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

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

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

Ultrastructural localization of GAT-1 in the barrel B3 hollow in trained side CS+UCS group.

<p>GAT-1+ terminal (white asterisk), which forms a symmetrical synaptic contact (arrowheads), and the terminal (black asterisk), which ends in asymmetrical synaptic contacts (arrowheads) are localized on the same dendritic spine. The adjacent terminal (black asterisk) with asymmetric specialization (arrowheads) is unlabeled. Scale bar 0.5 µm.</p

Location of polyribosomes in dendrites of B2 barrel.

<p>Electron micrographs of B2 barrel hollow showing polyribosomes (arrows) located in the dendritic spine and associated with excitatory synapse (<b>A</b>), located in the dendritic spine and associated with inhibitory synapse (<b>B</b>), located in the dendritic shaft and associated with excitatory synapse (<b>C</b>) and located in the dendritic shaft and associated with inhibitory synapse (<b>D</b>). Scale bar: 0.5 μm.</p

Density of polyribosomes in dendritic spines.

<p>Conditioning increases the density of polyribosomes located in spines and associated with excitatory (ANOVA, p<0.001) and inhibitory synapses (ANOVA, p<0.001). <b>A.</b> Total density of polyribosomes located in dendritic spines in: control, conditioned (CS+UCS) and pseudoconditioned (PSEUDO) group. <b>B.</b> Density of polyribosomes located in spines and associated with excitatory synapses. <b>C</b>. Density of polyribosomes located in spines and associated with inhibitory synapses. All graphs show means ± SD (**p<0.01, ***p<0.001).</p

Location of polyribosomes in heads/necks/bases of dendritic spines.

<p>Conditioning and pseudoconditioning do not change the location of polyribosomes in the dendritic spines. <b>A.</b> Electron micrograph of B2 barrel hollow showing polyribosomes (arrow) located in the head of single-synapse spine (s). <b>B.</b> Electron micrograph of B2 barrel hollow showing polyribosomes (arrows) located in the neck of single-synapse spine (s). Scale bar: 0.5 μm. <b>C.</b> Location of polyribosomes in dendritic spines in: control, conditioned (CS+UCS) and pseudoconditioned (PSEUDO) group. Graph shows means ± SD.</p

Postsynaptic density area.

<p><b>A.</b> Excitatory synapses located on spines containing polyribosomes have larger postsynaptic density area than those on spines without polyribosomes (two-way ANOVA, p<0.0001) and the synapses enlarge during conditioning (ANOVA, p<0.01). Synapses on spines without polyribosomes do not significantly differ in size in the control, in the conditioned group (CS+UCS) and in the pseudoconditioned group (PSEUDO). Graph shows means ± SD (*p<0.05, *p<0.01, ***p<0.001). <b>B</b>. Inhibitory synapses located on spines containing polyribosomes have larger postsynaptic density area of than those located on spines without polyribosomes (two-way ANOVA, p<0.0001) and the synapses enlarge during conditioning (ANOVA, p<0.05). Synapses on spines without polyribosomes do not significantly differ in size in the control, in the conditioned group (CS+UCS) and in the pseudoconditioned group (PSEUDO).</p