Interneurons in the basolateral amygdala

Neuropharmacology 60 (2011) 765e773

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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Invited review

Interneurons in the basolateral amygdala Jay Spampanato, Jai Polepalli 1, Pankaj Sah* The Queensland Brain Institute, University of Queensland, QBI Building (79), St Lucia, QLD 4072, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 September 2010 Received in revised form 1 November 2010 Accepted 10 November 2010

The amygdala is a temporal lobe structure that is the center of emotion processing in the mammalian brain. Recent interest in the amygdala arises from its role in processing fear and the relationship of fear to human anxiety. The amygdaloid complex is divided into a number of subnuclei that have extensive intra and extra nuclear connections. In this review we discuss recent findings on the physiology and plasticity of inputs to interneurons in the basolateral amygdala, the primary input station. These interneurons are a heterogeneous group of cells that can be separated on immunohistochemical and electrophysiological grounds. Glutamatergic inputs to these interneurons form diverse types of excitatory synapses. This diversity is manifest in both the subunit composition of the underlying NMDA receptors as well as their ability to show plasticity. We discuss these differences and their relationship to fear learning. This article is part of a Special Issue entitled ‘Synaptic Plasticity & Interneurons’. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.

Keywords: Learning and memory Anxiety GABA receptors NR2B subunits Ifenprodil LTP

1. Introduction The amygdala is a collection of associated nuclei located in the temporal lobe that is the center of emotion processing in the mammalian brain. Recent interest in the amygdala largely arises from its central role in a simple form of learning known as fear conditioning (Davis and Whalen, 2001; Fanselow and Poulos, 2005; LeDoux, 2003). This is a Pavlovian conditioning procedure in which an emotionally neutral stimulus (the conditioned stimulus, CS), such as a tone or light is contingently paired with an aversive one, typically a mild foot shock. After a small number of pairings subjects learn to associate the two stimuli and now respond with fear to the initially neutral CS. This learnt response, the conditioned response (CR), is rapidly acquired and long lasting (Davis, 1992; LeDoux, 2000). As such, the CR results from subjects learning the association between two sensory stimuli, storing this association and subsequent retrieval of the memory in response to sensing the CS. Once a fear memory has been established, it is generally stable for long periods of time, sometimes a lifetime. However, subsequent repeated presentation of the CS gradually reduces the CR, a process known as extinction (Myers and Davis, 2007; Quirk and Mueller, 2008). In extinction, rather than forgetting the initial learning, subjects form new associations that inform them that the * Corresponding author. Tel.: þ61 7 3346 6376. E-mail addresses: [email protected] (J. Spampanato), jaipolep@stanford. edu (J. Polepalli), [email protected] (P. Sah). 1 Present address: Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, CA 94304-5543, USA.

previously conditioned CS is no longer threatening. This therefore represents the learning and storage of new memory such that the original CS is no longer perceived as fearful, and the response to the CS is inhibited (Maren and Quirk, 2004; Pape and Pare, 2010; Quirk and Mueller, 2008). A large body of data has shown that the amygdala is critically involved in the learning, storage and retrieval of both conditioned fear and extinction (Herry et al., 2008; LeDoux, 1995; Pape and Pare, 2010; Sah et al., 2003). Thus, understanding the function of the amygdala, its intrinsic neural circuits, and the molecular mechanisms that underlie fear conditioning will firstly provide insight into the physiological mechanisms of learning and memory formation in the mammalian brain. Secondly, as there are clear similarities between learnt fear and anxiety in humans, it is believed that understanding the mechanisms that underlie fear conditioning and its dysfunction, may provide a window into the cellular mechanisms that underlie the genesis of disorders such as generalized anxiety, depression and post-traumatic stress (Davis and Whalen, 2001; Quirk and Gehlert, 2003). Moreover, treatments like exposure therapy that are used for a variety of anxiety disorders are largely based on extinction protocols (McNally, 2007). Thus, understanding the cellular mechanisms that underlie extinction inform us about the mechanisms that underpin the treatment of anxiety disorders, and suggest possible targets for the development of new therapies. The nuclei of the amygdaloid complex can be grouped in to three functionally relevant subdivisions: the centromedial, cortical and basolateral groups. These subdivisions can be identified based on their unique connectivity, immunohistochemical and

0028-3908/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2010.11.006

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cytoarchitectural profiles. The anatomy and physiology of the amygdala has been reviewed in detail previously (Alheid et al., 1995; McDonald, 1998; Sah et al., 2003; Swanson and Petrovich, 1998). A converging body of data has established that sensory information from both cortical and subcortical regions enters the amygdala at the level of the basolateral nucleus (BLA). This information is processed within the BLA and transmitted to the central nucleus (CeA). Projections from the CeA target hypothalamic and brainstem structures that evoke the conditioned response (Davis and Whalen, 2001; LeDoux, 2000). These two regions, the BLA and the CeA, and their connections play a central role in the formation and recall of memory traces during fear conditioning and extinction (Maren and Quirk, 2004; Pape and Pare, 2010; Pare et al., 2004). Learning and memory formation in the mammalian brain are generally thought to result from synaptic plasticity at glutamatergic synapses (Bliss and Collingridge, 1993). In agreement with this, both fear conditioning and extinction are accompanied by changes at glutamatergic synapses in the amygdala (Pape and Pare, 2010; Sah et al., 2008). However, emerging evidence indicates that GABAergic synapses within the amygdala also play key roles in both fear conditioning and extinction (Ehrlich et al., 2009). For example, enhancers of GABAergic inhibition in the BLA interfere with learning and expression of conditioned fear (Davis, 1979; Harris and Westbrook, 1998) whereas reduction in GABAergic inhibition has the opposite effect (Tang et al., 2007). Moreover, pharmacological agents that modulate anxiety levels are thought to produce their anxiolytic actions by acting at gaminobutyric-acid (GABA) receptors within the amygdala (Rudolph and Mohler, 2006).

hybridization for GABA and glutamic acid decarboxylase (GAD), the enzyme that converts glutamate to GABA (McDonald and Augustine, 1993; Pitkänen and Amaral, 1994; Sun and Cassell, 1993; Swanson and Petrovich, 1998). Neurons in the CeA have the same origin as striatal neurons (Medina et al., 2004; Garcia-Lopez et al., 2008) and are morphologically, histochemically and physiologically similar to striatal medium spiny projection neurons (Martina et al., 1999; McDonald and Augustine, 1993; Schiess et al., 1999). However, other than expression of GABA, these neurons share little in common with the local circuit GABAergic interneurons classically described in cortical areas (Ascoli et al., 2008; Freund and Buzsaki,1996; Markram et al., 2004). As these neurons are not thought of as local circuit interneurons, we will not consider them further in this review. In addition to the two main nuclei described above, the amygdaloid complex also contains small clusters of neurons called the intercalated cell masses that are distributed in several regions of the amygdaloid complex (McDonald and Augustine, 1993; Millhouse, 1986). In particular, several of these clusters form an interface between the BLA and CeA (Royer et al., 1999). These neurons are also GABAergic, thus providing feedforward inhibition to neurons in the CeA, and are thought to play a pivotal role in fear extinction (Amano et al., 2010; Likhtik et al., 2008). Excitatory inputs to this neuronal group also show synaptic plasticity (Royer and Paré, 2002), however, little is known about the properties of their synaptic inputs or the mechanisms that underlie LTP in these cells. We will therefore not consider these neurons any further in this review.

2. GABAergic neurons in the amygdala

Principal glutamatergic neurons in the BLA are the amygdaloid equivalent of hippocampal and cortical pyramidal neurons. They constitute the majority (80e85%) of the BLA neuronal population and display morphological features typical of cortical pyramidal neurons (McDonald, 1984, 1992; Sah et al., 2003). Interneurons form about 20% of the neuronal population (McDonald, 1985; McDonald and Augustine, 1993; Sah et al., 2003) and these cells have aspiny, smooth dendrites, axon collaterals restricted to the basolateral nuclei, and express calcium binding proteins or neuropeptides that are characteristic of local circuit GABAergic interneurons (McDonald, 1997; McDonald and Betette, 2001; McDonald and Mascagni, 2002, 2004). While they make up the smaller fraction of the total neuronal mass, interneurons tightly

The amygdaloid complex (Fig. 1) can be described as an interface of cortical and striatal cell lineages (Swanson and Petrovich, 1998; Waclaw et al., 2010). This feature makes a general discussion of GABAergic neurons in the amygdala a tale of two halves. Both developmentally (Waclaw et al., 2010), and structurally (McDonald, 1992), the BLA is a cortical like structure. As with other cortical regions, it contains two main types of neuron: glutamatergic principal neurons and GABAergic interneurons (McDonald, 1992; Pare and Smith, 1998). In contrast to the BLA, the CeA is entirely populated by GABAergic neurons. This has been demonstrated in both rodents and primates by immunohistochemical labeling or in situ

2.1. The basolateral Amygdala

Fig. 1. Interneurons of the amygdala. A coronal section from a GAD67-EGFP mouse with fluorescence enhanced by labeling with an antibody for EGFP. Genetic control of EGFP expression is achieved by the knock-in technique resulting in fluorescence restricted to GAD67 expressing interneurons. The density of EGFP expression can be used to easily identify the individual nuclei of the Amygdala. Abbreviations: CTX: Cortex, Pir CTX: Piriform Cortex, STR: Striatum, LA: Lateral Amygdala, BA: Basal Amygdala, CeL: Central Lateral Amygdala, CeM: Central Medial Amygdala, l-ITC: lateral Intercalated Cells, m-ITC: medial Intercalated Cells, ITC: Intercalated Cells.

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regulate principal cell excitability (Bissiere et al., 2003; Lang and Pare, 1997; Li et al., 1996; Woodruff and Sah, 2007a). Thus, within the BLA, principal cells have low resting firing rates (Pare and Gaudreau, 1996) and single interneurons can powerfully block activity in principal neurons (Woodruff and Sah, 2007a). As in neocortical areas and the hippocampus, interneurons in the BLA can be characterized by expression of specific calcium binding proteins or neuropeptides. Although the relative ratios of cell types differ between the lateral and basal nuclei, there is a similar trend between the two areas. The two primary nonoverlapping groups of interneurons are those that express calbindin and those that express calretinin. Calbindin (CB) expression accounts for approximately half of all the interneurons in the BLA and calretinin (CR) expression accounts for approximately 20% of those remaining (Kemppainen and Pitkanen, 2000; McDonald and Mascagni, 2001a). Each of these groups can be further sub-divided into more functionally relevant groups by neuropeptide expression or expression of the calcium binding protein parvalbumin (PV). Some CRþ interneurons express vasoactive intestinal peptide (VIP) and/or cholecystokinin (CCK) with some overlap between these two sub groups (Mascagni and McDonald, 2003; McDonald and Mascagni, 2002). CBþ interneurons can be sub-divided into nonoverlapping groups that express either PV, somatostatin (SOM) or CCK individually (Davila et al., 2008; Mascagni and McDonald, 2003; McDonald and Mascagni, 2002). These expression profiles share some similarities with those reported for cortical and hippocampal interneurons with the major exception being the expansion of CB expression throughout and in particular its overlap with the PV group (Markram et al., 2004; Somogyi and Klausberger, 2005). Interestingly, using single cell PCR, and unsupervised cluster analysis, mRNA expression of these markers does not appear to match the pattern seen using antibody labeling (Sosulina et al., 2010, 2006). The reason for this difference is not clear but likely relates to differences between mRNA and protein expression levels. The characteristic diversity of calcium binding protein and neuropeptide expression in BLA interneurons correlates with their anatomical post-synaptic target specificity and physiology (Table 1). The primary post-synaptic targets of each interneuron group are glutamatergic principal cells of the BLA. PVþ and CCKþ axon terminals form basket-type synapses with somata as well as proximal dendrites, while SOMþ terminals are more often found in contact with small caliber, presumably more distal dendrites (Katona et al., 2001; McDonald and Mascagni, 2001b; Muller et al., 2006, 2007). In addition, PVþ interneurons also form specialized axoaxonic synapses with principal cell axon initial segments (McDonald and Betette, 2001). VIPþ terminals are less specific in their post-

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synaptic target selection, though this represents a heterogeneous group of CCKþ and CCK terminals (Muller et al., 2003). Interneurons in the BLA also innervate other local circuit interneurons. VIPþ interneurons heavily target CBþ interneurons, which form a smaller reciprocal connection (Muller et al., 2003). A small percentage of SOMþ terminals target VIPþ or PVþ interneurons (Muller et al., 2007). Finally, each interneuron group (VIPþ, PVþ, SOMþ and CBþ) also forms synapses with other interneurons within their own group, with PVþ interneurons forming the largest portion of subtype homogeneous connections (Muller et al., 2003, 2005, 2007). Interneurons in the BLA have firstly been differentiated by their firing properties in response to somatic current injections. With respect to the immunohistochemical identity of these cells, recordings have been made from interneurons identified after the recording or from neurons expressing enhanced green fluorescent protein (EGFP) under the control of interneuron specific promoters. Electrophysiological analyses have identified at least 6 distinct types of firing properties (Fig. 2) (Jasnow et al., 2009; Mahanty and Sah, 1998; Rainnie et al., 2006; Sosulina et al., 2010; Woodruff and Sah, 2007b). Consistent with the idea that the BLA is a cortical like structure, these firing properties are similar to those found in cortical interneurons (Ascoli et al., 2008), but different from those of GABAergic neurons in the CeA (Dumont et al., 2002; Lopez de Armentia and Sah, 2004; Schiess et al., 1999). PVþ interneurons are easily identified by their firing properties. They fire short duration action potentials with a half-width measured at w0.5 ms (Rainnie et al., 2006; Woodruff and Sah, 2007b). Most are uniquely capable of firing long trains of action potentials at sustained or stuttered frequencies in excess of 100 Hz (Rainnie et al., 2006; Woodruff and Sah, 2007b). Because of these two phenotypes, these cells are commonly referred to as fast spiking (Fig. 2). However, a small subtype of PVþ interneurons also exhibits regular firing and accommodating phenotypes (Rainnie et al., 2006; Sosulina et al., 2010; Woodruff and Sah, 2007b). As interneurons with similar electrophysiological properties are preferentially connected by gap junctions, and make similar types of synaptic connections, four distinct subtypes of PVþ interneurons have been suggested (Woodruff and Sah, 2007b). Their gap junction coupling suggests that networks of similar PVþ cells may act in concert to modulate BLA activity. PVþ interneurons make strong connections with projection neurons and, rather than simply reducing the activity of principal cells (Windels et al., in press), these interneurons synchronize the firing of principal cells and are capable of altering the phase of this synchrony (Woodruff and Sah, 2007a). Like in the cortex and the hippocampus, CCKþ and PVþ basket cells in the BLA share post-synaptic somatic innervation, but differ

Table 1 Summary of interneuron characteristics in the BLA. Immunohistochemistry

Post-synaptic targets

Physiology

General

Specific

AP half-width

Firing patterns

CB

PV

Large caliber dendrites, somata, axon initial segments

0.5 ms

Fast spiking, stuttering, non-adapting, adapting

CCK (VIP-)

Large caliber dendrites, somata

1 ms

Non-adapting, burst adapting

SOM

Small caliber dendrites

CCK (VIP+)

Large caliber dendrites, somata

1 ms

Adapting

VIP

CB+ interneurons

CR

References

McDonald and Betette, 2001; McDonald and Mascagni, 2001a; McDonald et al., 2005; Muller et al., 2005, 2006; Rainnie et al., 2006; Woodruff and Sah, 2007b; Davila et al., 2008; Sosulina et al., 2010 Katona et al., 2001; Mascagni and McDonald, 2003; McDonald and Mascagni, 2001b; Jasnow et al., 2009; Sosulina et al., 2010 McDonald and Mascagni, 2002; Muller et al., 2007 Katona et al., 2001; McDonald and Mascagni, 2001b; Mascagni and McDonald, 2003; Jasnow et al., 2009; Sosulina et al., 2010 Mascagni and McDonald, 2003; Muller et al., 2003

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Regular Spiking

10mV 200pA

Irregular Spiking

10mV 200pA 100ms

100ms

Fast Spiking

10mV 200pA

Stuttering

10mV 200pA 100ms

100ms

Accomodating

10mV 200pA

Delayed

10mV 200pA 100ms

100ms

Fig. 2. Firing properties of interneurons in the BLA. Six representative traces of the firing properties observed in interneurons of the BLA recorded in in vitro slice preparations from the GAD67-EGFP mouse (Fig. 1). Action potentials were elicited by suprathreshold square pulse current injections shown below the recording for each cell. The firing properties demonstrated here closely resemble those observed in recordings from hippocampal and cortical local circuit interneurons.

significantly in their physiology (Freund and Katona, 2007). Thus, in contrast to PVþ interneurons, CCKþ interneurons fire broad action potentials at lower frequencies and most undergo significant spike frequency adaptation during sustained depolarizations (Jasnow et al., 2009; Sosulina et al., 2010). As with CCKþ interneurons outside the amygdala, immunoreactivity for the cannabinoid receptor CB1 is found to co-localize with somata and dendrites as well as presynaptic terminals of CCKþ interneurons where it’s activation produces a short duration reduction of the action potential evoked release of GABA and the subsequent inhibitory post-synaptic current (Katona et al., 2001; McDonald and Mascagni, 2001b). Interneurons identified by the presence of a single marker have so far only been studied in PV (Woodruff and Sah, 2007b) and CCK (Jasnow et al., 2009) expressing cells. Recently Sosulina et al. have made a detailed characterization of interneurons in the lateral

amygdala using mice expressing EGFP under the control of the GAD67 promoter (Sosulina et al., 2010; Tamamaki et al., 2003). In this study, 5 types of interneurons were identified based on electrophysiological measurements (Sosulina et al., 2010). As in PV-EGFP animals (Woodruff and Sah, 2007b), four electrophysiologically separable types of cells were found to express PV in combination with either CCK or CB (Sosulina et al., 2010). Correlation of mRNA levels of the known immunohistochemical markers with neuron firing properties did not reveal any clear separation of the different types of interneurons. Together, these results show that interneurons in the BLA share many properties with those in other brain regions; though it appears that in the BLA it is more difficult to definitively identify interneuron types on either electrophysiological properties or single marker immunohistochemistry alone.

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2.2. Synaptic physiology and plasticity Three sources of fast excitatory input: glutamatergic afferents from the cortex and thalamus as well as local circuit principal cell axons converge onto interneurons of the basolateral complex (Mahanty and Sah, 1998, 1999; Muller et al., 2006; Pare and Smith, 1998; Pare et al., 1995; Szinyei et al., 2000; Weisskopf and LeDoux, 1999). Some of these afferents have been found to undergo synaptic plasticity (see below). As described above, interneurons can be separated into distinct groups anatomically, using calcium binding protein and neuropeptide expression and, to a lesser extent by correlation with distinct firing properties. However, currently there are no clear correlations between the physiology and plasticity of glutamatergic synapses within or between these groups. Instead, glutamatergic inputs have been independently investigated and differentiated by the subunit composition of their post-synaptic ionotropic receptors. As in most forebrain pyramidal neurons, glutamatergic inputs to principal neurons of the BLA make dual-component synapses with both fast a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) and slower N-methyl-D-aspartate receptors (NMDAR) being present in the post-synaptic membrane (Mahanty and Sah, 1999; Weisskopf and LeDoux, 1999). AMPARs at these synapses show linear currentevoltage relationships (IeVs) consistent with the presence receptors assembled with GluA2 subunits (Farb and LeDoux, 1997; Farb and Ledoux, 1999; Mahanty and Sah, 1999) and NMDARs containing both GluN2A and GluN2B subunits are present at all synapses (Farb and LeDoux, 1997; Farb and Ledoux, 1999; Lopez de Armentia and Sah, 2003). Throughout the central nervous system, where NMDARs are present at glutamatergic synapses on pyramidal neurons, they play an essential role in the induction of synaptic plasticity (Malenka and Bear, 2004). Consistent with this finding, both fear learning and extinction are thought to require NMDAR-dependent synaptic plasticity of inputs to pyramidal neurons in the BLA (Rodrigues et al., 2004; Sah and Westbrook, 2008; Walker and Davis, 2002). In contrast, glutamatergic inputs to interneurons show two key differences. Firstly, all synapses on interneurons express AMPARs that have rapid kinetics and show IeVs with a strong inward rectification, indicative of the presence of calcium permeable AMPARs (CP-AMPARs) that lack GluA2 subunits (Fig. 3A, B) (Mahanty and Sah, 1998; Polepalli et al., 2010). This result is consistent with anatomical data showing that interneurons express lower levels of GluA2 than principal cells in the BLA (He et al., 1999; McDonald, 1996). Secondly, glutamatergic synapses on interneurons express a heterogeneous population of NMDARs (Fig. 3A) that can be separated into three groups (Fig. 3C). In one group, largely described in the lateral amygdala, glutamatergic synapses express only fast AMPARs but lack NMDARs (Mahanty and Sah, 1998; Polepalli et al., 2010). In a second group of interneurons, excitatory synapses express NMDARs with fast decay kinetics (weighted time constant w50 ms) that are insensitive to GluN1/GluN2B heterodimer selective antagonists such as ifenprodil (Williams, 1993), suggesting that they contain mostly GluN2A subunits (Fig. 3C) (Polepalli et al., 2010). Finally, a third group has synapses that express NMDARs that are kinetically slow (weighted time constant w100 ms) and sensitive to GluN2B selective antagonists showing that NMDARs at these synapses are largely GluN1/GluN2B heterodimers (Polepalli et al., 2010; Szinyei et al., 2003). In interneurons that lack synaptic NMDARs, tetanic stimulation of cortical inputs results in long-term potentiation (LTP) that is blocked when post-synaptic calcium is buffered suggesting that a post-synaptic calcium rise is necessary to trigger LTP (Mahanty and Sah, 1998). As expected from the lack of NMDARs at these synapses, this form of LTP is not blocked when NMDARs are inhibited (Mahanty and Sah, 1998; Sosulina et al., 2006; Szinyei et al., 2007). In

A

769

+40 mV

+40 mV

50 µM D-AP5 + 10 µM NBQX +40 mV

-60 mV

-60 mV

-60 mV

50 µM D-AP5

20 pA 50 ms

B

C

No NMDA

+40 -60

Fast NMDA

20 pA 1 ms

+40

τ ~ 50ms

-60

40 Slow NMDA

-80 -60 -40 -20 -40

20 40 60 Vm (mV)

τ ~100 ms +40

-80

-60

-120 I (pA) 100 ms Fig. 3. Interneurons of the amygdala express rectifying AMPARs and can be separated on the type of NMDARs present. Evoked post-synaptic currents demonstrate the receptor composition of the glutamatergic synapses. A. The slow outward current recorded at þ40 mV can be almost entirely blocked by the NMDA antagonist D-AP5, while the remaining fast inward current recorded at 60 mV is blocked by the AMPA antagonist NBQX. B. The traces on the upper panel show AMPAR mediated EPSCs recorded at holding potentials from 80 mV to þ40 mV in 20 mV steps. The peak currentevoltage relationship is shown below. The IeV is strongly inwardly rectifying showing that these receptors lack GluA2 subunits. C. Three types of interneurons can be identified based on the contribution of synaptic NMDARs. The first class of interneurons contains no detectable NMDAR mediated current (top panel). The outward current recorded at þ40 mV is mediated entirely by calcium permeable, rectifying AMPARs. The second class of interneurons expresses fast NMDARs (middle panel). The outward current recorded at þ40 mV has a weighted time constant of w50 ms and is mediated by GluN2A containing receptors. The third class of interneurons expresses slow NMDARs (bottom panel). The outward current recorded at þ40 mV has a decay time constant of w100 ms and is mediated by GluN2B containing receptors. In each case the AMPAR mediated inward current is recorded at 60 mV. Traces are shown normalized to the peak AMPA mediated current at 60 mV.

interneurons that express synaptic NMDARs, repetitive stimulation of cortical afferents also leads to LTP. However, LTP at these synapses has the same pharmacological properties as that seen at inputs that lack NMDARs. Thus, it does not require calcium influx through NMDARs, but is blocked when neurons are loaded with a calcium buffer indicating that it requires a rise in post-synaptic calcium (Polepalli et al., 2010). In both cell types, synaptic AMPARs show strong inward rectification indicating that these receptors are calcium permeable (Polepalli et al., 2010). Moreover, LTP does not require activation of voltage dependent calcium channels or release of calcium from intracellular stores (Polepalli et al., 2010). As this form of LTP is inhibited when AMPARs are blocked during induction (Polepalli et al., 2010), it is likely that, as in hippocampal interneurons that express CP-AMPARs (Kullmann and Lamsa, 2007; Lei and

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McBain, 2002; Nissen et al., 2010; Oren et al., 2009), LTP is triggered by calcium influx via these receptors. In hippocampal interneurons, LTP also shows input specificity. That is, only the synapses in the repetitively stimulated afferent pathway show plasticity (Lamsa et al., 2007). Similarly, at inputs to interneurons in the amygdala, LTP is also input specific (Fig. 4) (Polepalli et al., 2010; Szinyei et al., 2000). While LTP is synapse-specific at the single cell level, there are conflicting reports on specificity at the input level. Thus, one report found that LTP is restricted to cortical inputs (Polepalli et al., 2010) while another suggests it is equally present at thalamic and cortical inputs (Polepalli et al., 2010; Szinyei et al., 2000). It has also been reported that heterosynaptic plasticity can occur at synapses onto interneurons in the BLA (Bauer and LeDoux, 2004). Interestingly, this heterosynaptic LTP has been found to be NMDAR dependent (Bauer and LeDoux, 2004), however, properties of the interneurons that mediate this form of plasticity have not been determined. In interneurons that have dual-component synapses, both AMPA and NMDA receptors are clearly present, and both are activated by

A

Cortex tetanised Thalamus

4 Tetanised Non tetanised

Normalised EPSC

3.25

2.5

1.75

1

0.25 -5

B

0

5 Time (mins)

10

Cortex (tetanised)

Thalamus

PN IN

IN

Normalised IPSC

2.5

Cortical pathway Thalamic pathway

2

1.5

1

0.5 -5

0

5

10

15

20

Time-min Fig. 4. Long-term potentiation of interneurons in the BLA. A. LTP at glutamatergic inputs to interneurons of the BLA is input specific. Tetanisation of the cortical input results in LTP at cortical synapses while the thalamic synapses remain at baseline level. B. Potentiation of the feedforward IPSC is also input specific. Tetanisation of the cortical input results in a feedforward potentiation of the disynaptic IPSC recorded in a BLA principal cell. The disynaptic IPSC recorded as a result of stimulation of the thalamic input is not potentiated.

synaptically released glutamate (Polepalli et al., 2010). At these synapses, tetanic stimulation induces LTP that requires a rise in postsynaptic calcium. However, unlike in pyramidal neurons, calcium influx though NMDARs is not required to trigger LTP. As discussed above, two classes of interneurons can be defined based on the subunit composition of synaptic NMDARs present: one type expresses NMDARs that largely contain GluN1/GluN2B heterodimers, whereas the other expresses NMDARs that contain GluN2A subunits with little or no GluN2B.Curiously, LTP appears to be restricted to the class of cells that do not express synaptic GluN2B subunits (Polepalli et al., 2010). As calcium influx via synaptic NMDARs is not required for LTP induction, this finding raises the question e what is role of synaptic NMDARs in these interneurons? The subunit composition of NMDARs at most synapses undergoes developmental regulation, with GluN2B being expressed prenatally whereas GluN2A expression begins shortly after birth, and progressively increases (Monyer et al., 1994; Sheng et al., 1994). Thus, early in development, synapses express NMDARs composed of GluN1/GluN2B heterodimers while at mature synapses, GluN2B subunits are replaced by GluN2A subunits in an activity-dependent manner (Barria and Malinow, 2002; Philpot et al., 2001). While the developmental regulation of NMDARs at interneuron synapses has not been specifically examined, it is clear at one set of interneurons in the BLA, there is a developmental switch with GluN2A subunits being present in adult synapses. However, in a different population of interneurons, synapses continue to express GluN2B subunits. These findings raise two possibilities. First, it is possible that the lack of change in subunit composition may simply be a genetic marker that identifies a population of cells that does not show LTP. Alternatively, it is possible that interneurons that express synaptic GluN2B subunits represent a developmentally delayed subset of cells, and the lack of LTP at inputs to these interneurons reflects their developmental immaturity. In both hippocampal pyramidal (Hayashi et al., 2000) and LA principal neurons (Rumpel et al., 2005), post-synaptic NMDARdependent-LTP results from activation of CaMKII and subsequent trafficking of new AMPARs to the synapse (Malinow and Malenka, 2002). Thus, in this form of LTP, both its induction and maintenance are post-synaptic. In BLA interneurons, LTP induction is clearly post-synaptic (Mahanty and Sah, 1998; Polepalli et al., 2010) but these cells do not express CaMKII (McDonald et al., 2002); therefore induction of this form of LTP must engage fundamentally different post-synaptic mechanisms. This form of LTP does not require NMDARs, but is triggered by a rise in post-synaptic calcium. The increase in synaptic transmission in these interneurons is not associated with a change in paired pulse facilitation suggesting that there is no change in presynaptic release probability. Moreover, LTP at these inputs is blocked when the actin-cytoskeleton is disrupted, suggesting that in amygdala interneurons, LTP requires trafficking of post-synaptic AMPARs (Polepalli et al., 2010). Thus, as in principal neurons, both the induction and expression of LTP in BLA interneurons is post-synaptic. Different local circuit interneurons provide feedforward and feedback inhibition and both types are present in the BLA (Lang and Pare, 1998; Pare and Smith, 1998; Woodruff and Sah, 2007b) although the identities of these two cell types is not known. Recordings in the lateral amygdala show that tetanic stimulation of glutamatergic cortical fibers results in potentiation of the feedforward disynaptic inhibitory post-synaptic current (IPSC) onto principal cells (Mahanty and Sah, 1998; Szinyei et al., 2007). This LTP of the disynaptic IPSC is resistant to NMDAR blockade, is input specific (Fig. 4), and requires activity of CP-AMPARs (Mahanty and Sah, 1998; Szinyei et al., 2007). As tetanic stimulation of the GABAergic inputs to pyramidal neurons in the BLA does not induce LTP (Mahanty and Sah, 1998), LTP of the disynaptic IPSC is likely due to

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potentiation of excitatory inputs to the interneurons in this circuit. As described above, LTP of inputs to BLA interneurons is restricted to cells that do not express GluN2B containing NMDARs. Thus, at least some of these interneurons must form a pool of feedforward cells in the LA. These results suggest that the lack of GluN2B expression in one type of BLA interneuron may be a marker for this class of feedforward interneurons. Functionally, enhancement of the feedforward IPSC reduces the ability of cortical inputs to drive principal neurons (Polepalli et al., 2010). In feedforward circuits, the balance between excitation and inhibition maintains the temporal fidelity of information processing (Pouille and Scanziani, 2001). Following fear conditioning, pyramidal neurons in the BLA show enhanced activation by the CS (Herry et al., 2008; Quirk et al., 1995). This enhanced response is thought to result from LTP of excitatory inputs to pyramidal neurons, and drives enhanced fear response to the CS. Following extinction training, the behavioural response to the CS is reduced and is correlated with a similar reduction in CS induced activity of BLA pyramidal neurons (Herry et al., 2008). Extinction training clearly engages the medial prefrontal cortex (mPFC) (Quirk and Mueller, 2008), and its activity is required for expression of extinction. Projections from the infralimbic mPFC to the medial intercalated cells that then inhibit the output of CeA neurons has been proposed as one mechanism for expression of extinction (Likhtik et al., 2008). However, the infralimbic cortex also projects to the BLA (Pape and Pare, 2010). As the cortically evoked feedforward IPSC in LA principal neurons is potentiated following repetitive stimulation of cortical afferents, it is possible that the reduction in BLA neuron activity following extinction training is due to plasticity of cortical inputs (perhaps those from infralimbic cortex) to feedforward interneurons in the BLA. 2.3. Axo-axonic interneurons In addition to the feedforward inhibition provided by interneurons in the BLA, one specialized type of interneuron also generates a feedforward excitation of principal cells. In paired recordings from PVþ interneurons, Woodruff et al. (Woodruff et al., 2006), demonstrated that single action potentials in the presynaptic PVþ interneuron are capable of producing both a monosynaptic IPSC as well as a disynaptic excitatory post-synaptic current (EPSC) in the post-synaptic PVþ interneuron (Woodruff et al., 2006). The disynaptic EPSC is blocked by the GABAA receptor antagonist bicuculline as well as the AMPA/kainate receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX), implying that the GABA release produces a post-synaptic excitation in a glutamatergic principal cell that is excited to threshold to produce the observed NBQX sensitive feedback EPSC (Woodruff et al., 2006). In some cases the presynaptic interneuron also receives direct feedback excitation. A similar circuit has been described in the human neocortex where axo-axonic cells that innervate the axon initial segment of pyramidal cells are capable of firing the post-synaptic cell in a one-to-one spike coupled manner (Molnar et al., 2008; Szabadics et al., 2006). Though the circuit is similar, it appears that the axo-axonic cells in the BLA of the rodent are a basket cell axo-axonic cell hybrid, whereas their human cortical counterparts are specialized as one or the other (Howard et al., 2005; Molnar et al., 2008; Szabadics et al., 2006; Woodruff et al., 2006). It remains to be determined exactly what role this feedback excitation circuit plays in amygdala-based learning and behaviour. However, the large amplitude of the feedback EPSC and the unitary EPSC in fast spiking and stuttering PVþ interneurons combined with the high fidelity of the excitatory loop (our unpublished observations) suggest this circuit could act as a powerful amplifier of BLA information processing (Woodruff et al., 2006; Woodruff and Sah, 2007a). While

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the presence of axo-axonic excitation in the BLA is clear, a functional role for this circuit is not known. The therapeutic actions of benzodiazepines in the BLA are generally thought to result from their actions in enhancing inhibitory transmission. However, the fact that some GABAergic circuits in the BLA are excitatory, indicate that actions of these agents on circuit activity are not that clear. 3. Conclusions The amygdala is a temporal lobe structure that is involved in emotional processing. In particular it plays a key role in fear learning and extinction. The BLA is the main route by which sensory information enters the amygdala and much of the plasticity that underlies fear learning is thought to occur at synapses in the BLA. The BLA is a cortical like structure that contains glutamatergic principal neurons and local circuit interneurons; however, the local circuitry of the BLA is just beginning to be elucidated. Interneurons within the BLA are separable into distinct populations, with some areas of overlap, based on their firing properties, protein expression and synaptic inputs that they receive. Glutamatergic inputs to interneurons activate synapses that uniformly express CP-AMPARs, however, neurons can be divided into three populations based on the properties of synaptic NMDARs. One population has no NMDARs at their synapses. A second population expresses NMDARs that largely contain GluN2A subunits and a third population largely expresses GluN2B subunits. Repetitive stimulation of cortical inputs to interneurons in the lateral amygdala results in LTP that is initiated by a rise in post-synaptic calcium but does not require NMDARs. This LTP is absent in cells were synapses express GluN2B containing NMDARs. We suggest that expression of GluN2B containing NMDA receptors may define a specific pool of feedforward interneurons. The identity of these interneurons with respect to their immunohistochemical signature remains unknown. Clearly, this issue needs to be addressed in future studies of BLA interneurons. Interneurons in the BLA form only about 20% of the total cell population but maintain a tight control of principal cell excitability and modulation of local inhibition has dramatic effects on fear related learning and anxiety states. In the hippocampus, single unit recordings in vivo have shown that interneurons rarely fire more than a few action potentials at a time but are responsible for shaping the network oscillations that correlate with different behavioural states (Klausberger et al., 2003, 2005). Moreover, anatomically and immunohistochemically identifiable groups of interneurons participate differentially in each type of oscillation, with the timing of their firing correlated to a specific phase of the network oscillation (Klausberger et al., 2003, 2005). For interneurons in the BLA, however, there is little data linking the firing of specific interneuron populations with different brain states or activities. Thus while there is clear electrophysiological heterogeneity within immunohistochemical groups, there is no correlation of these different populations with the specific roles of the amygdala in shaping behaviour. In future experiments it will be important to determine the properties of interneurons in the BLA in vivo during amygdala coupled network oscillations (Popescu et al., 2009) to identify the roles that the different groups of cells play in amygdala-based emotional facilitation of learning and memory. Understanding the local circuitry of the amygdala and the properties of these interneurons will provide insights into the neurobiological changes that cause many anxiety related disorders. References Alheid, G.F., de Olmos, J., Beltramino, C.A., 1995. Amygdala and extended amygdala. In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, California, pp. 495e578.

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