Fear learning and aversive stimuli differentially change excitatory synaptic transmission in perisomatic inhibitory cells of the basal amygdala.

Inhibitory circuits in the basal amygdala (BA) have been shown to play a crucial role in associative fear learning. How the excitatory synaptic inputs received by BA GABAergic interneurons are influenced by memory formation, a network parameter that may contribute to learning processes, is still largely unknown. Here, we investigated the features of excitatory synaptic transmission received by the three types of perisomatic inhibitory interneurons upon cue-dependent fear conditioning and aversive stimulus and tone presentations without association. Acute slices were prepared from transgenic mice: one group received tone presentation only (conditioned stimulus, CS group), the second group was challenged by mild electrical shocks unpaired with the CS (unsigned unconditioned stimulus, unsigned US group) and the third group was presented with the CS paired with the US (signed US group). We found that excitatory synaptic inputs (miniature excitatory postsynaptic currents, mEPSCs) recorded in distinct interneuron types in the BA showed plastic changes with different patterns. Parvalbumin (PV) basket cells in the unsigned US and signed US group received mEPSCs with reduced amplitude and rate in comparison to the only CS group. Coupling the US and CS in the signed US group caused a slight increase in the amplitude of the events in comparison to the unsigned US group, where the association of CS and US does not take place. Excitatory synaptic inputs onto cholecystokinin (CCK) basket cells showed a markedly different change from PV basket cells in these behavioral paradigms: only the decay time was significantly faster in the unsigned US group compared to the only CS group, whereas the amplitude of mEPSCs increased in the signed US group compared to the only CS group. Excitatory synaptic inputs received by PV axo-axonic cells showed the least difference in the three behavioral paradigm: the only significant change was that the rate of mEPSCs increased in the signed US group when compared to the only CS group. These results collectively show that associative learning and aversive stimuli unpaired with CS cause different changes in excitatory synaptic transmission in BA perisomatic interneuron types, supporting the hypothesis that they play distinct roles in the BA network operations upon pain information processing and fear memory formation.

Structural Organization of Perisomatic Inhibition in the Mouse Medial Prefrontal Cortex.

Perisomatic inhibition profoundly controls neural function. However, the structural organization of inhibitory circuits giving rise to the perisomatic inhibition in the higher-order cortices is not completely known. Here, we performed a comprehensive analysis of those GABAergic cells in the medial prefrontal cortex (mPFC) that provide inputs onto the somata and proximal dendrites of pyramidal neurons. Our results show that most GABAergic axonal varicosities contacting the perisomatic region of superficial (layer 2/3) and deep (layer 5) pyramidal cells express parvalbumin (PV) or cannabinoid receptor type 1 (CB1). Further, we found that the ratio of PV/CB1 GABAergic inputs is larger on the somatic membrane surface of pyramidal tract neurons in comparison with those projecting to the contralateral hemisphere. Our morphologic analysis of in vitro labeled PV+ basket cells (PVBC) and CCK/CB1+ basket cells (CCKBC) revealed differences in many features. PVBC dendrites and axons arborized preferentially within the layer where their soma was located. In contrast, the axons of CCKBCs expanded throughout layers, although their dendrites were found preferentially either in superficial or deep layers. Finally, using anterograde trans-synaptic tracing we observed that PVBCs are preferentially innervated by thalamic and basal amygdala afferents in layers 5a and 5b, respectively. Thus, our results suggest that PVBCs can control the local circuit operation in a layer-specific manner via their characteristic arborization, whereas CCKBCs rather provide cross-layer inhibition in the mPFC.SIGNIFICANCE STATEMENT Inhibitory cells in cortical circuits are crucial for the precise control of local network activity. Nevertheless, in higher-order cortical areas that are involved in cognitive functions like decision-making, working memory, and cognitive flexibility, the structural organization of inhibitory cell circuits is not completely understood. In this study we show that perisomatic inhibitory control of excitatory cells in the medial prefrontal cortex is performed by two types of basket cells endowed with different morphologic properties that provide inhibitory inputs with distinct layer specificity on cells projecting to disparate areas. Revealing this difference in innervation strategy of the two basket cell types is a key step toward understanding how they fulfill their distinct roles in cortical network operations.

CaMKIIα Promoter-Controlled Circuit Manipulations Target Both Pyramidal Cells and Inhibitory Interneurons in Cortical Networks.

A key assumption in studies of cortical functions is that excitatory principal neurons, but not inhibitory cells express calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) resulting in a widespread use of CaMKIIα promoter-driven protein expression for principal cell manipulation and monitoring their activities. Using neuroanatomical and electrophysiological methods we demonstrate that in addition to pyramidal neurons, multiple types of cortical GABAegic cells are targeted by adeno-associated viral vectors (AAV) driven by the CaMKIIα promoter in both male and female mice. We tested the AAV5 and AAV9 serotype of viruses with either Channelrhodopsin 2 (ChR2)-mCherry or Archaerhodopsin-T-green fluorescent protein (GFP) constructs, with different dilutions. We show that in all cases, the reporter proteins can visualize a large fraction of different interneuron types, including parvalbumin (PV), somatostatin (SST), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and cholecystokinin (CCK)-containing GABAergic cells, which altogether cover around 60% of the whole inhibitory cell population in cortical structures. Importantly, the expression of the excitatory opsin Channelrhodopsin 2 in the interneurons effectively drive spiking of infected GABAergic cells even if the immunodetectability of reporter proteins is ambiguous. Thus, our results challenge the use of CaMKIIα promoter-driven protein expression as a selective tool in targeting cortical glutamatergic neurons using viral vectors.

Total Number and Ratio of GABAergic Neuron Types in the Mouse Lateral and Basal Amygdala.

GABAergic neurons are key circuit elements in cortical networks. Despite growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types has been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22%) is significantly higher than in the LA (16%) in both male and female mice. No difference was observed between the right and left hemispheres in either sex. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5%-6%), basket cells expressing parvalbumin (17%-20%) or cholecystokinin (7%-9%), dendrite-targeting inhibitory cells expressing somatostatin (10%-16%), NPY-containing neurogliaform cells (14%-15%), VIP and/or calretinin-expressing interneuron-selective interneurons (29%-38%), and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (5.5%-8%). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathologic conditions, and to model functioning amygdalar networks in health and disease.SIGNIFICANCE STATEMENT GABAergic cells in cortical structures, as in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyperexcitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions, including anxiety, post-traumatic stress disorder, schizophrenia, and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathologic brain states.

Associative and plastic thalamic signaling to the lateral amygdala controls fear behavior.

Decades of research support the idea that associations between a conditioned stimulus (CS) and an unconditioned stimulus (US) are encoded in the lateral amygdala (LA) during fear learning. However, direct proof for the sources of CS and US information is lacking. Definitive evidence of the LA as the primary site for cue association is also missing. Here, we show that calretinin (Calr)-expressing neurons of the lateral thalamus (Calr+LT neurons) convey the association of fast CS (tone) and US (foot shock) signals upstream from the LA in mice. Calr+LT input shapes a short-latency sensory-evoked activation pattern of the amygdala via both feedforward excitation and inhibition. Optogenetic silencing of Calr+LT input to the LA prevents auditory fear conditioning. Notably, fear conditioning drives plasticity in Calr+LT neurons, which is required for appropriate cue and contextual fear memory retrieval. Collectively, our results demonstrate that Calr+LT neurons provide integrated CS-US representations to the LA that support the formation of aversive memories.

Differential excitatory control of 2 parallel basket cell networks in amygdala microcircuits.

Information processing in neural networks depends on the connectivity among excitatory and inhibitory neurons. The presence of parallel, distinctly controlled local circuits within a cortical network may ensure an effective and dynamic regulation of microcircuit function. By applying a combination of optogenetics, electrophysiological recordings, and high resolution microscopic techniques, we uncovered the organizing principles of synaptic communication between principal neurons and basket cells in the basal nucleus of the amygdala. In this cortical structure, known to be critical for emotional memory formation, we revealed the presence of 2 parallel basket cell networks expressing either parvalbumin or cholecystokinin. While the 2 basket cell types are mutually interconnected within their own category via synapses and gap junctions, they avoid innervating each other, but form synaptic contacts with axo-axonic cells. Importantly, both basket cell types have the similar potency to control principal neuron spiking, but they receive excitatory input from principal neurons with entirely diverse features. This distinct feedback synaptic excitation enables a markedly different recruitment of the 2 basket cell types upon the activation of local principal neurons. Our data suggest fundamentally different functions for the 2 parallel basket cell networks in circuit operations in the amygdala.

Perisomatic GABAergic synapses of basket cells effectively control principal neuron activity in amygdala networks.

Efficient control of principal neuron firing by basket cells is critical for information processing in cortical microcircuits, however, the relative contribution of their perisomatic and dendritic synapses to spike inhibition is still unknown. Using in vitro electrophysiological paired recordings we reveal that in the mouse basal amygdala cholecystokinin- and parvalbumin-containing basket cells provide equally potent control of principal neuron spiking. We performed pharmacological manipulations, light and electron microscopic investigations to show that, although basket cells innervate the entire somato-denditic membrane surface of principal neurons, the spike controlling effect is achieved primarily via the minority of synapses targeting the perisomatic region. As the innervation patterns of individual basket cells on their different postsynaptic partners show high variability, the impact of inhibitory control accomplished by single basket cells is also variable. Our results show that both basket cell types can powerfully regulate the activity in amygdala networks predominantly via their perisomatic synapses.

Synaptic Organization of Perisomatic GABAergic Inputs onto the Principal Cells of the Mouse Basolateral Amygdala.

Spike generation is most effectively controlled by inhibitory inputs that target the perisomatic region of neurons. Despite the critical importance of this functional domain, very little is known about the organization of the GABAergic inputs contacting the perisomatic region of principal cells (PCs) in the basolateral amygdala. Using immunocytochemistry combined with in vitro single-cell labeling we determined the number and sources of GABAergic inputs of PCs at light and electron microscopic levels in mice. We found that the soma and proximal dendrites of PCs were innervated primarily by two neurochemically distinct basket cell types expressing parvalbumin (PVBC) or cholecystokinin and CB1 cannabinoid receptors (CCK/CB1BC). The innervation of the initial segment of PC axons was found to be parceled out by PVBCs and axo-axonic cells (AAC), as the majority of GABAergic inputs onto the region nearest to the soma (between 0 and 10 µm) originated from PVBCs, while the largest portion of the axon initial segment was innervated by AACs. Detailed morphological investigations revealed that the three perisomatic region-targeting interneuron types significantly differed in dendritic and axonal arborization properties. We found that, although individual PVBCs targeted PCs via more terminals than CCK/CB1BCs, similar numbers (15-17) of the two BC types converge onto single PCs, whereas fewer (6-7) AACs innervate the axon initial segment of single PCs. Furthermore, we estimated that a PVBC and a CCK/CB1BC may target 800-900 and 700-800 PCs, respectively, while an AAC can innervate 600-650 PCs. Thus, BCs and AACs innervate ~10 and 20% of PC population, respectively, within their axonal cloud. Our results collectively suggest, that these interneuron types may be differently affiliated within the local amygdalar microcircuits in order to fulfill specific functions in network operation during various brain states.

Strategically positioned inhibitory synapses of axo-axonic cells potently control principal neuron spiking in the basolateral amygdala.

Axo-axonic cells (AACs) in cortical regions selectively innervate the axon initial segments (AISs) of principal cells (PCs), where the action potentials are generated. These GABAergic interneurons can alter the activity of PCs, but how the efficacy of spike control correlates with the number of output synapses remains unclear. Moreover, the relationship between the spatial distribution of GABAergic synapses and the action potential initiation site along the AISs is not well defined. Using paired recordings obtained in the mouse basolateral amygdala, we found that AACs powerfully inhibited or delayed the timing of PC spiking by 30 ms, if AAC output preceded PC spiking with no more than 80 ms. By correlating the number of synapses and the probability of spiking, we revealed that larger numbers of presynaptic AAC boutons giving rise to larger postsynaptic responses provided more effective inhibition of PC spiking. At least 10-12 AAC synapses, which could originate from 2-3 AACs on average, were necessary to veto the PC firing under our recording conditions. Furthermore, we determined that the threshold for the action potential generation along PC axons is the lowest between 20 and 40 µm from soma, which axonal segment received the highest density of GABAergic inputs. Single AACs preferentially innervated this narrow portion of the AIS where action potentials were generated with the highest likelihood, regardless of the number of synapses forming a given connection. Our results uncovered a fine organization of AAC innervation maximizing their inhibitory efficacy by strategically positioning synapses along the AISs.

Feedforward inhibition underlies the propagation of cholinergically induced gamma oscillations from hippocampal CA3 to CA1.

Gamma frequency (30-80 Hz) oscillations are implicated in memory processing. Such rhythmic activity can be generated intrinsically in the CA3 region of the hippocampus from where it can propagate to the CA1 area. To uncover the synaptic mechanisms underlying the intrahippocampal spread of gamma oscillations, we recorded local field potentials, as well as action potentials and synaptic currents in anatomically identified CA1 and CA3 neurons during carbachol-induced gamma oscillations in mouse hippocampal slices. The firing of the vast majority of CA1 neurons and all CA3 neurons was phase-coupled to the oscillations recorded in the stratum pyramidale of the CA1 region. The predominant synaptic input to CA1 interneurons was excitatory, and their discharge followed the firing of CA3 pyramidal cells at a latency indicative of monosynaptic connections. Correlation analysis of the input-output characteristics of the neurons and local pharmacological block of inhibition both agree with a model in which glutamatergic CA3 input controls the firing of CA1 interneurons, with local pyramidal cell activity having a minimal role. The firing of phase-coupled CA1 pyramidal cells was controlled principally by their inhibitory inputs, which dominated over excitation. Our results indicate that the synchronous firing of CA3 pyramidal cells rhythmically recruits CA1 interneurons and that this feedforward inhibition generates the oscillatory activity in CA1. These findings identify distinct synaptic mechanisms underlying the generation of gamma frequency oscillations in neighboring hippocampal subregions.

Cannabinoids attenuate hippocampal γ oscillations by suppressing excitatory synaptic input onto CA3 pyramidal neurons and fast spiking basket cells.

CB(1) cannabinoid receptor (CB(1)R) activation by exogenous ligands can impair memory processes, which critically depend on synchronous neuronal activities that are temporarily structured by oscillations. In this study, we aimed to reveal the mechanisms underlying the cannabinoid-induced decrease in gamma oscillations. We first verified that cannabinoids (CP55,940 and WIN55,212-2) readily suppressed carbachol-induced gamma oscillations in the CA3 region of hippocampal slices via activation of CB(1)Rs. The cannabinoid-induced decrease in the peak power of oscillations was accompanied by reduced and less precise firing activity in CA3 pyramidal cells and fast spiking basket cells. By examining the cannabinoid sensitivity of synaptic inputs we found that the amplitude of evoked excitatory postsynaptic currents was significantly suppressed upon CB(1)R activation in both CA3 pyramidal cells and fast spiking basket cells. In contrast, evoked inhibitory postsynaptic currents in CA3 pyramidal cells were unaltered. Furthermore, we observed that a CB(1)R agonist-induced decrease in the oscillation power at the beginning of the drug application was accompanied primarily by the reduced discharge of fast spiking basket cells, while pyramidal cell firing was unaltered. This result implies that the dampening of cholinergically induced gamma oscillations in the hippocampus by cannabinoids can be explained by a reduced excitatory input predominantly onto fast spiking basket cells, which leads to a reduction in neuronal firing frequency and precision, and thus to smaller field potentials. In addition, we uncovered that the spontaneously occurring sharp wave-ripple activities in hippocampal slices could also be suppressed by CB(1)R activation suggesting that cannabinoids profoundly reduce the intrinsically generated oscillatory activities at distinct frequencies in CA3 networks by reducing synaptic neurotransmission.

Nitric oxide signaling modulates synaptic transmission during early postnatal development.

Early γ-aminobutyric acid mediated (GABAergic) synaptic transmission and correlated neuronal activity are fundamental to network formation; however, their regulation during early postnatal development is poorly understood. Nitric oxide (NO) is an important retrograde messenger at glutamatergic synapses, and it was recently shown to play an important role also at GABAergic synapses in the adult brain. The subcellular localization and network effect of this signaling pathway during early development are so far unexplored, but its disruption at this early age is known to lead to profound morphological and functional alterations. Here, we provide functional evidence--using whole-cell recording--that NO signaling modulates not only glutamatergic but also GABAergic synaptic transmission in the mouse hippocampus during the early postnatal period. We identified the precise subcellular localization of key elements of the underlying molecular cascade using immunohistochemistry at the light--and electron microscopic levels. As predicted by these morpho-functional data, multineuron calcium imaging in acute slices revealed that this NO-signaling machinery is involved also in the control of synchronous network activity patterns. We suggest that the retrograde NO-signaling system is ideally suited to fulfill a general presynaptic regulatory role and may effectively fine-tune network activity during early postnatal development, while GABAergic transmission is still depolarizing.