Rapid, learning-induced inhibitory synaptogenesis in murine barrel field

The structure of neurons changes during development and in response to injury or alteration in sensory experience. Changes occur in the number, shape, and dimensions of dendritic spines together with their synapses. However, precise data on these changes in response to learning are sparse. Here, we show using quantitative transmission electron microscopy that a simple form of learning involving mystacial vibrissae results in approximately 70% increase in the density of inhibitory synapses on spines of neurons located in layer IV barrels that represent the stimulated vibrissae. The spines contain one asymmetrical (excitatory) and one symmetrical (inhibitory) synapse (double-synapse spines), and their density increases threefold as a result of learning with no apparent change in the density of asymmetrical synapses. This effect seems to be specific for learning because pseudoconditioning (in which the conditioned and unconditioned stimuli are delivered at random) does not lead to the enhancement of symmetrical synapses but instead results in an upregulation of asymmetrical synapses on spines. Symmetrical synapses of cells located in barrels receiving the conditioned stimulus also show a greater concentration of GABA in their presynaptic terminals. These results indicate that the immediate effect of classical conditioning in the "conditioned" barrels is rapid, pronounced, and inhibitory

Axonal Dynamics of Excitatory and Inhibitory Neurons in Somatosensory Cortex

Electrophysiology-delivery of fluorescent viral vectors-and two-photon microscopy were used to demonstrate the rapidity of axonal restructuring of both excitatory and inhibitory neurons in rodent cortical layer II/III following alterations in sensory experience

Assessment of changes in structure of dried tissue of sour cherry pretreated using ultrasound-assisted osmotic dehydration

Celem pracy było określenie wpływu oddziaływania ultradźwięków na strukturę wewnętrzną owoców wiśni poddawanych obróbce osmotycznej i suszeniu. Owoce wiśni odmiany ‘Nefris’ odwadniano w 60-procentowym roztworze sacharozy przez 120 min (40 ºC), w łaźni wodnej wyposażonej w przetwornik ultradźwiękowy (25 kHz, 0,4 W/cm²) i platformę wytrząsającą (30 rpm). Całkowity czas obróbki osmotycznej wynosił 120 min, w trakcie którego zastosowano różne warianty czasu traktowania ultradźwiękami (ang. ultrasound – US) i wytrząsania (ang. shaking – S). W pierwszej kolejności próbki traktowano US przez 0, 30, 60, 90 lub 120 min, po czym wytrząsano (S) przez odpowiednio: 120, 90, 60, 30 lub 0 min. Odwodnione owoce poddawano suszeniu konwekcyjnemu. Strukturę suszu osmo-konwekcyjnego owoców odwadnianych oraz surowca analizowano mikroskopowo. Potwierdzono, że zastosowanie ultradźwięków powoduje zmiany struktury tkanek. Dłuższy okres ekspozycji tkanki wiśni na działanie US sprzyjał zmniejszaniu się zwartości komórek bezpośrednio sąsiadujących z epidermą. Jednocześnie obserwowano zwiększanie deformacji komórek miękiszu dośrodkowego i zanikanie przestrzeni międzykomórkowych. Najbardziej ujednoliconą strukturą (komórki najmniej wydłużone i komórki okrągłe) charakteryzowały się wysuszone owoce poddane obróbce osmotycznej i równocześnie poddane przez 60 min: oddziaływaniu US i wytrząsaniu (60US+60S), co wskazuje na istnienie optymalnego stanu sprzyjającego redukcji naprężenia wewnętrznego. Suszenie konwekcyjne powoduje znaczny skurcz tkanki. W owocach suszonych, które nie były poddane działaniu ultradźwięków, ale były wytrząsane przez 120 min (0US+120S), zaobserwowano występowanie wolnych przestrzeni międzykomórkowych na przemian z dużymi zagęszczeniami. Zastosowanie US do 60 min sprzyjało zwiększeniu jednorodności struktury suszonego materiału (wariant optymalny – 60US+60S). Oddziaływanie ultradźwięków powyżej 60 min wyraźnie potęgowało efekt zagęszczania struktury, w związku z czym próbki traktowane US przez 120 min (120US+0S) charakteryzowały się największym skurczem tkanki.The objective of the research study was to determine the effect of ultrasounds on the microstructure of osmo-treated and dried sour cherry fruits. The ‘Nefris’ sour cherry fruit variety was osmotically dehydrated using a 60 % sucrose solution for 120 min. (40 ºC) in an ultrasonic bath equipped with a transducer (25 kHz, 0.4 W/cm²) and a shaking platform (30 rpm). The total time of osmotic dehydration was 120 min; during that time period, diverse time variants of ultrasound (US) treatment and shaking (S) were applied. First, the samples were treated by ultrasounds (US) for 0, 30, 60, 90, and 120 min.; next, they were shaken for 120, 90, 60, 30, or 0 min., respectively. The dehydrated fruits were convectively dried. The structure of osmo-convectively dried samples of dehydrated fruits and raw material was microscopically analyzed. It was confirmed that the application of ultrasounds caused changes in the structure of tissues. A longer time of exposing sour cherry tissue to ultrasounds resulted in a decrease in the density of cells directly adjacent to the epidermis. Concurrently, it was found that the deformation of parenchyma cells increased and the intercellular spaces faded away. By the most homogenous structure (the most elongated and circular cells) were characterized the dried fruits that were osmo-treated and, simultaneously, ultrasound-treated and shaken for 60 min. (60US+60S); thus the presence of optimal state was proved that supported the reduction in internal tension. The convective drying causes the tissue to considerably contract. In the dried fruits, which were not treated by ultrasounds, but only shaken for 120 min. (0US+120S), alternating free intercellular and highly compact spaces were found. The application of ultrasounds for max 60 min. caused the homogeneity of structure of the material being dried to increase (optimal variant: 6US+60S)

Increases in the Numerical Density of GAT-1 Positive Puncta in the Barrel Cortex of Adult Mice after Fear Conditioning

<div><p>Three days of fear conditioning that combines tactile stimulation of a row of facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) expands the representation of “trained” vibrissae, which can be demonstrated by labeling with 2-deoxyglucose in layer IV of the barrel cortex. We have also shown that functional reorganization of the primary somatosensory cortex (S1) increases GABAergic markers in the hollows of “trained” barrels of the adult mouse. This study investigated how whisker-shock conditioning (CS+UCS) affected the expression of puncta of a high-affinity GABA plasma membrane transporter GAT-1 in the barrel cortex of mice 24 h after associative learning paradigm. We found that whisker-shock conditioning (CS+UCS) led to increase expression of neuronal and astroglial GAT-1 puncta in the “trained” row compared to controls: Pseudoconditioned, CS-only, UCS-only and Naïve animals. These findings suggest that fear conditioning specifically induces activation of systems regulating cellular levels of the inhibitory neurotransmitter GABA.</p></div

CB1 Cannabinoid Receptor Expression in the Barrel Field Region Is Associated with Mouse Learning

We found previously that fear conditioning by combined stimulation of a row B facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) leads to expansion of the cortical representation of the “trained” row, labeled with 2-deoxyglucose (2DG), in the layer IIIb/IV of the adult mouse the primary somatosensory cortex (S1) 24 h later. We have observed that these learning-dependent plastic changes are manifested by increased expression of somatostatin, cholecystokinin (SST+, CCK+) but not parvalbumin (PV+) immunopositive interneurons We have expanded this research and quantified a numerical value of CB1-expressing and PV-expressing GABAergic axon terminals (CB1+ and PV+ immunopositive puncta) that innervate different segments of postsynaptic cells in the barrel hollows of S1 cortex. We used 3D microscopy to identify the CB+ and PV+ puncta in the barrel cortex “trained” and the control hemispheres CS+UCS group and in controls: Pseudoconditioned, CS-only, UCS-only, and naive animals. We have identified that (i) the association between whisker-shock “trained” barrel B hollows and CB1+, but not PV+ puncta expression remained significant after Bonferroni correction, (ii) CS+UCS has had a significant increasing effect on expression of CB1+ but not PV+ puncta in barrel cortex “trained” hemisphere, and (iii) the pseudoconditioning had a significant decreasing effect on expression of CB1+, but not on PV+ puncta in barrel cortex, both trained and untrained hemispheres. It is correlated to disturbing behaviors. The results suggest that CB1+ puncta regulation is specifically linked with mechanisms leading to learning-dependent plasticity in S1 cortex

GAT-1 and GFAP in a tangential section taken from layer IV of the SI cortex.

<p>(A) Nuclear staining with Hoechst 33258 delineates the barrel cortex. Letters A–E denote rows of barrels: the arrow indicates the hollow of barrel B3. (B) Nuclear staining with Hoechst 33258 of the outline of the barrel B3 hollow. Photomicrographs C, D, E, F, depict the same field covering the hollow of barrel B3. (C) shows GAT-1 immunopositive puncta (green); (D) shows GFAP - immunopositive astrocyte (red); (E) shows nuclear staining with Hoechst 33258 dye (blue); (F) overlays images C, D, and E; (G) confocal images of immunostaining for GFAP, GAT-1 and Hoechst 33258. A GFAP+ astrocytic processes (red) contains GAT-1 (red and yellow, indicated by arrow), as in the xz and yz orthogonal views and G1–G3 higher magnification images. The images are comprised of 15 optical sections of 1000 nm thickness. White asterisks in C–G denote the same blood vessel. Scale bar: A = 100 µm, B = 20 µm, C–G = 10 µm.</p

Tangential sections of the mouse barrel field immunostained for GABA transporter GAT-1.

<p>(A) An example of a tangential section of the mouse barrel field immunostained for GAT-1, letters A-E denotes rows of barrels. Scale bar 100 µm. (B) GAT-1+ puncta were observed throughout the neuropil in the barrel hollow. GAT-1+ puncta were numerous around unlabeled neuronal perikarya (asterisk). Fibers running obliquely or radially (arrowed) show irregularly spaced varicose swellings. Scale bar 10 µm. (C) High magnification micrographs from the trained side barrel B3 hollow in comparison with the control side barrel B3 hollow in the group of animals receiving CS+UCS (D). Note that CS+UCS induced an increased density of GAT-1+ puncta. Scale bar 20 µm. Immunocytochemical staining for GAT-1 was performed as described previously Minelli and co-workers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110493#pone.0110493-Minelli1" target="_blank">[29]</a>.</p

Effect of Associative Learning on Memory Spine Formation in Mouse Barrel Cortex

Associative fear learning, in which stimulation of whiskers is paired with mild electric shock to the tail, modifies the barrel cortex, the functional representation of sensory receptors involved in the conditioning, by inducing formation of new inhibitory synapses on single-synapse spines of the cognate barrel hollows and thus producing double-synapse spines. In the barrel cortex of conditioned, pseudoconditioned, and untreated mice, we analyzed the number and morphological features of dendritic spines at various maturation and stability levels: sER-free spines, spines containing smooth endoplasmic reticulum (sER), and spines containing spine apparatus. Using stereological analysis of serial sections examined by transmission electron microscopy, we found that the density of double-synapse spines containing spine apparatus was significantly increased in the conditioned mice. Learning also induced enhancement of the postsynaptic density area of inhibitory synapses as well as increase in the number of polyribosomes in such spines. In single-synapse spines, the effects of conditioning were less pronounced and included increase in the number of polyribosomes in sER-free spines. The results suggest that fear learning differentially affects single- and double-synapse spines in the barrel cortex: it promotes maturation and stabilization of double-synapse spines, which might possibly contribute to permanent memory formation, and upregulates protein synthesis in single-synapse spines

Changes in the numerical density of GAT-1+ puncta in the barrel B3 hollows in all groups.

<p>The values represent the mean numerical densities of the GAT-1+ puncta (x10⁸/mm³± SE. ANOVA, followed by Huynh-Feldt (H–F) <i>post hoc</i> test ***<i>p</i><0.001). Whisker-shock conditioning (CS+UCS <i>n</i> = 8), pseudoconditioning (PSEUDO <i>n</i> = 7), whisker stimulation alone (CS-only <i>n</i> = 7), tail shock alone (UCS-only <i>n</i> = 6) and control (NAIVE <i>n</i> = 10). Black bars represent trained side GAT-1 expression including GAT-1+/GFAP+ (white checkered pattern) in the trained barrel B3 hollow in all group of mice. Gray bars represent control side GAT-1 expression including GAT-1+/GFAP+ (white checkered pattern) in the control barrel B3 hollow in all group of mice.</p