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 mum 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

Investigation of the Performance of Thermally Generated Au/Ag Nanoislands for SERS and LSPR Applications

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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

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

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

<div><p>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.</p></div

Principle neurons (PNs) give rise to distinct excitatory synaptic inputs onto the 2 basket cell (BC) types.

<p>(A) Panoramic images showing a cholecystokinin-expressing basket cell (CCKBC)→PN (left) and a parvalbumin-containing basket cell (PVBC)→PN (right) pair in the anterior part of the basal amygdala (BA). Interneurons were filled with Cascade Blue and PNs with biocytin. Insets show a higher magnification of the cell pairs recorded. Scales: 250 μm. (B) Representative traces of unitary excitatory postsynaptic currents (EPSCs) evoked by action potentials in a PN→CCKBC (upper traces) and a PN→PVBC pair (lower traces). Ten superimposed consecutive traces are in gray, and averages are in blue and orange. Scales: x: 2 ms; PN→CCKBC, y: 80 mV/8 pA; PN→PVBC, y: 90 mV/30 pA. (C) PVBCs receive unitary excitatory postsynaptic currents (uEPSCs) from local PNs with larger amplitude and lower failure rate than CCKBCs (potency, CCKBC: 21.5 ± 1.6 pA, PVBC: 94.25 ± 15.08 pA; failure rate, CCKBC: 0.58 ± 0.04, PVBC: 0.25 ± 0.04; Mann–Whitney <i>U</i> test, ***<i>p</i> < 0.001) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001421#pbio.2001421.s016" target="_blank">S6 Data</a>). Each data point on the plots represents an average obtained in a pair recording, and lines represent means. (D) Schematic illustration of the experimental design to excite PNs using blue light. mCherry-containing construct was injected into the BA of transgenic mice (bottom drawing illustrates a horizontal slice showing the expression site in red). Among adeno-associated virus (AAV)-infected PNs (red circles), a CCKBC (blue) was recorded in whole-cell mode, while its excitatory input from neighboring PNs was tested by light illumination and/or whole-cell recording. BA, basal amygdala; Hip, hippocampus. (E) Connectivity map. Concentric circles indicate Δ100 μm distance from the interneuron in the center (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001421#pbio.2001421.s016" target="_blank">S6 Data</a>). Each dot represents a tested PN soma location. (F) PVBCs receive excitatory synaptic inputs from their neighboring PNs with high probability, while CCKBCs are innervated by PNs via their local collaterals with low probability, independently of the inter-somatic distance (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001421#pbio.2001421.s016" target="_blank">S6 Data</a>).</p