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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.

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.

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.

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.

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

Summary 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., Graphical abstract, Highlights • Nutritional state shapes mitochondrial cristae and OPA1 expression in POMC neurons • OPA1 deletion in POMC neurons alters mitochondrial Ca2+ handling and α-MSH release • POMC OPA1-deficient mice show impaired fat lipolysis and metabolic health • Pharmacological restoration of Ca2+ recovered molecular and metabolic alterations, Gómez-Valadés et al. report that mitochondrial cristae dynamically reshape in POMC neurons in response to nutritional state. With genetic targeting, they reveal that the cristae-remodeling protein OPA1 is essential to sustain cristae structure and mitochondrial Ca2+ homeostasis in POMC neurons, ensuring appropriate modulation of α-MSH release, the lipolytic program in adipose tissue and metabolic health.

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.

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.

Summary: 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. : Lazaro et al. demonstrate a decrease in synaptic inputs onto mPFC L2/3 pyramidal neurons of Cntnap2 KO mice, concurrent with reduced spines and synapses. These lead to perturbed network activity, with mPFC cells exhibiting reduced phase locking and altered preferred phases to slow LFP oscillations, which may underlie autism-related phenotypes. Keywords: oscillation, delta, theta, phase-locking, inhibition, brain state, connectivity, functional, biomarker, EEG

Loss of function mutations in CNTNAP2 cause a syndromic form of autism spectrum disorder (ASD) in humans and produce social deficits, repetitive behaviors, and seizures in mice. Yet, the functional effects of these mutations at the cellular and circuit level remain elusive. Using laser scanning photostimulation, whole-cell recordings, and electron microscopy, we found a dramatic decrease in functional excitatory and inhibitory synaptic inputs in L2/3 medial prefrontal cortex (mPFC) of Cntnap2 knock-out (KO) mice. In accordance with decreased synaptic input, KO mice displayed reduced spine and synapse densities, despite normal intrinsic excitability and dendritic complexity. To determine how this decrease in synaptic inputs alters coordination of neuronal firing patterns in vivo, we recorded mPFC local field potentials (LFP) and unit spiking in head-fixed mice during locomotion and rest. In KO mice, LFP power was not significantly altered at all tested frequencies, but inhibitory neurons showed delayed phase-firing and reduced phase-locking to delta and theta oscillations during locomotion. Excitatory neurons showed similar changes but only to delta oscillations. These findings suggest that profound ASD-related alterations in synaptic inputs can yield perturbed temporal coordination of cortical ensembles.

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.

Consumption of high-energy diets may compromise health and may also impair cognition; these impairments have been linked to tasks that require hippocampal function. Conversely, food restriction has been shown to improve certain aspects of hippocampal function, including spatial memory and memory persistence. These diet-dependent functional changes raise the possibility that the synaptic structure underlying hippocampal function is also affected. To examine how short-term food restriction (FR) alters the synaptic structure of the hippocampus, we used quantitative electron microscopy to analyze the organization of neuropil in the CA1 stratum radiatum of the hippocampus in young rats, consequent to reduced food. While four weeks of FR did not modify the density, size, or shape of postsynaptic spines, the synapses established by these spines were altered, displaying increased mean length, and more frequent perforations of postsynaptic densities. That the number of perforated synapses (believed to be an indicator of synaptic enhancement) increased, and that the CA1 spine population had on average significantly longer PSDs suggests that synaptic efficacy of axospinous synapses also increased in the CA1. Taken together, our ultrastructural data reveal previously unrecognized structural changes at hippocampal synapses as a function of food restriction, supporting a link between metabolic balance and synaptic plasticity.

PKD regulates the stabilization of the F-actin network within dendritic spines upon chemically induced plasticity changes and is needed for proper hippocampal LTP and spatial memory formation., Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.

Despite evidence for a strong genetic contribution to several major psychiatric disorders, individual candidate genes account for only a small fraction of these disorders, leading to the suggestion that multigenetic pathways may be involved. Several known genetic risk factors for psychiatric disease are related to the regulation of actin polymerization, which plays a key role in synaptic plasticity. To gain insight into and test the possible pathogenetic role of this pathway, we designed a conditional knock-out of the Arp2/3 complex, a conserved final output for actin signaling pathways that orchestratesde novoactin polymerization. Here we report that postnatal loss of the Arp2/3 subunit ArpC3 in forebrain excitatory neurons leads to an asymmetric structural plasticity of dendritic spines, followed by a progressive loss of spine synapses. This progression of synaptic deficits corresponds with an evolution of distinct cognitive, psychomotor, and social disturbances as the mice age. Together, these results point to the dysfunction of actin signaling, specifically that which converges to regulate Arp2/3, as an important cellular pathway that may contribute to the etiology of complex psychiatric disorders.

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 H2O2 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 NFκB pathway were phosphorylated upon H2O2 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 GFP-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 NFκB signaling and suggest a potential mitochondrial function for PKD in cultured neurons. This article is protected by copyright. All rights reserved.

Glutamatergic axons in the mammalian forebrain terminate predominantly onto dendritic spines. Long-term changes in the efficacy of these excitatory synapses are tightly coupled to changes in spine morphology. The reorganization of the actin cytoskeleton underlying this spine "morphing" involves numerous proteins that provide the machinery needed for adaptive cytoskeletal remodeling. Here, we review recent literature addressing the chemical architecture of the spine, focusing mainly on actin-binding proteins (ABPs). Accumulating evidence suggests that ABPs are organized into functionally distinct microdomains within the spine cytoplasm. This functional compartmentalization provides a structural basis for regulation of the spinoskeleton, offering a novel window into mechanisms underlying synaptic plasticity.

Changes in the morphology of a dendritic spine require remodeling of its actin-based cytoskeleton. Biochemical mechanisms underlying actin remodeling have been studied extensively, but little is known about the physical organization of the actin-binding proteins that mediate remodeling in spines. Long-term potentiation-inducing stimuli trigger expansion of the spine head, suggesting local extension and branching of actin filaments. Because filament branching requires the Arp2/3 complex, we used quantitative immunoelectron microscopy to elucidate the organization of ARPC-2 (Arp2/3 complex subunit 2), an essential component of the complex. Our data from CA1 hippocampus indicate that Arp2/3 concentrates within spines in a previously unrecognized torroidal domain, apparently specialized to mediate actin filament branching.

Consumption of high-energy diets may compromise health and may also impair cognition; these impairments have been linked to tasks that require hippocampal function. Conversely, food restriction has been shown to improve certain aspects of hippocampal function, including spatial memory and memory persistence. These diet-dependent functional changes raise the possibility that the synaptic structure underlying hippocampal function is also affected. To examine how short-term food restriction (FR) alters the synaptic structure of the hippocampus, we used quantitative electron microscopy to analyze the organization of neuropil in the CA1 stratum radiatum of the hippocampus in young rats, consequent to reduced food. While four weeks of FR did not modify the density, size, or shape of postsynaptic spines, the synapses established by these spines were altered, displaying increased mean length, and more frequent perforations of postsynaptic densities. That the number of perforated synapses (believed to be an indicator of synaptic enhancement) increased, and that the CA1 spine population had on average significantly longer PSDs suggests that synaptic efficacy of axospinous synapses also increased in the CA1. Taken together, our ultrastructural data reveal previously unrecognized structural changes at hippocampal synapses as a function of food restriction, supporting a link between metabolic balance and synaptic plasticity.

SummaryProper growth of dendrites is critical to the formation of neuronal circuits, but the cellular machinery that directs the addition of membrane components to generate dendritic architecture remains obscure. Here, we demonstrate that post-Golgi membrane trafficking is polarized toward longer dendrites of hippocampal pyramidal neurons in vitro and toward apical dendrites in vivo. Small Golgi outposts partition selectively into longer dendrites and are excluded from axons. In dendrites, Golgi outposts concentrate at branchpoints where they engage in post-Golgi trafficking. Within the cell body, the Golgi apparatus orients toward the longest dendrite, and this Golgi polarity precedes asymmetric dendrite growth. Manipulations that selectively block post-Golgi trafficking halt dendrite growth in developing neurons and cause a shrinkage of dendrites in mature pyramidal neurons. Further, disruption of Golgi polarity produces neurons with symmetric dendritic arbors lacking a single longest principal dendrite. These results define a novel polarized organization of neuronal secretory trafficking and demonstrate a mechanistic link between directed membrane trafficking and asymmetric dendrite growth.

Pre-embedding light microscopic immunocytochemistry, using a monoclonal antibody (mAb-KA8) raised against a frog brain kappa receptor preparation, recognising selectively the kappa-opioid receptor, was used for studying the occurrence, distribution, and species-specificity of the kappa-opioid receptor in the hippocampal formation of four rodent species (rat, guinea pig, hamster and gerbil). MAb-KA8 immunoreactivity was detectable in the rat, hamster and gerbil hippocampus, however the distribution of the labelled structures was heterogeneous. In the rat and hamster the hilus of dentate gyrus and the stratum oriens of the CA1 area contained immunoreactive cell bodies and proximal dendrites. In the gerbil mAb-KA8 immunopositive cell bodies were recognisable in the stratum radiatum of the CA1 and CA3 areas and in the subiculum. In the hamster varicose axon-like elements were also detected in the CA3 pyramidal layer. With the mAb-KA8 antibody there was no detectable kappa opioid receptor labelling in the hippocampus of the guinea pig. The results confirm the high degree of species-specific heterogeneity characterising the distribution of opioid peptides and their receptors in the hippocampal formation. The receptor was found in most cases postsynaptically, however in the hamster the immunopositive axons may refer to a presynaptic localisation.

Psychiatric and neurodevelopmental disorders may arise from anomalies in long-range neuronal connectivity downstream of pathologies in dendritic spines. However, the mechanisms that may link spine pathology to circuit abnormalities relevant to atypical behavior remain unknown. Using a mouse model to conditionally disrupt a critical regulator of the dendritic spine cytoskeleton, the actin-related protein 2/3 complex (Arp2/3), we report here a molecular mechanism that unexpectedly reveals the inter-relationship of progressive spine pruning, elevated frontal cortical excitation of pyramidal neurons and striatal hyperdopaminergia in a cortical-to-midbrain circuit abnormality. The main symptomatic manifestations of this circuit abnormality are psychomotor agitation and stereotypical behaviors, which are relieved by antipsychotics. Moreover, this antipsychotic-responsive locomotion can be mimicked in wild-type mice by optogenetic activation of this circuit. Collectively these results reveal molecular and neural-circuit mechanisms, illustrating how diverse pathologies may converge to drive behaviors relevant to psychiatric disorders.

Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists. Here, we investigated whether a complex of septin cytoskeletal GTPases localized at the base of the spine neck regulates diffusion across the spine neck. We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane. We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck. Finally, when Sept7 expression was suppressed by RNA interference, membrane molecules explored larger membrane areas. Our findings indicate that Sept7 regulates membrane protein access to spines., Human Frontier Science Program (Strasbourg, France) (Short-Term Fellowship)

A local GABA-system is known to have a mediatory function between several afferents and the principal cells of the hippocampus. This study examines the distribution and fine structure of kappa opioid receptor-immunoreactive elements in the CA1 subfield and reveals some new aspects concerning the structural basis of opioid-GABA interaction in the rat hippocampal formation. Kappa receptors were visualized immunocytochemically with a previously produced and characterized monoclonal antibody, the mAb KA8 (Maderspach, K., Nemeth, K., Simon, J., Benyhe, S., Szucs, M., Wollemann, M., 1991. A monoclonal antibody recognizing kappa-, but not mu- and delta-opioid receptors. J. Neurochem. 56, 1897–1904). The antibody selectively recognizes the kappa opioid receptor with preference to the kappa 2 subtype. Neuronal cell bodies, proximal dendrites and occasionally glial processes surrounding neuronal perikarya were labelled in the CA1 area. The immunopositive cells were present mainly in the stratum oriens, followed by the stratum pyramidale in a rostrocaudally increasing number. Their shape was fusiform, or multipolar. Occasionally kappa receptor-immunoreactive boutons surrounding weakly immunopositive somata were also observed. Electron microscopy of immunopositive neurons showed that the DAB labelling was intensive in the perinuclear cytoplasm. The widths and electron densities of the postsynaptic densities of some axosomatic synapses were remarkably increased. Similar increase of postsynaptic densities were observable at some axodendritic and axospinous synapses. On the basis of their location and fine structural properties the labelled cells are suggested to be GABAergic inhibitory interneurons, probably belonging to the somatostatinergic sub-population. The axons of these inhibitory interneurons are known to arborize in the stratum lacunosum-moleculare where the entorhinal afferents terminate. A modulatory effect of opioids on the entorhinal input, mediated by somatostatinergic interneurons is suggested

USING correlated light and electron microscopic pre-embedding enkephalin immunocytochemistry combined with post-embedding GABA immunogold staining, we found morphological evidence of a direct connection between the enkephalinergic and GABAergic systems in the rat hippocampus. Enkephalin-immunoreactive boutons were found to be presynaptic to GABA-immunoreactive postsynaptic profiles, establishing type 2 symmetrical synapses on GABA-positive cell bodies and dendritic shafts in strata radiatum and lacunosum moleculare of the CA1 region. Thirty-six percent of all studied postsynaptic targets (n = 40) were non-pyramidal, including all somatic (n = 7) and 47% of the dendritic (n = 13) postsynaptic targets. The remaining 64% consisted of pyramidal dendritic shafts and spines. These results support previous physiological experiments suggesting that the opioidergic system takes part in disinhibitory processes in the hippocampal formation.

By conveying signals from the small GTPase family of proteins to the Arp2/3 complex, proteins of the WAVE family facilitate actin remodeling. The WAVE-1 isoform is expressed at high levels in brain, where it plays a role in normal synaptic processing, and is implicated in hippocampus-dependent memory retention. We used electron microscopy to determine whether synaptic structure is modified in the hippocampus of WAVE-1 knockout mice, focusing on the neuropil of CA1 stratum radiatum. Mice lacking WAVE-1 exhibited alterations in the morphology of both axon terminals and dendritic spines; the relationship between the synaptic partners was also modified. The abnormal synaptic morphology we observed suggests that signaling through WAVE-1 plays a critical role in establishing normal synaptic architecture in the rodent hippocampus.

The metabolic state has long been shown to affect reproduction. Peripheral signals and hormones from the reproductive organs are also known to regulate energy metabolism and feeding and energy expenditure. Much attention has been paid to determine the signaling flow from key hypothalamic neuronal populations, including those producing the anorexigenic proopiomelanocortin (POMC) derivate, α-melanocyte stimulating hormone (α-MSH), to the medial preoptic area gonadotropin-releasing hormone (GnRH) neurons, cells that are the drivers of ovulation and reproduction in general. In this study, the authors explored whether a reverse signaling modality may also exist. Specifically, the authors analyzed GnRH efferents in the arcuate nucleus with particular emphasis on their anatomical proximity to arcuate nucleus melanocortin perikarya. Using correlated light and electron microscopy, the authors observed direct apposition between GnRH-containing axon terminals and POMC cell bodies. These data provide the first experimental evidence to suggest that GnRH may have a direct influence on feeding, energy expenditure, and glucose homeostasis, independent of the activity of the gonadal axis.

Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin assembly adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission and plasticity. However, the molecular mechanisms that situate the EZ near the PSD, and the role of local spine endocytosis in synaptic transmission, are unknown. Here we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses devoid of postsynaptic clathrin. This loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.

The effect of 40% food deprivation for 1 week on the immunohistochemically detectable amount of neuropeptide Y (NPY) was studied in the lateral septum (LS) of intact and ovariectomized (OVX) female rats. Animals were either fed ad libitum or 40% food-deprived. Densitometric analysis of immunostained material showed a significant decrease in NPY-immunoreactivity (NPY-IR) in OVX rats compared to the control group. Food deprivation increased the density of punctate NPY-IR profiles in both intact and OVX animals, however, the density in food-deprived OVX animals was increased compared to baseline but remained reduced compared to intact rats. Our study indicates that the lack of gonadal hormones - most likely estrogen - results in a decrease in the density of NPY-IR axonal fibers within the LS, while food deprivation induced considerable elevation in NPY density. Food restriction-induced changes in the density of NPY-containing neural elements suggest that the LS may play a crucial role in the regulation of food intake and energy balance, in concert with the relevant hypothalamic areas.

Synaptic plasticity is associated with morphological changes in dendritic spines. The actin-based cytoskeleton plays a key role in regulating spine structure, and actin reorganization in spines is critical for the maintenance of long term potentiation. To test the hypothesis that a stable pool of F-actin rests in the spine "core," while a dynamic pool lies peripherally in its "shell," we performed immunoelectron microscopy in the stratum radiatum of rat hippocampus to elucidate the subcellular distribution of cofilin, an actin-depolymerizing protein that mediates reorganization of the actin cytoskeleton. We provide direct evidence that cofilin in spines avoids the core, and instead concentrates in the shell and within the postsynaptic density. These data suggest that cofilin may link synaptic plasticity to the actin remodeling that underlies changes in spine morphology.

The small GTPases Rac1 and Cdc42 are key regulators of the morphogenesis of actin-rich dendritic spines in neurons. However, little is known about how activated Rac1/Cdc42 regulates dendritic spines. Insulin receptor substrate 53 (IRSp53), which is highly expressed in the postsynaptic density (PSD), is known to link activated Rac1/Cdc42 to downstream effectors for actin regulation in non-neural cells. Here, we report that IRSp53 interacts with two specific members of the PSD-95 family, PSD-95 and chapsyn-110/PSD-93, in brain. An IRSp53 mutant lacking the C-terminal PSD-95-binding motif shows significant loss of synaptic localization in cultured neurons. Overexpression of IRSp53 in cultured neurons increases the density of dendritic spines but does not affect their length or width. Conversely, short-interfering RNA-mediated knock-down of IRSp53 reduces the density, length, and width of spines. In addition, the density and size of spines are decreased by a dominant-negative IRSp53 with a point mutation in the Src homology 3 (SH3) domain and a dominant-negative proline-rich region of WAVE2 (Wiskott-Aldrich syndrome protein family Verprolin-homologous protein), a downstream effector of IRSp53 that binds to the SH3 domain of IRSp53. These results suggest that PSD-95 interaction is an important determinant of synaptic IRSp53 localization and that the SH3 domain of IRSp53 links activated Rac1/Cdc42 to downstream effectors for the regulation of spine morphogenesis.

Spines may undergo rapid, activity-dependent changes in shape and size, reflecting reorganization of the actin cytoskeleton. This remodeling is implicated in development and also in the late phase of long-term potentiation. However, the cellular mechanisms that convert activity into morphological change remain poorly understood, and little is known about the anatomical distribution of the actin-regulating proteins that mediate this remodeling. Using immunocytochemistry, we demonstrate here that cortactin (a protein implicated in actin filament nucleation, branching, and stabilization) is concentrated in hippocampal spines, where it colocalizes with F-actin. Cortactin has a Shank-binding domain; recent studies report that synaptic activity may trigger actin remodeling via this interaction with Shank. However, our immunogold electron microscopic data show that cortactin concentrates within the spine core, 100-150 nm away from the postsynaptic density (PSD); only a small fraction of the cortactin in spines lies adjacent to the PSD. These data suggest that the adult dendritic spine contains two functional pools of cortactin: a large pool in the spine core that may help to mediates changes in spine shape and a small synaptic pool that may modify the PSD in response to synaptic activity.

In our previous studies (J. Chem. Neuroanat. 2000;19:233-241), kappa opioid receptors were immunocytochemically identified in inhibitory interneurons of the dentate hilus and CA1 area of the rat hippocampus. From among the known interneuron subtypes, somatostatin- (SOM) and neuropeptide Y- (NPY) immunoreactive (IR) hippocampal interneurons show morphology and distribution similar to the kappa opioid receptor (KOR) immunopositive cells. In the present study, with the help of double immunocytochemical labelling, we provide direct evidence that the majority of the interneurons immunoreactive for SOM and/or NPY also express the kappa opioid receptor. The receptor was localized on the perikaryal and proximal dendritic region of the SOM- and NPY-immunopositive neurons in the dentate hilus and the CA1 region. From among the SOM-immunoreactive cells, 77% in the dentate hilus and 51% in the CA1 stratum oriens was double labelled. In the case of NPY-immunoreactive neurons this proportion was 56 and 65%, respectively. The co-expression of KOR and SOM/NPY suggests that hippocampal interneurons can selectively be activated by the different opioids under different physiological circumstances.

Postsynaptic membrane trafficking plays an important role in synaptic plasticity, but the organization of trafficking machinery within dendritic spines is poorly understood. We use immunocytochemical analysis of rat hippocampal neurons to show that proteins mediating endocytosis are systematically arrayed within dendritic spines, tangential to the synapse. Thus, previously unrecognized lateral domains of the spine organize endocytic protein machinery at sites removed from the postsynaptic density.