Presynaptic Rac1 in the hippocampus selectively regulates working memory.

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory is selectively impaired following the expression of a genetically encoded Rac1-inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

Effects of Lactobacillus rhamnosus DSM7133 on Intestinal Porcine Epithelial Cells.

Antimicrobial resistance is one of the biggest health challenges nowadays. Probiotics are promising candidates as feed additives contributing to the health of the gastrointestinal tract. The beneficial effect of probiotics is species/strain specific; the potential benefits need to be individually assessed for each probiotic strain or species. We established a co-culture model, in which gastrointestinal infection was modeled using Escherichia coli (E. coli) and Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium). Using intestinal porcine epithelial cells (IPEC-J2), the effects of pre-, co-, and post-treatment with Lactobacillus (L.) rhamnosus on the barrier function, intracellular (IC) reactive oxygen species (ROS) production, proinflammatory cytokine (IL-6 and IL-8) response, and adhesion inhibition were tested. E. coli- and S. Typhimurium-induced barrier impairment and increased ROS production could be counteracted using L. rhamnosus (p < 0.01). S. Typhimurium-induced IL-6 production was reduced via pre-treatment (p < 0.05) and post-treatment (p < 0.01); increased IL-8 secretion was decreased via pre-, co-, and post-treatment (p < 0.01) with L. rhamnosus. L. rhamnosus demonstrated significant inhibition of adhesion for both S. Typhimurium (p < 0.001) and E. coli (p < 0.001 in both pre-treatment and post-treatment; p < 0.05 in co-treatment). This study makes a substantial contribution to the understanding of the specific benefits of L. rhamnosus. Our findings can serve as a basis for further in vivo studies carried out in pigs and humans.

Morphological and histological investigation of the conduction system in the equine atrial muscle sleeve of pulmonary veins.

BACKGROUND: Atrial fibrillation is the most common arrhythmia in horses causing poor performance. The role of pulmonary vein triggers in the pathogenesis has been identified in horses. Ablation methods have been investigated, but the available information on anatomical, histological and immunohistochemical assessment of the pulmonary vein ostia and the conduction system of the myocardial sleeve is still limited. OBJECTIVES: The aim of the study was to describe the morphological properties of the myocardial sleeve in healthy horses. STUDY DESIGN: Cross-sectional. METHODS: Eighty-three equine hearts were dissected. The number and diameters of pulmonary vein ostia were determined, and anatomical localisation was described. Fifty-eight tissue samples were collected for routine histology and 12 of these were used for immunohistochemistry (connexin 43, 45, S100, and tyrosine hydroxylase antibodies). RESULTS: The mean number of pulmonary vein ostia was 4.5 (4 veins: 46 horse, 5 veins: 31 horses, 6 veins: 6 horses). Diameters (mean ± SD) of the main ostia were as follows: vein I: 20.2 ± 7.0 mm, vein II: 32.7 ± 7.1 mm, vein III: 33.4 ± 5.9 mm, vein IV: 18.1 ± 4.5 mm. Diameters of supernumerary vein ostia varied between 3.0 and 28.0 mm (11.5 ± 5.5 mm). Early branching was found in 26 horses (31.3%) and 30 veins (vein I: 14, vein II: 9, vein III: 5, vein IV: 2). Histology confirmed the presence of a muscle sleeve composed of myocardial tissue in each pulmonary vein. S100 and TH positivity was detected in each vein, and it confirmed the presence of adrenergic and non-adrenergic nerve fibres within the myocardial sleeve. Cx43 and 45 positivity were also found in each vein indicating the presence of gap junctions. MAIN LIMITATIONS: The effect of bodyweight on pulmonary vein dimensions is unknown. CONCLUSIONS: Future ablation techniques should consider that conductive tissue is present in the entire myocardial sleeve in all pulmonary vein ostia.

Comparison of Police Data on Animal Cruelty and the Perception of Animal Welfare NGOs in Hungary.

Animal cruelty has been a criminal offence in Hungary since 2004 and the legislator has tightened and differentiated the regulations in several waves since then. However, it is not an exaggeration to say that the public is often impatient and dissatisfied with the actions of the authorities in relation to animal cruelty. In our research, based on the data of the Criminal Investigation Department of the National Police Headquarters, we examined the opinions of 99 out of a total of 155 police stations in Hungary whose staff currently working there had experience in dealing with animal cruelty. The investigators gave their opinion on a total of 1169 cases in which some kind of police action was taken, either following a report to the police or as a result of their own investigative actions. In another survey, we questioned those members of society who are most committed to animal protection using a self-completion questionnaire. The questionnaire sent to the 116 Hungarian animal welfare non-governmental organisations (NGOs) on the publicly available lists was also posted for a short period on the social networking site of NGO activists. Among those who responded, a total of 150 identified as active participants in the animal protection work of these NGOs. The picture of the police treatment of animal cruelty, as perceived by NGOs working in the field of animal protection, is significantly less favourable than suggested by the police data. According to the official data, 77.7% of reports initiated an investigation, while the vast majority of animal welfare activists (81.3%) suspects that only 25% of the reports result in action by the prosecuting authority.

Annual and seasonal trends in cow's milk quality determined by FT-MIR spectroscopy in Hungary between 2011 and 2020.

We analysed and monitored the major chemical composition of cow's bulk milk by Fourier transform mid-infrared (FT-MIR) spectroscopy over a 10-year period in the whole territory of Hungary. In addition, the two most important key parameters for milk quality assessment, total bacterial count (TBC) and somatic cell count (SCC) were also followed. Production parameters showed significant seasonal and yearly changes. The overall mean fat, protein, lactose and solids-non-fat (SNF) contents of cow's milk were 3.81%, 3.32%, 4.74% and 8.76%, respectively. A circannual variation was observed in the chemical composition and yield of milk components of samples examined between 2011 and 2020. Concerning milk fat, milk protein and SNF, the values were the lowest in summer and the highest in winter. In the case of lactose, the minimum values were measured in autumn and the maximum values in spring. An obvious trend of long-term elevation of lactose and SNF was found in the raw cow milk samples over the observed period. The overall mean TBC and SCC of cow's milk were 52 × 103 CFU ml-1 and 270 × 103 cells/ml, respectively. Although there were differences in the monthly average values, no seasonal cyclicality was observed.

AgRP neurons control structure and function of the medial prefrontal cortex.

Hypothalamic agouti-related peptide and neuropeptide Y-expressing (AgRP) neurons have a critical role in both feeding and non-feeding behaviors of newborn, adolescent, and adult mice, suggesting their broad modulatory impact on brain functions. Here we show that constitutive impairment of AgRP neurons or their peripubertal chemogenetic inhibition resulted in both a numerical and functional reduction of neurons in the medial prefrontal cortex (mPFC) of mice. These changes were accompanied by alteration of oscillatory network activity in mPFC, impaired sensorimotor gating, and altered ambulatory behavior that could be reversed by the administration of clozapine, a non-selective dopamine receptor antagonist. The observed AgRP effects are transduced to mPFC in part via dopaminergic neurons in the ventral tegmental area and may also be conveyed by medial thalamic neurons. Our results unmasked a previously unsuspected role for hypothalamic AgRP neurons in control of neuronal pathways that regulate higher-order brain functions during development and in adulthood.

Changes in the Dentition of Small Dogs up to 4 Months of Age.

It is common practice in EU member states to permit the entry of dogs vaccinated against rabies at the age of at least 3 months. In the absence of easily applicable comparative data, subjective disputes emerge around age. The aim of our study was to observe the development of dog teeth. During birth, an abnormally lying Yorkshire Terrier fetus was stuck in the birth canal, which led to a caesarean section, hence, the exact date of birth was known. For the next 4 months, two puppies were examined weekly, and they showed the same development. The dogs were born without teeth. At the age of 4.5 weeks, the canines I appeared, together with the adjacent incisors (i3), and the second incisor (i2) also erupted at the age of 6 weeks. A week later, a first incisor (i1) also appeared. From the age of 2.5 months, the distance between the teeth was increasing, especially on the upper dental arch. At 3.5 months of age, only the bottom front incisors (i1) had not grown in a row, and the significant distance between the top incisors, comparable to the width of the tooth, was striking. Since only two dogs of one breed were involved in this case study, the observations cannot be generalized.

AgRP neurons control feeding behaviour at cortical synapses via peripherally derived lysophospholipids.

Phospholipid levels are influenced by peripheral metabolism. Within the central nervous system, synaptic phospholipids regulate glutamatergic transmission and cortical excitability. Whether changes in peripheral metabolism affect brain lipid levels and cortical excitability remains unknown. Here, we show that levels of lysophosphatidic acid (LPA) species in the blood and cerebrospinal fluid are elevated after overnight fasting and lead to higher cortical excitability. LPA-related cortical excitability increases fasting-induced hyperphagia, and is decreased following inhibition of LPA synthesis. Mice expressing a human mutation (Prg-1R346T) leading to higher synaptic lipid-mediated cortical excitability display increased fasting-induced hyperphagia. Accordingly, human subjects with this mutation have higher body mass index and prevalence of type 2 diabetes. We further show that the effects of LPA following fasting are under the control of hypothalamic agouti-related peptide (AgRP) neurons. Depletion of AgRP-expressing cells in adult mice decreases fasting-induced elevation of circulating LPAs, as well as cortical excitability, while blunting hyperphagia. These findings reveal a direct influence of circulating LPAs under the control of hypothalamic AgRP neurons on cortical excitability, unmasking an alternative non-neuronal route by which the hypothalamus can exert a robust impact on the cortex and thereby affect food intake.

Effects of Bacillus licheniformis and Bacillus subtilis on Gut Barrier Function, Proinflammatory Response, ROS Production and Pathogen Inhibition Properties in IPEC-J2-Escherichia coli/Salmonella Typhimurium Co-Culture.

The emergence of antimicrobial resistance raises serious concerns worldwide. Probiotics offer a promising alternative to enhance growth promotion in farm animals; however, their mode of action still needs to be elucidated. The IPEC-J2 cell line (porcine intestinal epithelial cells) is an appropriate tool to study the effect of probiotics on intestinal epithelial cells. In our experiments, IPEC-J2 cells were challenged by two gastrointestinal (GI) infection causing agents, Escherichia coli (E. coli) or Salmonella enterica ser. Typhimurium (S. Typhimurium). We focused on determining the effect of pre-, co-, and post-treatment with two probiotic candidates, Bacillus licheniformis or Bacillus subtilis, on the barrier function, proinflammatory cytokine (IL-6 and IL-8) response, and intracellular reactive oxygen species (ROS) production of IPEC-J2 cells, in addition to the adhesion inhibition effect. Bacillus licheniformis (B. licheniformis) and Bacillus subtilis (B. subtilis) proved to be anti-inflammatory and had an antioxidant effect under certain treatment combinations, and further effectively inhibited the adhesion of pathogenic bacteria. Interestingly, they had little effect on paracellular permeability. Based on our results, Bacillus licheniformis and Bacillus subtilis are both promising candidates to contribute to the beneficial effects of probiotic multispecies mixtures.

Effect of Enterococcus faecium NCIMB 10415 on Gut Barrier Function, Internal Redox State, Proinflammatory Response and Pathogen Inhibition Properties in Porcine Intestinal Epithelial Cells.

In farm animals, intestinal diseases caused by Salmonella spp. and Escherichia coli may lead to significant economic loss. In the past few decades, the swine industry has largely relied on the prophylactic use of antibiotics to control gastrointestinal diseases. The development of antibiotic resistance has become an important issue both in animal and human health. The use of antibiotics for prophylactic purposes has been banned, moreover the new EU regulations further restrict the application of antibiotics in veterinary use. The swine industry seeks alternatives that are capable of maintaining the health of the gastrointestinal tract. Probiotics offer a promising alternative; however, their mode of action is not fully understood. In our experiments, porcine intestinal epithelial cells (IPEC-J2 cells) were challenged by Salmonella Typhimurium or Escherichia coli and we aimed at determining the effect of pre-, co-, and post-treatment with Enterococcus faecium NCIMB 10415 on the internal redox state, paracellular permeability, IL-6 and IL-8 secretion of IPEC-J2 cells. Moreover, the adhesion inhibition effect was also investigated. Enterococcus faecium was able to reduce oxidative stress and paracellular permeability of IPEC-J2 cells and could inhibit the adhesion of Salmonella Typhimurium and Escherichia coli. Based on our results, Enterococcus faecium is a promising candidate to maintain the health of the gastrointestinal tract.

Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca2+ homeostasis with adipose tissue lipolysis.

Appropriate cristae remodeling is a determinant of mitochondrial function and bioenergetics and thus represents a crucial process for cellular metabolic adaptations. Here, we show that mitochondrial cristae architecture and expression of the master cristae-remodeling protein OPA1 in proopiomelanocortin (POMC) neurons, which are key metabolic sensors implicated in energy balance control, is affected by fluctuations in nutrient availability. Genetic inactivation of OPA1 in POMC neurons causes dramatic alterations in cristae topology, mitochondrial Ca2+ handling, reduction in alpha-melanocyte stimulating hormone (α-MSH) in target areas, hyperphagia, and attenuated white adipose tissue (WAT) lipolysis resulting in obesity. Pharmacological blockade of mitochondrial Ca2+ influx restores α-MSH and the lipolytic program, while improving the metabolic defects of mutant mice. Chemogenetic manipulation of POMC neurons confirms a role in lipolysis control. Our results unveil a novel axis that connects OPA1 in POMC neurons with mitochondrial cristae, Ca2+ homeostasis, and WAT lipolysis in the regulation of energy balance.

Action potential-coupled Rho GTPase signaling drives presynaptic plasticity.

In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with synaptic vesicle membranes in adult mice. Using three independent approaches to alter presynaptic Rac1 activity (genetic knockout, spatially restricted inhibition, and temporal optogenetic manipulation), we discover that this pathway negatively regulates synaptic vesicle replenishment at both excitatory and inhibitory synapses, bidirectionally sculpting short-term synaptic depression. Finally, we use two-photon fluorescence lifetime imaging to show that presynaptic Rac1 activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across excitatory and inhibitory terminals. It also provides a new proteomic framework for better understanding presynaptic physiology, along with a blueprint of experimental strategies to isolate the presynaptic effects of ubiquitously expressed proteins.

Dietary spermidine improves cognitive function.

Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.

Distribution of cortactin in cerebellar Purkinje cell spines.

Dendritic spines are the primary sites of excitatory transmission in the mammalian brain. Spines of cerebellar Purkinje Cells (PCs) are plastic, but they differ from forebrain spines in a number of important respects, and the mechanisms of spine plasticity differ between forebrain and cerebellum. Our previous studies indicate that in hippocampal spines cortactin-a protein that stabilizes actin branch points-resides in the spine core, avoiding the spine shell. To see whether the distribution of cortactin differs in PC spines, we examined its subcellular organization using quantitative preembedding immunoelectron microscopy. We found that cortactin was enriched in the spine shell, associated with the non-synaptic membrane, and was also situated within the postsynaptic density (PSD). This previously unrecognized distribution of cortactin within PC spines may underlie structural and functional differences in excitatory spine synapses between forebrain, and cerebellum.

Perisomatic Inhibition and Its Relation to Epilepsy and to Synchrony Generation in the Human Neocortex.

Inhibitory neurons innervating the perisomatic region of cortical excitatory principal cells are known to control the emergence of several physiological and pathological synchronous events, including epileptic interictal spikes. In humans, little is known about their role in synchrony generation, although their changes in epilepsy have been thoroughly investigated. This paper demonstraits how parvalbumin (PV)- and type 1 cannabinoid receptor (CB1R)-positive perisomatic interneurons innervate pyramidal cell bodies, and their role in synchronous population events spontaneously emerging in the human epileptic and non-epileptic neocortex, in vitro. Quantitative electron microscopy showed that the overall, PV+ and CB1R+ somatic inhibitory inputs remained unchanged in focal cortical epilepsy. On the contrary, the size of PV-stained synapses increased, and their number decreased in epileptic samples, in synchrony generating regions. Pharmacology demonstrated-in conjunction with the electron microscopy-that although both perisomatic cell types participate, PV+ cells have stronger influence on the generation of population activity in epileptic samples. The somatic inhibitory input of neocortical pyramidal cells remained almost intact in epilepsy, but the larger and consequently more efficient somatic synapses might account for a higher synchrony in this neuron population. This, together with epileptic hyperexcitability, might make a cortical region predisposed to generate or participate in hypersynchronous events.

Spermidine protects from age-related synaptic alterations at hippocampal mossy fiber-CA3 synapses.

Aging is associated with functional alterations of synapses thought to contribute to age-dependent memory impairment (AMI). While therapeutic avenues to protect from AMI are largely elusive, supplementation of spermidine, a polyamine normally declining with age, has been shown to restore defective proteostasis and to protect from AMI in Drosophila. Here we demonstrate that dietary spermidine protects from age-related synaptic alterations at hippocampal mossy fiber (MF)-CA3 synapses and prevents the aging-induced loss of neuronal mitochondria. Dietary spermidine rescued age-dependent decreases in synaptic vesicle density and largely restored defective presynaptic MF-CA3 long-term potentiation (LTP) at MF-CA3 synapses (MF-CA3) in aged animals. In contrast, spermidine failed to protect CA3-CA1 hippocampal synapses characterized by postsynaptic LTP from age-related changes in function and morphology. Our data demonstrate that dietary spermidine attenuates age-associated deterioration of MF-CA3 synaptic transmission and plasticity. These findings provide a physiological and molecular basis for the future therapeutic usage of spermidine.

Reduced Prefrontal Synaptic Connectivity and Disturbed Oscillatory Population Dynamics in the CNTNAP2 Model of Autism.

Loss-of-function mutations in CNTNAP2 cause a syndromic form of autism spectrum disorder in humans and produce social deficits, repetitive behaviors, and seizures in mice. However, the functional effects of these mutations at cellular and circuit levels remain elusive. Using laser-scanning photostimulation, whole-cell recordings, and electron microscopy, we found a dramatic decrease in excitatory and inhibitory synaptic inputs onto L2/3 pyramidal neurons of the medial prefrontal cortex (mPFC) of Cntnap2 knockout (KO) mice, concurrent with reduced spines and synapses, despite normal dendritic complexity and intrinsic excitability. Moreover, recording of mPFC local field potentials (LFPs) and unit spiking in vivo revealed increased activity in inhibitory neurons, reduced phase-locking to delta and theta oscillations, and delayed phase preference during locomotion. Excitatory neurons showed similar phase modulation changes at delta frequencies. Finally, pairwise correlations increased during immobility in KO mice. Thus, reduced synaptic inputs can yield perturbed temporal coordination of neuronal firing in cortical ensembles.

Quantitative Electron Microscopic Assay Using Random Sampling from Single Sections to Test Plastic Synaptic Changes in Hippocampus.

Studies over several decades on the organization of the CA1 hippocampus-a particularly favorable model for learning, memory and certain forms of cognition-have shown that the synaptic network in this brain region is plastic ( Fortin et al., 2012 ). Recent evidence suggests that a number of environmental and endogenous stimuli may have a substantial effect on hippocampus-dependent cognitive function, implying enhanced synaptic plasticity in this brain region. Stimuli (e.g., food restriction, enriched environment, social interaction, gene-loss [knock-out animals], etc.) can trigger structural and functional plasticity (e.g., spine formation, increased expression of neurotrophic factors, synaptic function and neurogenesis) in the hippocampus ( Stewart et al., 1989 ; Andrade et al., 2002 ; Babits et al., 2016 ). Using quantitative electron microscopy, we can study the synaptic neuropil of CA1 hippocampus in rodents during short- or long-term treatments and/or stimuli. Within the scope of this electron microscopic methodological construct, the density of various synaptic connections, the morphology and internal structure of excitatory spine synapses (e.g., the mean length and width of postsynaptic densities) can be quantified. Such quantitative ultrastructural measurement using high-resolution electron-microscopy may be applied to observe structural manifestations of synaptic plasticity in rodent brain tissue. The presented ultrastructural protocol may empower researchers to reveal details and synaptic changes which may not be obvious using only light microscopy. Ultrastructural data may provide substantial advances in our understanding of the changes in hippocampal synaptic architecture under different conditions.

Protein kinase D exerts neuroprotective functions during oxidative stress via nuclear factor kappa B-independent signaling pathways.

Members of the protein kinase D (PKD) family of serine/threonine kinases are known to exert diverse roles in neuronal stress responses. Here, we show the transient activation and nuclear translocation of endogenous PKD upon oxidative stress induced by H2 O2 treatment in primary neuronal cultures. Using pharmacological inhibition, we show that PKD activity protects neurons from oxidative stress-induced cell death. Although members of the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF kappaB) pathway were phosphorylated upon H2 O2 treatment, it was found that the neuronal response to oxidative stress is not executed through the nuclear translocation and activity of RelA. On the other hand, we demonstrate for the first time in neuronal cells, the association of green fluorescent protein-tagged kinase inactive PKD1 with mitochondrial membranes in vivo and the presence of PKD activity in the close vicinity of mitochondria in vitro. Our findings thus support the notion that the neuroprotective role of PKD is exerted independently from NF kappaB signaling and suggest a potential mitochondrial function for PKD in cultured neurons.

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.