Ewa Siucinska

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 learning - dependent these 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) 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 CB1+ but not PV+ puncta in barrel cortex "trained" hemisphere, (iii) the pseudoconditioning has 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.

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.

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.

Associative fear learning, resulting from whisker stimulation paired with application of a mild electric shock to the tail in a classical conditioning paradigm, changes the motor behavior of mice and modifies the cortical functional representation of sensory receptors involved in the conditioning. It also induces the formation of new inhibitory synapses on double-synapse spines of the cognate barrel hollows. We studied density and distribution of polyribosomes, the putative structural markers of enhanced synaptic activation, following conditioning. By analyzing serial sections of the barrel cortex by electron microscopy and stereology, we found that the density of polyribosomes was significantly increased in dendrites of the barrel activated during conditioning. The results revealed fear learning-induced increase in the density of polyribosomes associated with both excitatory and inhibitory synapses located on dendritic spines (in both single- and double-synapse spines) and only with the inhibitory synapses located on dendritic shafts. This effect was accompanied by a significant increase in the postsynaptic density area of the excitatory synapses on single-synapse spines and of the inhibitory synapses on double-synapse spines containing polyribosomes. The present results show that associative fear learning not only induces inhibitory synaptogenesis, as demonstrated in the previous studies, but also stimulates local protein synthesis and produces modifications of the synapses that indicate their potentiation.

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 ∼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.