Effect of Combined High-Pressure Homogenization and Biotechnological Processes on Chitin, Protein, and Antioxidant Activity of Cricket Powder-Based Ingredients.

The main objective of this work was to evaluate the combined effect of a biotechnology process, based on selected yeast strains, and a high-pressure homogenization (HPH) treatment on the microbiological quality, structural organization of proteins, chitin content, and antioxidant activity of a mixture of cricket powder (Acheta domesticus) and water. Compared to untreated samples, the cricket matrix treated with HPH four times at 180 MPa promoted the growth of the inoculated Yarrowia lipolytica and Debaryomyces hansenii strains. HPH did not affect the concentration of chitin; however, the combination with microorganisms tended to reduce the content. Although the antioxidant activity increased from 0.52 to 0.68 TAC mM/TE after a 48 h incubation in the control, it was further improved by the combination of HPH and D. hansenii metabolism, reaching a value of 0.77 TAC mM/TE. The combination of the two approaches also promoted a reduction in the intensity of bands with molecular weights between 31 and 21.5 kDa in favor of bands with a lower molecular weight. In addition, HPH treatment reduced the number of accessible thiols, suggesting protein structure changes that may further impact the technological properties of cricket powder.

Quality evaluation of house cricket flour processed by electrohydrodynamic drying and pulsed electric fields treatment.

House crickets are expected to play a significant role in the future food sector. Electrohydrodynamic (EHD) drying offers an environmentally friendly alternative to conventional drying methods. Pulsed electric fields (PEF) is a non-thermal process that facilitates conventional processes. EHD was applied to house crickets with and without PEF pretreatment, and the effect of PEF and EHD on the quality of the insects was evaluated. PEF pretreatment positively affected the oven drying at 60 °C by reducing its duration and thus decreasing the energy consumption by 14.22%. Moisture removal of EHD was not sufficient to replace oven drying, but when combined with oven drying, the overall energy consumption was reduced by >50%. PEF processing also increased the protein solubility (53.07% higher than the respective control) and antioxidant activity (24.05% higher than the respective control) of the oven-dried samples and reduced the histamine content of the EHD-dried samples (25.87% lower than the respective control).

Cue- versus reward-encoding basolateral amygdala projections to nucleus accumbens.

In substance use disorders, drug use as unconditioned stimulus (US) reinforces drug taking. Meanwhile, drug-associated cues (conditioned stimulus [CS]) also gain incentive salience to promote drug seeking. The basolateral amygdala (BLA) is implicated in both US- and CS-mediated responses. Here, we show that two genetically distinct BLA neuronal types, expressing Rspo2 versus Ppp1r1b, respectively, project to the nucleus accumbens (NAc) and form monosynaptic connections with both dopamine D1 and D2 receptor-expressing neurons. While intra-NAc stimulation of Rspo2 or Ppp1r1b presynaptic terminals establishes intracranial self-stimulation (ICSS), only Ppp1r1b-stimulated mice exhibit cue-induced ICSS seeking. Furthermore, increasing versus decreasing the Ppp1r1b-to-NAc, but not Rspo2-to-NAc, subprojection increases versus decreases cue-induced cocaine seeking after cocaine withdrawal. Thus, while both BLA-to-NAc subprojections contribute to US-mediated responses, the Ppp1r1b subprojection selectively encodes CS-mediated reward and drug reinforcement. Such differential circuit representations may provide insights into precise understanding and manipulation of drug- versus cue-induced drug seeking and relapse.

Fresh aquaculture sludge management with black soldier fly (Hermetia illucens L.) larvae: investigation on bioconversion performances.

Aquaculture solid waste (ASW) is a nutrient rich material that can pose a significant environment challenge if not properly managed. This study investigated the potential of black soldier fly (BSF) larvae in converting this waste into biomass. Five substrates comprising chicken feed supplemented with varying proportions of fresh ASW (0%, 25%, 50%, 75%, 100%) were formulated and evaluated for larval growth and waste bioconversion efficiency. High nutrients retention (N: 23.25 ± 1.40%; C: 21.94 ± 0.99%; S: 12.20 ± 1.33%) and feed conversion ratio (1.78 ± 0.08) were detected on substrate 100ASW, although the limited feeding rate (114.54 ± 5.38 mg dry substrate/larvae) and the high amount of indigestible fibres (ADF = 15.87 ± 0.24%; ADL = 6.36 ± 0.17%) were translated to low larval growth (final larval average weight: 66.17 ± 1.81 mg). Decreasing ASW content resulted in reduced fibres and ash, increase in non-fibrous carbohydrates and C/N ratio, and improved larval growth and substrate utilization. However, high larval metabolic activity suggested higher nutrients loss to the environment. Substrate 75ASW demonstrated the best performances in terms of larval production (final larval average weight: 176.30 ± 12.12 mg), waste reduction (substrate reduction corrected by percentage of ASW: 26.76 ± 0.86%) and nutrients assimilation (N: 22.14 ± 1.14%; C: 15.29 ± 0.82%; S: 15.40 ± 0.99%). This substrate closely aligned with optimal BSF rearing substrates reported in literature. Overall, this study highlights the potential of BSF larvae in managing fresh ASW, offering a dual benefit of waste reduction and insect biomass production.

Evaluating an emerging technology-based biorefinery for edible house crickets.

Introduction: Edible insects, specifically house crickets, are expected to play an important role in the future food systems due to their rich nutritional profile, low environmental impact and growing consumer acceptance as food. Their content of proteins, lipids, chitin and phenolics offer great potential for the valorization of their biomass into nutritional end products and fractions. Furthermore, emerging food processing technologies and green solvents are relevant for improving the valorization process. Materials and methods: High pressure (HP) and ultrasound (US) processing were implemented in an insect biorefinery system, where a hexane/methanol/water solvent was used to separate fat, phenolics and a solid fraction containing proteins and chitin. Subsequently, a deep eutectic solvent of betaine and urea (B/U) was used to for protein and chitin isolation. Results: A maximum of 15% of fat was isolated, with no positive effect from the US or HP treatments. The US treatment enhanced the phenolic extraction yield by 38.69%, while HP negatively affected the antioxidant capacity. B/U was efficient in separating proteins and chitin, resulting in a protein concentrate with a protein content ≥80% and a chitinous fraction with a chitin content ≥70%. Conclusion: House cricket biomass can be refined into valuable fractions with a quick and simple method, making the process industrially relevant.

TRKB interaction with PSD95 is associated with latency of fluoxetine and 2R,6R-hydroxynorketamine.

Brain derived neurotrophic factor (BDNF) and its receptor tropomyosin kinase receptor B (TRKB) are key regulators of activity-dependent plasticity in the brain. TRKB is the target for both slow- and rapid-acting antidepressants and BDNF-TRKB system mediates the plasticity-inducing effects of antidepressants through their downstream targets. Particularly, the protein complexes that regulate the trafficking and synapse recruitment of TRKB receptors might be crucial in this process. In the present study, we investigated the interaction of TRKB with the postsynaptic density protein 95 (PSD95). We found that antidepressants increase the TRKB:PSD95 interaction in adult mouse hippocampus. Fluoxetine, a slow-acting antidepressant, increases this interaction only after a long-term (7 days) treatment, while (2R,6R)-hydroxynorketamine (RHNK), an active metabolite of rapid-acting antidepressant ketamine, achieves this within a short treatment regimen (3 days). Moreover, the drug-induced changes of TRKB:PSD95 interaction correlate with drug latency in behaviour, observed in mice subjected to an object location memory test (OLM). While silencing of PSD95 by viral delivery of shRNA in hippocampus abolished the RHNK-induced plasticity in mice in OLM, overexpression of PSD95 shortened the fluoxetine latency. In summary, changes in the TRKB:PSD95 interaction contribute to differences observed in drug latency. This study sheds a light on a novel mechanism of action of different classes of antidepressants.

Effect of Narrowband UV-B Irradiation on the Growth Performance of House Crickets.

Indoor co-cultivation systems can answer to the need for sustainable and resilient food production systems. Rearing organisms under light-emitting diodes (LEDs) irradiation provides the possibility to control and shape the emitted light spectra. UV-B-irradiation (280-315 nm) can positively affect the nutritional composition of different plants and other organisms, whereas information on edible insects is scarce. To evaluate the potential effect of the photosynthetically active radiation (PAR) and LED-emitting LEDs on the rearing and nutritional quality of edible insects, house crickets (Acheta domesticus) were reared from the age of 21 days under controlled LED spectra, with an additional UV-B (0.08 W/m2) dose of 1.15 KJm2 d-1 (illuminated over a period for 4 h per day) for 34 days. UV-B exposure showed no harm to the weight of the crickets and significantly increased their survival by ca. 10% under narrowband UV-B treatment. The nutritional composition including proteins, fat and chitin contents of the insects was not affected by the UV-B light and reached values of 60.03 ± 10.41, 22.38 ± 2.12 and 9.33 ± 1.21%, respectively, under the LED irradiation. Therefore, house crickets can grow under LED irradiation with a positive effect of narrowband UV-B application on their survival.

Chitin Isolation and Chitosan Production from House Crickets (Acheta domesticus) by Environmentally Friendly Methods.

Alternative methods were evaluated for chitin isolation from Acheta domesticus. Chemical demineralization was compared to fermentation with Lactococcus lactis, citric acid treatment, and microwave treatment, leading to a degree of demineralization of 91.1 ± 0.3, 97.3 ± 0.8, 70.5 ± 3.5, and 85.8 ± 1.3%, respectively. Fermentation with Bacillus subtilis, a deep eutectic solvent, and enzymatic digestion were tested for chitin isolation, generating materials with less than half the chitin content when compared to alkaline deproteinization. Chitosan was produced on a large scale by deacetylation of the chitinous material obtained from two selected processes: the chemical treatment and an alternative process combining L. lactis fermentation with bromelain deproteinization. The chemical and alternative processes resulted in similar chitosan content (81.9 and 88.0%), antioxidant activity (59 and 49%), and degree of deacetylation (66.6 and 62.9%), respectively. The chitosan products had comparable physical properties. Therefore, the alternative process is appropriate to replace the chemical process of chitin isolation for industrial applications.

Visual-area-specific tonic modulation of GABA release by endocannabinoids sets the activity and coordination of neocortical principal neurons.

Perisomatic inhibition of pyramidal neurons (PNs) coordinates cortical network activity during sensory processing, and this role is mainly attributed to parvalbumin-expressing basket cells (BCs). However, cannabinoid receptor type 1 (CB1)-expressing interneurons are also BCs, but the connectivity and function of these elusive but prominent neocortical inhibitory neurons are unclear. We find that their connectivity pattern is visual area specific. Persistently active CB1 signaling suppresses GABA release from CB1 BCs in the medial secondary visual cortex (V2M), but not in the primary visual cortex (V1). Accordingly, in vivo, tonic CB1 signaling is responsible for higher but less coordinated PN activity in the V2M than in the V1. These differential firing dynamics in the V1 and V2M can be captured by a computational network model that incorporates visual-area-specific properties. Our results indicate a differential CB1-mediated mechanism controlling PN activity, suggesting an alternative connectivity scheme of a specific GABAergic circuit in different cortical areas.

Contingent Amygdala Inputs Trigger Heterosynaptic LTP at Hippocampus-To-Accumbens Synapses.

The nucleus accumbens shell (NAcSh) is a key brain region where environmental cues acquire incentive salience to reinforce motivated behaviors. Principal medium spiny neurons (MSNs) in the NAcSh receive extensive glutamatergic projections from limbic regions, among which, the ventral hippocampus (vH) transmits information enriched in contextual cues, and the basolateral amygdala (BLA) encodes real-time arousing states. The vH and BLA project convergently to NAcSh MSNs, both activated in a time-locked manner on a cue-conditioned motivational action. In brain slices prepared from male and female mice, we show that co-activation of the two projections induces long-term potentiation (LTP) at vH-to-NAcSh synapses without affecting BLA-to-NAcSh synapses, revealing a heterosynaptic mechanism through which BLA signals persistently increase the temporally contingent vH-to-NAcSh transmission. Furthermore, this LTP is more prominent in dopamine D1 receptor-expressing (D1) MSNs than D2 MSNs and can be prevented by inhibition of either D1 receptors or dopaminergic terminals in NAcSh. This heterosynaptic LTP may provide a dopamine-guided mechanism through which vH-encoded cue inputs that are contingent to BLA activation acquire increased circuit representation to reinforce behavior.SIGNIFICANCE STATEMENT In motivated behaviors, environmental cues associated with arousing stimuli acquire increased incentive salience, processes mediated in part by the nucleus accumbens (NAc). NAc principal neurons receive glutamatergic projections from the ventral hippocampus (vH) and basolateral amygdala (BLA), which transmit information encoding contextual cues and affective states, respectively. Our results show that co-activation of the two projections induces long-term potentiation (LTP) at vH-to-NAc synapses without affecting BLA-to-NAc synapses, revealing a heterosynaptic mechanism through which BLA signals potentiate the temporally contingent vH-to-NAc transmission. Furthermore, this LTP is prevented by inhibition of either D1 receptors or dopaminergic axons. This heterosynaptic LTP may provide a dopamine-guided mechanism through which vH-encoded cue inputs that are contingent to BLA activation acquire increased circuit representation to reinforce behavior.

Factors affecting consumer choice of novel non-thermally processed fruit and vegetables products: Evidence from a 4-country study in Europe.

A wide variety of novel non-thermal processing technologies (NTPTs) are under development to meet the increasing consumer demand for high-quality fruit and vegetable (F&V) products. Understanding consumers' needs and possible barriers to acceptance of these technologies is however essential to assess the commercial feasibility of mildly processed F&Vs. Situated within this context, and extending previous work on the topic, in this paper we present results from a large-scale choice-based conjoint analysis consumer survey to investigate consumers' choice behavior towards NTPT-processed F&V products in four European countries - Denmark, Italy, Serbia, and Spain, using three model products - orange juice, iceberg salad, and cherry tomatoes respectively processed via three NTPT - mild processing, novel washing, and active packaging, compared to three conventional processing techniques - pasteurization, conventional washing, and conventional packaging, respectively. Images of the three product categories were developed to systematically vary in three key attributes: stated benefits (health and nutrition, natural taste, shelf-life), information on processing (conventional, NTPT), and price point (reference, premium price). The results showed that, out of the three attributes considered, "stated benefit" was the most important driver of consumer choice - in all countries and across product categories. Benefits relevant to health and nutrition, and to natural taste were more positively received, compared to extension of shelf-life. Information on processing and price levels had a similar influence on consumer choice of iceberg salad and cherry tomatoes, whilst for orange juice processing had a larger effect than price, suggesting that information on processing may be more impactful for F&V-derived products than for fresh produce. Individual differences among consumers according to country, age, gender, and dietary status, appeared small and transient. The most consequential individual characteristic was consumers' level of food technology neophobia (FTN), with results showing that high FTN consumers (17% of the sample) were less likely to choose F&V treated with NTPT, compared to consumers with medium or low FTN. Overall, this research suggests that products treated with NTPT may have a broad appeal across European consumers, and that targeted communication explicitly and efficiently focusing on health and taste benefits has the greatest chance to meet consumer preferences.

Ca2+-permeable AMPA receptors set the threshold for retrieval of drug memories.

Frequent relapse prevents the successful treatment of substance use disorders and is triggered in part by retrieval of drug-associated memories. Drug-conditioned behaviours in rodents are reinstated upon drug memory retrieval following re-exposure to cues previously associated with the drug, or the drug itself. Therapies based on mechanistic insights from rodent studies have focused on amnesic procedures of cue-drug associations but with so far limited success. Conversely, more recent studies propose that inhibiting drug memory retrieval offers improved anti-relapse efficacy. However, mechanisms of memory retrieval are poorly understood. Here, we used a conditioned place preference (CPP) procedure in mice to investigate the cellular and molecular underpinnings of drug-induced memory retrieval. After extinction training of CPP, Ca2+-permeable AMPA receptors (CP-AMPARs) accumulated at drug-generated silent synapses of nucleus accumbens (NAc) medium spiny neurons. The NAc CP-AMPARs regulated the retrieval mechanism of drug memories after extinction. Specifically, we used different priming doses of cocaine, fentanyl, or a cue associated with drug exposure to reinstate CPP, providing different memory retrieval conditions. Although both high and low doses of these two drugs induced CPP reinstatement, compromising CP-AMPAR accumulation impaired CPP reinstatement, induced by low doses of each drug or the cue. This threshold effect was mediated by NAc CP-AMPARs as region specific knock-down of PSD-95 prevented low-dose cocaine-induced retrieval selectively. These results demonstrate the NAc as a brain region and CP-AMPARs as key synaptic substrates that govern the threshold for drug-induced retrieval and behavioural expression of drug memories.

Direct Evidence for a Radial Gradient in Age of the Apple Fruit Cuticle.

The pattern of cuticle deposition plays an important role in managing strain buildup in fruit cuticles. Cuticular strain is the primary trigger for numerous fruit-surface disorders in many fruit crop species. Recent evidence indicates a strain gradient may exist within the apple fruit cuticle. The outer layers of the cuticle are more strained and thus more susceptible to microcracking than the inner layers. A radial gradient in cuticle age is the most likely explanation. Our study aimed to establish whether (or not) deposition of new cutin in a developing apple fruit occurs on the inner surface of the cuticle, i.e., immediately abutting the outward-facing epidermal cell wall. Developing apples were fed with 13C oleic acid through the skin. Following a 14-d period for incorporation, the fruit was harvested and the cuticular membranes (CMs) isolated enzymatically. The CMs were then ablated to varying extents from the inner or the outer surfaces, using a cold atmospheric pressure plasma (CAPP). Afterwards, the ablated CMs were dewaxed and the 13C contents were determined by mass spectrometry. The incorporation of 13C in the cutin fraction was higher than in the wax fraction. The 13C content was highest in non-ablated, dewaxed CM (DCM) and decreased as ablation depth from the inner surface increased. There was no change in 13C content when ablation was carried out from the outer surface. As fruit development proceeded, more 13C label was found towards the middle of the DCM. These results offered direct evidence for deposition of cutin being on the inner surface of the cuticle, resulting in a radial gradient in cuticular age-the most recent deposition (youngest) being on the inner cuticle surface (abutting the epidermal cell wall) and the earliest deposition (oldest) being on the outer surface (abutting the atmosphere).

A Molecular Survey of Bacterial Species in the Guts of Black Soldier Fly Larvae (Hermetia illucens) Reared on Two Urban Organic Waste Streams in Kenya.

Globally, the expansion of livestock and fisheries production is severely constrained due to the increasing costs and ecological footprint of feed constituents. The utilization of black soldier fly (BSF) as an alternative protein ingredient to fishmeal and soybean in animal feed has been widely documented. The black soldier fly larvae (BSFL) used are known to voraciously feed and grow in contaminated organic wastes. Thus, several concerns about their safety for inclusion into animal feed remain largely unaddressed. This study evaluated both culture-dependent sequence-based and 16S rDNA amplification analysis to isolate and identify bacterial species associated with BSFL fed on chicken manure (CM) and kitchen waste (KW). The bacteria species from the CM and KW were also isolated and investigated. Results from the culture-dependent isolation strategies revealed that Providencia sp. was the most dominant bacterial species detected from the guts of BSFL reared on CM and KW. Morganella sp. and Brevibacterium sp. were detected in CM, while Staphylococcus sp. and Bordetella sp. were specific to KW. However, metagenomic studies showed that Providencia and Bordetella were the dominant genera observed in BSFL gut and processed waste substrates. Pseudomonas and Comamonas were recorded in the raw waste substrates. The diversity of bacterial genera recorded from the fresh rearing substrates was significantly higher compared to the diversity observed in the gut of the BSFL and BSF frass (leftovers of the rearing substrates). These findings demonstrate that the presence and abundance of microbiota in BSFL and their associated waste vary considerably. However, the presence of clinically pathogenic strains of bacteria in the gut of BSFL fed both substrates highlight the biosafety risk of potential vertical transmission that might occur, if appropriate pre-and-postharvest measures are not enforced.

Aqueous and gaseous plasma applications for the treatment of mung bean seeds.

Sprouts are particularly prone to microbial contamination due to their high nutrient content and the warm temperatures and humid conditions needed for their production. Therefore, disinfection is a crucial step in food processing as a means of preventing the transmission of bacterial, parasitic and viral pathogens. In this study, a dielectric coplanar surface barrier discharge (DCSBD) system was used for the application of cold atmospheric plasma (CAP), plasma activated water (PAW) and their combination on mung bean seeds. Germination assessments were performed in a test tube set-up filled with glass beads and the produced irrigation water. Overall, it was found that the combined seed treatment with direct air CAP (350 W) and air PAW had no negative impact on mung bean seed germination and growth, nor the concentration of secondary metabolites within the sprouts. These treatments also reduced the total microbial population in sprouts by 2.5 log CFU/g. This research reports for first time that aside from the stimulatory effect of plasma discharge on seed surface disinfection, sustained plasma treatment through irrigation of treated seeds with PAW can significantly enhance seedling growth. The positive outcome and further applications of different forms, of plasma i.e., gaseous and aqueous, in the agro-food industry is further supported by this research.

Effect of Cereal α-Amylase/Trypsin Inhibitors on Developmental Characteristics and Abundance of Digestive Enzymes of Mealworm Larvae (Tenebrio molitor L.).

The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.

Aspects of high hydrostatic pressure food processing: Perspectives on technology and food safety.

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety-relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non-HHP-treated food, the allergenic potential of HHP-treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data.

Spine dynamics of PSD-95-deficient neurons in the visual cortex link silent synapses to structural cortical plasticity.

Critical periods (CPs) are time windows of heightened brain plasticity during which experience refines synaptic connections to achieve mature functionality. At glutamatergic synapses on dendritic spines of principal cortical neurons, the maturation is largely governed by postsynaptic density protein-95 (PSD-95)-dependent synaptic incorporation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors into nascent AMPA-receptor silent synapses. Consequently, in mouse primary visual cortex (V1), impaired silent synapse maturation in PSD-95-deficient neurons prevents the closure of the CP for juvenile ocular dominance plasticity (jODP). A structural hallmark of jODP is increased spine elimination, induced by brief monocular deprivation (MD). However, it is unknown whether impaired silent synapse maturation facilitates spine elimination and also preserves juvenile structural plasticity. Using two-photon microscopy, we assessed spine dynamics in apical dendrites of layer 2/3 pyramidal neurons (PNs) in binocular V1 during ODP in awake adult mice. Under basal conditions, spine formation and elimination ratios were similar between PSD-95 knockout (KO) and wild-type (WT) mice. However, a brief MD affected spine dynamics only in KO mice, where MD doubled spine elimination, primarily affecting newly formed spines, and caused a net reduction in spine density similar to what has been observed during jODP in WT mice. A similar increase in spine elimination after MD occurred if PSD-95 was knocked down in single PNs of layer 2/3. Thus, structural plasticity is dictated cell autonomously by PSD-95 in vivo in awake mice. Loss of PSD-95 preserves hallmark features of spine dynamics in jODP into adulthood, revealing a functional link of PSD-95 for experience-dependent synapse maturation and stabilization during CPs.

AMPA and NMDA Receptor Trafficking at Cocaine-Generated Synapses.

Cocaine experience generates AMPA receptor (AMPAR)-silent synapses in the nucleus accumbens (NAc), which are thought to be new synaptic contacts enriched in GluN2B-containing NMDA receptors (NMDARs). After drug withdrawal, some of these synapses mature by recruiting AMPARs, strengthening the newly established synaptic transmission. Silent synapse generation and maturation are two consecutive cellular steps through which NAc circuits are profoundly remodeled to promote cue-induced cocaine seeking after drug withdrawal. However, the basic cellular processes that mediate these two critical steps remains underexplored. Using a combination of electrophysiology, viral-mediated gene transfer, and confocal imaging in male rats as well as knock-in (KI) mice of both sexes, our current study characterized the dynamic roles played by AMPARs and NMDARs in generation and maturation of silent synapses on NAc medium spiny neurons after cocaine self-administration and withdrawal. We report that cocaine-induced generation of silent synapses not only required synaptic insertion of GluN2B-containing NMDARs, but also, counterintuitively, involved insertion of AMPARs, which subsequently internalized, resulting in the AMPAR-silent state on withdrawal day 1. Furthermore, GluN2B NMDARs functioned to maintain these cocaine-generated synapses in the AMPAR-silent state during drug withdrawal, until they were replaced by nonGluN2B NMDARs, a switch that allowed AMPAR recruitment and maturation of silent synapses. These results reveal dynamic interactions between AMPARs and NMDARs during the generation and maturation of silent synapses after cocaine experience and provide a mechanistic basis through which new synaptic contacts and possibly new neural network patterns created by these synapses can be manipulated for therapeutic benefit.SIGNIFICANCE STATEMENT Studies over the past decade reveal a critical role of AMPA receptor-silent, NMDA receptor-containing synapses in forming cocaine-related memories that drive cocaine relapse. However, it remains incompletely understood how AMPA and NMDA receptors traffic at these synapses during their generation and maturation. The current study characterizes a two-step AMPA receptor trafficking cascade that contributes to the generation of silent synapses in response to cocaine experience, and a two-step NMDA receptor trafficking cascade that contributes to the maturation of these synapses after cocaine withdrawal. These results depict a highly regulated cellular procedure through which nascent glutamatergic synapses are generated in the adult brain after drug experience and provide significant insight into the roles of glutamate receptors in synapse formation and maturation.

Neuropathic pain generates silent synapses in thalamic projection to anterior cingulate cortex.

ABSTRACT: Pain experience can change the central processing of nociceptive inputs, resulting in persistent allodynia and hyperalgesia. However, the underlying circuit mechanisms remain underexplored. Here, we focus on pain-induced remodeling of the projection from the mediodorsal thalamus (MD) to the anterior cingulate cortex (ACC), a projection that relays spinal nociceptive input for central processing. Using optogenetics combined with slice electrophysiology, we detected in male mice that 7 days of chronic constriction injury (CCI; achieved by loose ligation of the sciatic nerve) generated AMPA receptor (AMPAR)-silent glutamatergic synapses within the contralateral MD-to-ACC projection. AMPAR-silent synapses are typically GluN2B-enriched nascent glutamatergic synapses that mediate the initial formation of neural circuits during early development. During development, some silent synapses mature and become "unsilenced" by recruiting and stabilizing AMPARs, consolidating and strengthening the newly formed circuits. Consistent with these synaptogenic features, pain-induced generation of silent synapses was accompanied by increased densities of immature dendritic spines in ACC neurons and increased synaptic weight of GluN2B-containing NMDA receptors (NMDARs) in the MD-to-ACC projection. After prolonged (∼30 days) CCI, injury-generated silent synapses declined to low levels, which likely resulted from a synaptic maturation process that strengthens AMPAR-mediated MD-to-ACC transmission. Consistent with this hypothesis, viral-mediated knockdown of GluN2B in ACC neurons, which prevented pain-induced generation of silent synapses and silent synapse-mediated strengthening of MD-to-ACC projection after prolonged CCI, prevented the development of allodynia. Taken together, our results depict a silent synapse-mediated mechanism through which key supraspinal neural circuits that regulate pain sensitivity are remodeled to induce allodynia and hyperalgesia.