irregular burst firing (2–3 spikes) was often
observed, as reflected in high coefficients of variation of firing (CVs, which quantify
irregularity of spike trains, 1.95 ± 0.13). Noteworthy, we found that principal cells
fired longer-lasting spikes than all four types of interneurons (Figure S8; Table
S5). Unsupervised cluster analysis could differentiate principal cells and interneurons
(Figure S8C).
We verified the identity of 15 recorded neurons after labeling. They showed large
dendrites covered with spines (Figure 6A), typical of principal neurons (Faber et al.,
2001; McDonald, 1982). All were identified as glutamatergic by the expression of the
vesicular glutamate transporter 1 (Figure 6B). They coexpressed CaMKIIα (n = 14/14
tested; Figure 6C; Table S4). Of the remaining eight neurons, three were weakly Neurobiotin-filled
cells expressing CaMKIIα, whereas the other five were unlabeled (see Supplemental
Experimental Procedures).
The firing of 39% (9/23) of principal neurons was strongly modulated in phase with
dCA1 θ oscillations (mean r = 0.17; Figure 6D; Table S4). The majority of BLA principal
neurons thus fired independently of dCA1 θ. Theta-modulated cells did not form a tightly
synchronized group (R′ = 0.72, R0.05,9 = 1.053, Moore test; Figure 6D), in line with
the weak ensemble (LFP) θ activity observed in the BLA. Importantly, the proportion
of θ-modulated neurons and the preferred phase distribution (Figure 6E) were both
consistent with previous studies in nonanesthetized animals (Paré and Gaudreau, 1996;
Popa et al., 2010).
Modulation with Ventral Hippocampal Theta
The BLA receives dense innervation from the ventral hippocampal formation (McDonald,
1998; Pitkänen et al., 2000), but not from dCA1. However, dCA1 θ oscillations represent
a more reliable reference signal compared with ventral hippocampal θ. In dCA1, the
θ rhythm is regular, reproducible across animals and it has been suggested to indirectly
but accurately reflect ventral hippocampal activities (Royer et al., 2010). Indeed,
θ oscillations recorded from dorsal and ventral CA1 are coherent in both urethane-anesthetized
and drug-free rats (Adhikari et al., 2010; Hartwich et al., 2009; Royer et al., 2010),
and many ventral hippocampal neurons fire phase-locked to dCA1 θ (Hartwich et al.,
2009; Royer et al., 2010). In contrast, LFP θ in ventral hippocampus would have been
an unsuitable reference. LFP θ phase in ventral hippocampus varies dramatically between
recordings, preventing a reliable comparison of phase locking between animals (Hartwich
et al., 2009; Table S6). Moreover, ventral hippocampal θ oscillations have low amplitude
and occur only transiently (Adhikari et al., 2010; Hartwich et al., 2009; Royer et al.,
2010), compromising the isolation of θ epochs using unbiased methods (Csicsvari et al.,
1999; Klausberger et al., 2003) and the calculation of θ phases.
To validate that dCA1 signal predicted spike timing of BLA neurons relative to ventral
hippocampal θ, we performed experiments that included a vCA1-subiculum electrode (n =
3 animals, 6 neurons). Ventral stratum radiatum LFP signal was used as second reference.
Theta oscillations were intermittent and had generally low amplitude, as reported
in behaving rodents (Figure S9; Adhikari et al., 2010; Royer et al., 2010).
As expected, dCA1 signal predicted BLA unit firing modulation with ventral hippocampal
θ. Differences between the phases of dCA1 and vCA1-subiculum LFP θ oscillations were
similar to, and correlated with the difference between the preferred phases of neuron
firing calculated with the two references (Pearson's correlation r = 0.975, p = 0.025
and circular-circular correlation: Fisher and Lee's method, Oriana software, p < 0.05,
n = 4: 3 principal cells, 1 PV+ basket cell; Figures 7 and S9). Moreover, θ modulation
strengths of units calculated with dorsal and ventral hippocampal references were
similar and linearly correlated (Pearson's correlation r = 0.976, p = 0.024; n = 4;
Figure 7D). These results establish that dCA1 is a suitable and sensitive reference
to study the coupling of BLA neuron firing to hippocampal θ.
Discussion
This study defines several types of BLA interneurons and their role in shaping BLA
activity in relation to dCA1 θ oscillations and noxious stimuli, two processes critical
in forming emotional memories. The key findings are the following: dendrite-targeting
CB+ interneurons provide inhibition to BLA principal cells in phase with hippocampal
θ oscillations. The firing of PV+ basket cells is not tightly synchronized with θ
oscillations. Axo-axonic cells consistently and dramatically increase their firing
in response to noxious stimuli. In addition, we discovered a GABAergic cell type well
placed to coordinate spontaneous and sensory-related BLA-AStria interactions. Our
results support the hypothesis that interneurons are critical in regulating timing
in the BLA, and that they operate in a cell-type-specific manner. We demonstrate that
this principle is not limited to firing relationships with ongoing oscillations, but
also applies to the integration of sensory information.
GABAergic Cell Types of the BLA
Defining cell types requires the correlated analysis of molecular markers, full dendritic
and axonal patterns and postsynaptic targets at ultrastructural level (Somogyi, 2010).
The present study unambiguously defines four interneuron types of the BLA.
First, we demonstrate that axo-axonic and PV+ basket cells are two distinct cell types
in the rat BLA. Indeed, PV+ basket cells target somata and dendrites of principal
neurons, whereas axo-axonic cells innervate almost exclusively axon initial segments.
Thus, the hypothesis that axo-axonic and PV+ basket cells of BLA are a single cell
type (Woodruff et al., 2006) should be rejected, at least in adult rats. The present
report of an extensive coexpression of PV, CB, and/or GABAAR-α1 in BLA interneurons
is consistent with earlier studies (McDonald and Betette, 2001; McDonald and Mascagni,
2004). Our data suggest that the coexpression of moderate to high levels of PV, CB,
and GABAA-Rα1 may be specific to basket cells.
Second, we identified a CB+ dendrite-targeting cell type. The existence in the BLA
of such PV+ interneurons specifically targeting dendrites has been inferred (Muller
et al., 2006; Woodruff et al., 2006; Woodruff and Sah, 2007), but never directly demonstrated.
The target selectivity of basket and dendrite-targeting cells demonstrates a clear
separation, and precludes their grouping into a single population.
Third, we report a specific GABAergic cell type, that we named AStria-projecting,
for its axon reaching outside the BLA.
The BLA most likely comprises additional GABAergic cell types (Ehrlich et al., 2009).
Indeed, Golgi staining has revealed BLA interneurons with axo-dendritic patterns distinct
from those presented here (e.g., neurogliaform-like cells, McDonald, 1982). Moreover,
populations of BLA GABAergic neurons lacking PV have been shown to express markers
such as calretinin, cholecystokinin, neuropeptide Y, or somatostatin (Spampanato et al.,
2011). Recent in vitro studies have elucidated the firing characteristics, dendritic
and axonal patterns, expression of neurochemical markers, and functional connectivity
of some of these neurons (Jasnow et al., 2009; Rainnie et al., 2006; Sosulina et al.,
2010). However, the lack of a comprehensive anatomical strategy has so far prevented
a clear characterization of these interneuron types.
We demonstrated that different BLA interneuron types make GABAergic synapses with
specific domains of principal cells. This appears of key significance in light of
their distinct firing activities.
Firing Relationship with Hippocampal Oscillations
The firing relation of BLA interneurons to hippocampal θ differed between cell types.
This is consistent with only a subset of putative BLA interneurons firing in phase
with hippocampal θ in behaving cats (Paré and Gaudreau, 1996). Importantly, the modulation
strength of interneuron activity was independent from the power and frequency of dCA1
θ oscillations (Experimental Procedures).
Dendrite-targeting CB+ cells showed the most consistent firing modulation. The dendritic
inhibition they provide could modulate the integration of glutamatergic inputs and
limit action potential back-propagation, thereby rendering synaptic plasticity onto
principal neurons dependent on hippocampal θ. This is particularly important in the
BLA, where synaptic plasticity on dendritic spines is thought to underlie fear memory
encoding (Humeau et al., 2005; Ostroff et al., 2010).
We found weak and inconsistent θ-modulation of PV+ basket and axo-axonic cell firing,
which both innervate the perisomatic domain of target cells. At the population level,
these cells appear to provide constant perisomatic inhibition of principal neurons.
We cannot rule out that synchronization is limited to subpopulations of these neurons.
Somata of BLA principal cells are innervated by ∼60 PV+ boutons and their axon initial
segment by ∼20 boutons (Muller et al., 2006). Terminals of PV+ fast-spiking cells
release GABA with high fidelity (Hefft and Jonas, 2005). Together with our results,
this suggests that ∼900 boutons release GABA around each BLA principal cell soma every
second. Such powerful inhibition likely contributes to the very low firing rates of
principal neurons, provided axo-axonic cells chiefly inhibit postsynaptic cells (Woodruff
et al., 2011). Our finding of weakly θ-related activity of perisomatic-innervating
cells constitutes a major difference from what has been reported in neocortex and
hippocampus (Hartwich et al., 2009; Klausberger et al., 2003). Individual AStria-projecting
cells might provide θ-modulated perisomatic inhibition to their target neurons in
BLA and AStria, but they do not seem to play such a role as a population.
Interneurons might adjust their relationship with θ rhythms on a fine time-scale,
possibly depending on behavioral states. The present analysis assumes relatively stationary
activities and was not designed to capture specific bouts of dynamic synchronization.
The juxtacellular method used here restricts sample sizes. It is possible that large
assemblies of interneurons whose activity is weakly synchronized can still have a
large net effect on principal neuron populations.
None of the recorded interneurons showed modulation in phase with dCA1 γ oscillations.
This held true for the analysis of θ-nested γ oscillations and for entire γ oscillation
periods. Our findings are consistent with γ oscillations being generated locally and
indicate that BLA interneurons are more likely to participate in amygdalo-hippocampal
synchrony at θ frequencies.
The firing of ∼40% of principal cells was strongly modulated in phase with hippocampal
θ. Modulated cells could correspond to the so-called fear neurons, which selectively
receive inputs from ventral hippocampus (Herry et al., 2008). As found in behaving
rats, preferred θ phases of principal cells were dispersed (Popa et al., 2010). Phase-modulation
heterogeneity may result from the convergence at heterogeneous phases of perisomatic
inhibition (as our data suggest) and of excitatory inputs from several brain regions.
For example, perirhinal and entorhinal cortices also innervate the BLA (McDonald,
1998; Pitkänen et al., 2000) and contain neuronal assemblies oscillating at θ frequencies
(Collins et al., 1999).
Firing Responses to Noxious Stimuli
Salient sensory events recruit the amygdala to attach emotional significance to coincident
neutral stimuli (LeDoux, 2000). Previous work suggests that phasic GABAergic inhibition
may be instrumental in integrating noxious stimuli, by increasing synchrony in the
BLA (Crane et al., 2009; Windels et al., 2010). Diversity in roles played by interneuron
types could be expected not only during spontaneous activity, but also in integrating
salient sensory stimuli. Indeed, we found cell-type-dependent responses to noxious
stimuli.
AStria-projecting neurons responded with a long-lasting inhibition of firing. Their
target neurons in amygdala and AStria should be concomitantly disinhibited, perhaps
promoting Hebbian synaptic plasticity. While the functions of AStria neurons are unknown,
they might be involved in appetitive behavior and potentially participate in a parallel
circuit controlling emotional responses.
In contrast, the firing of axo-axonic cells increased systematically and dramatically
upon noxious stimuli presentation. Inputs from extrinsic afferents might mediate this
effect. The responses of axo-axonic cells to noxious events may trigger the stimulus-induced
GABAergic currents recorded in principal cells, thus generating synchrony in the BLA
(Windels et al., 2010). Axo-axonic cells could provide temporal precision to large
principal cell assemblies for the encoding of associations with unconditioned stimuli,
in two ways: (1) by synchronizing principal neurons for glutamatergic inputs subsequently
reaching the BLA; (2) by limiting the synaptic integration time window (Pouille and
Scanziani, 2001), thus controlling spike-timing-dependent plasticity (Humeau et al.,
2005). Activation of GABAB receptors, specifically expressed on glutamatergic inputs
to BLA principal neurons (Pan et al., 2009), might also reinforce the temporal precision
of synaptic plasticity (Humeau et al., 2003). Alternatively, the response of axo-axonic
cells might restrict principal cell firing to those most strongly excited by noxious
stimuli.
The stimuli used in this study closely resemble those employed in classical fear conditioning
experiments. Therefore, our results predict how BLA interneurons might be involved
in fear learning. The present results were obtained from urethane-anaesthetized rats.
We cannot rule out that firing patterns of BLA neurons are different in behaving animals.
However, reports on responses of single units to visual or auditory cues in different
brain regions and species have found strong similarities between awake and urethane
anesthesia states (Niell and Stryker, 2010; Schumacher et al., 2011). Spontaneous
firing frequencies appear decreased by urethane, whereas direction and magnitude of
sensory-evoked responses seem unaffected. Urethane treatment induces brain states
comparable to those observed in natural conditions (Clement et al., 2008). Hippocampal
θ oscillations display patterns resembling those in the unanaesthetized state (Lubenov
and Siapas, 2009, and our results). In addition, we found that BLA principal neurons
fired similarly phase-locked to hippocampal θ as previously reported in behaving animals.
In hippocampus, groups of putative interneurons recorded in behaving rats appear similarly
θ-modulated to the main GABAergic cell classes recorded under urethane (Czurkó et al.,
2011). Overall, it is likely that firing patterns of BLA neurons reported here recapitulate
their main characteristics in drug-free conditions.
BLA Theta Genesis
BLA-hippocampal theta synchronization increases after fear conditioning. This might
facilitate the cortical transfer of emotional memories for long term storage (Paré
et al., 2002; Popa et al., 2010). How may specific firings of GABAergic interneurons
contribute to this? Convergent excitatory inputs onto principal cells during sensory
stimuli can trigger synaptic plasticity (Humeau et al., 2003). Dendrite-targeting
interneurons, such as those CB+ cells, could provide powerful inhibitory control of
such excitatory inputs (Lovett-Barron et al., 2012). Calbindin+ interneurons preferentially
fire before the peak of dCA1 θ. Therefore, excitatory inputs active around the θ trough
are more likely to increase their synaptic weight during intense sensory stimulation.
Axo-axonic cells may ensure that synaptic potentiation is restricted to inputs concomitantly
active with the salient stimulus. Assuming that some extrinsic inputs are θ-modulated,
the net effect could be a stronger θ modulation of excitatory input to BLA principal
neurons. This potentiation would create synchrony in large cell assemblies in synergy
with the intrinsic membrane potential resonance of BLA principal neurons (Paré et al.,
1995). Consistent with this, LFP θ power increases in BLA following fear conditioning
(Paré and Collins, 2000; Seidenbecher et al., 2003), and BLA principal neurons become
more θ modulated and synchronous after fear conditioning (Paré and Collins, 2000).
These changes are made possible by the fact that in naive animals, only 20%–40% (Popa
et al., 2010, and our findings) of BLA principal neurons are θ-modulated, and at dispersed
phases. BLA θ oscillations increase after fear conditioning with a delay (Pape et al.,
2005; Paré et al., 2002), which may be explained by the induction of structural plasticity
(Ostroff et al., 2010).
The present results suggest that PV+ basket and axo-axonic cells play minor roles
in θ increase. However, they might modify their activities with emotional learning
and later support BLA θ oscillations. Futures work in behaving animals is needed to
examine the activities of BLA interneurons after fear conditioning and, most critically,
to address how they change during learning. Our finding of cell-type-dependent firing
could be used to facilitate the classification of putative BLA interneurons recorded
in behaving animals.
Conclusion
Modulation of neuronal synchrony in the BLA is critical for the formation of emotional
memories. This study provides insights into the cell type-specific contribution of
GABAergic cells to BLA synchrony. Timed release of GABA on specific domains of BLA
principal neuron is likely important for emotional information processing. We propose
that the cooperation between precise spike-timing of various interneuron types is
necessary for the encoding and persistence of emotional memories. Future studies could
build on our findings to manipulate specific interneuron populations during behavior
and directly test this hypothesis.
Experimental Procedures
In Vivo Electrophysiological Recordings
All procedures involving experimental animals were performed in accordance with the
Animals (Scientific Procedures) Act, 1986 (UK) and associated regulations, under approved
project and personal licenses. Seventy adult male Sprague-Dawley rats (250–350 g)
were anesthetized with intraperitoneal injections of urethane (1.30 g.kg−1 body weight)
plus supplemental doses of ketamine and xylazine, (10–15 and 1–1.5 mg.kg−1, respectively)
as needed. The rectal temperature was maintained at 37°C with a homeothermic heating
device. Craniotomies-duratomies were performed over the right hippocampus and amygdala.
Neuronal activities in the BLA and dCA1 (stratum oriens-pyramidale) were recorded
with independent electrodes made of silver-chloride wires loaded in glass pipettes
filled with 1.5% Neurobiotin (Vector Laboratories) in 0.5 M NaCl (12–18 MΩ resistance
in vivo, tip diameter ∼1.1 μm). Glass electrode signals were referenced against a
wire implanted subcutaneously in the neck. The electrocorticogram (ECoG) was recorded
via a 1 mm diameter steel screw juxtaposed to the dura mater above the right prefrontal
cortex (Bregma AP: 4.5 mm, ML: 2.0 mm), and was referenced against a screw implanted
above the ipsilateral cerebellum.
Pinches of 15 s duration were delivered to the hindpaw controlateral to recording
sites using pneumatically driven forceps that delivered a pressure of 183 g.mm−2.
Similar mechanical stimuli have been shown to be noxious by eliciting an escape response
in behaving rats, as well as by recruiting nociceptive brain circuits in urethane-anesthetized
rats (Cahusac et al., 1990). Electrical stimuli (single current pulses of 5 mA intensity
and 2 ms duration) were delivered at 0.5 Hz through 2 wires implanted on the ventral
face of the controlateral hindpaw, for at least 100 trials. The timing of stimuli
delivery was controlled by an external pulse generator (Master-8; A.M.P.I.) and synchronously
recorded. Identical electrical shocks have been shown to activate spinal cord nociceptive
neurons in urethane-anesthetized rats (Coizet et al., 2006).
Residual 50 Hz noise and its harmonics were reduced in all signals using Humbugs (Quest
Scientific). Glass electrode signals were amplified (10×, Axoprobe 1A, Molecular Devices
Inc.), bifurcated, further amplified (100×), and differentially filtered (DPA-2FS
filter/amplifier; Scientifica) to extract local field potentials (LFPs, 0.3–5,000 Hz)
and unit activities (300–5,000 Hz). Raw ECoG signal was band-pass filtered (0.3–1,500 Hz)
and amplified (2,000×). All signals were digitized online at 16.67 kHz using a Power
1401 analog-digital converter (Cambridge Electronic Design) and stored on a PC running
Spike2 software (versions 6.08 and 6.09, Cambridge Electronic Design). GABAergic cell
recordings lasted 15–105 min (typically ∼45 min). The juxtacellular recording mode
(rather than, for example, a quasi-intracellular mode), was assured by only including
for analysis neurons that (1) had stable spontaneous firing rates/patterns and stable
spike widths; (2) did not display any “injury discharge”; and (3) were recorded in
the absence of spurious “baseline noise” or hyperpolarizing shifts in the electrode
potential.
Labeling of Single Neurons and Reference Sites
After recordings, neurons were selectively filled with Neurobiotin using juxtacellular
labeling (Pinault, 1996). Spike shape and amplitude were monitored throughout recording
and labeling to ensure that the same neuron was recorded and labeled. In order to
verify the location of the reference electrode, an extracellular Neurobiotin deposit
was made in the dorsal CA1 (100 nA anodal current 1 s, 50% duty cycle for 20–30 min).
Electrophysiological Data Analysis
Only data acquired before labeling and obtained from unequivocally identified cells
were analyzed. All data were analyzed off-line using Spike2 built-in functions and
custom scripts (Tukker et al., 2007). Spikes were detected with an amplitude threshold
in the BLA unit channel. Occasionally, additional smaller amplitude units were present
in the recording. Spike2 clustering function supervised manually was used to isolate
single units, and identity of labeled neurons was systematically ensured as described
above. Spike sorting was always checked using autocorrelograms, which showed clear
refractory periods (≥2 ms).
Hippocampal theta oscillation epochs were detected by calculating the theta (3–6 Hz)
to delta (2–3 Hz) power ratio in 2 s windows of the dCA1 LFP (Csicsvari et al., 1999;
Klausberger et al., 2003). Ratio >4 in at least three consecutive windows marked theta
episodes. We excluded from this analysis periods of noxious stimuli and the following
20 s. Every theta episode was visually checked. Selected periods always consisted
of robust theta oscillations. They exclusively occurred during persistently activated
brain state (Figure S9). After theta episodes detection, the dCA1 LFP was downsampled
to 1.04 kHz, digitally filtered (3–6 Hz) and the troughs were determined (Spike2).
Each spike was assigned an angle relative to surrounding theta troughs (Tukker et al.,
2007; Klausberger et al., 2003). The precision of our electrode placements (mediolateral
and antero-posterior ranges ∼400 μm) ensured phase consistency between experiments
(i.e., ∼8.5 degrees error, assuming a phase shift of 21°/mm; Lubenov and Siapas, 2009).
Mean firing frequency was calculated over 100 s continuous periods of robust theta
activity. Coefficient of variation (S.D./mean, CV) of interspike intervals during
these periods was used as a measure of firing regularity. CV greater than 1 indicated
the cell fired in an irregular pattern.
Responses to noxious stimuli were assessed by constructing peristimulus histograms
(bin size 20 ms for electrical footshocks, 200 ms for hindpaw pinches). Responses
were analyzed only if the brain state corresponded to stable global activation before,
during, and after the noxious stimulus. This allowed for the distinction of sensory-driven
responses from effects on the brain state (e.g., change from slow wave to activation).
In addition, we verified that hindpaw pinches did not induce changes in the power
of the LFP oscillations recorded in dCA1 or BLA (θ and γ bands; p > 0.05, Wilcoxon
signed-rank test, n = 25 cells).
Statistical Testing
Relation to hippocampal theta oscillations: all 833–20,522 (average 6,906) spike angle
values from single interneuron units were exported for testing with circular statistics
(Oriana v. 2.0, Kovac Computing Services). Modulation in phase with dCA1 theta oscillations
was tested for significance using Rayleigh's uniformity test (significance p < 0.005).
If p < 0.005, the sum vector of all spikes was computed and normalized by the number
of spikes. Its orientation determined the mean angle of spike firing, with respect
to the trough (0°) of dCA1 theta oscillation (180° represents the theta peak). The
length r of the normalized vector determined modulation depth. Phase modulation homogeneity
within neuron groups (only modulated cells included) was tested with Moore's non parametric
test (Zar, 1999). The null hypothesis was the absence of directionality in the group.
If p < 0.05, cells of the group fired at consistent phases and Batschelet's method
was used to calculate the population mean angle (Zar, 1999). This ensured the statistical
reliability of our conclusions on population modulation. Furthermore, we established
that the depth of modulation of BLA interneurons activity was not correlated with
either the power or the mean frequency of dCA1 theta oscillations (Pearson correlation,
R = 0.03, p = 0.896; R = 0.216, p = 0.335; respectively, n = 22).
Significance of responses to noxious stimuli was tested using thresholds. Footshocks:
significance was accepted if at least 3 consecutive bins differed from the preonset
300 ms mean by 2 SD or any bin by 4 SD. Pinches: for 1–2 trials, significance was
accepted if at least 3 consecutive bins differed from the preonset 10 s mean by 1
SD or any 1 bin by 4 SD. For 3 trials and more, significance was accepted if at least
3 consecutive bins differed from the preonset mean by 1.5 SD or any 1 bin by 4 SD.
Latency was defined as the starting time of the first bin meeting these criteria.
The peak time was the starting time of the largest change in the first significant
series.
Differences in postsynaptic dendrite diameter between cell subgroups were evaluated
using the Kruskal-Wallis test followed by Dunn's post hoc analysis.
Data are expressed as mean ± SEM, unless otherwise stated.
Tissue Processing and Anatomical Analysis
Details on brains fixation, immunofluorescence, electron microscopy, and camera lucida
reconstructions are given in the Supplemental Information.