CB1 cannabinoid receptor expression in the barrel ... - ACS Publications

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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CB1 Cannabinoid Receptor Expression in the Barrel Field Region Is Associated with Mouse Learning Ewa Siucinska,*,† Wojciech Brutkowski,‡ and Tytus Bernas‡ †

Laboratory of Neuroplasticity, Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology of Polish Academy of Sciences, 3 Pasteur Str., 02-093 Warsaw, Poland ‡ Laboratory of Imaging Tissue Structure and Function Neurobiology Center, Nencki Institute of Experimental Biology of Polish Academy of Sciences, 3 Pasteur Str., 02-093 Warsaw, Poland S Supporting Information *

ABSTRACT: 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 learningdependent plasticity in S1 cortex. KEYWORDS: Barrel cortex, CB1+/PV+ puncta, fear conditioning, pseudoconditioning, plasticity

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detected that the coordination of the “trained” barrel hollow located neuronal ensembles is carried out by somatostatin (SST +)21 and cholecystokinin (CCK+)22 but not parvalbumin (PV +)23 immunopositive interneurons in the mouse somatosensory cortex. However, in the brain, PV+ interneuron function concentrates on synchronization the electrical activity of pyramidal neurons.24−26 CCK+ interneurons express presynaptic CB1 cannabinoid receptors (CB1Rs), CB1 immunopositive puncta (CB1+).27,28 Endogenous cannabinoid signaling pathways have been implicated in promoting neuronal survival after cerebral ischemia, coordination, vasoregulation, thermoregulation, inflammation, pain, and trauma.29 High CB1Rs expression has been detected in caudate putamen, hippocampus, and neocortex in rats.30,31 Moreover, high density of CB1Rs has been found in songbird telencephalon, notably within regions known to be involved in

INTRODUCTION It has been known that 3 days (only 10 min/day) of whiskershock fear conditioning learning paradigm expands the representation of “trained” row B vibrissae, which can be demonstrated by labeling with 2-deoxyglucose (2DG) in layer IIIb/IV of the primary somatosensory cortex (S1) of adult mice.1 During whisker-shock training, a greater density of GABAergic cells concentrates in the hollow of the “trained row” B barrels compared to the hollows in the barrel field of the opposite hemisphere in the same mouse.2 This ultimately leads to “rewiring” of neuronal networks. On the subcellular level, altered neuronal activity induces activation of systems regulating cellular levels of the inhibitory neurotransmitter GABA,3 supporting the concept that the functional organization of adult sensory cortices can be modified by learning.4−12 Indeed, structural plasticity of synapses on spines and axonal boutons excitatory and inhibitory neurons is well documented in the adult brain.13−19 Likewise, remodeling of synapses, induced by whisker-shock increased sensory input of an adult barrel field, has been demonstrated.20 Previously, we © XXXX American Chemical Society

Received: December 13, 2017 Accepted: March 14, 2018 Published: March 14, 2018 A

DOI: 10.1021/acschemneuro.7b00500 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 1. Mixed spatial profiles, obtained by staining with Hoechst 33258 dye and CB1+/PV+ immunofluorescence in the mouse barrel cortex. (A) Nuclear staining with Hoechst 33258 dye delineating the barrel cortex.63 Photomicrograph of the barrel cortex whole mounts of a tangential section. Letters A−E denote rows of the barrels.63 (B) Photomicrograph shows the same tangential section of barrels of S1 mice nuclear staining with Hoechst 33258 dye. Black line delineates the barrel hollows analyzed regions row A (A1h−A4h), row B (B1h−B4h), row C (C1h−C4h), row D (D1h−D4h), and row E (E1h−E4h). White arrow indicated hollow of barrel B3. (C) 3D locations of the spatial profiles located in hollow of barrel B3. Subsection volume depth 10 μm, image of CB1+ puncta (red) and PV+ puncta (green) in the hollows of barrel B3 of the control hemisphere as reconstructed with 3D software. CB1+ and PV+ puncta were immunolabeled as described in a previous double label immunofluorescence staining procedure. (D) 3D locations of the spatial profiles located in the same region hollow of the barrel B3 described in (C); green profiles indicate PV+ interneuron, blue profiles indicate PV− and CB1− neurons, CB1+ puncta (red), PV+ puncta (green). Scale bars represent 100 μm (A,B), 5 μm (C), and 10 μm (D).

song learning and production. 32 On the other hand, cannabinoid-induced deficits correlated with impairment of memory storage33 and spatial memory tasks.34,35 These data suggest that expression of CB1Rs is involved in control of learning, memory, and cognitive functions. Therefore, we hypothesized that endogenous cannabinoid signaling mechanisms representing a key component of CB1+ puncta mobilization contributed to fear-learning-dependent changes in a barrel cortex 24 h after whisker-shock training. However, restoring the function of the PV+ inhibitory circuits normalized network synchrony and basic cortical processing in the barrel cortex 24 h after induction of plastic changes by fear learning. Parvalbumin positive inhibitory neurons are a nonplastic

neuronal population that are indispensable for basic cortical processing in hippocampus,24 and for normal synaptic dynamics in the mouse barrel cortex.36 It may be noted that the patterns of CB1+ and PV+ puncta in the mice S1 cortex do not overlap. Thus, we used confocal microscopy immunofluorescence stereology approaches37,38 to add new data to the knowledge pool concerning the endocannabinoid system and its physiological functions and translational potential for treating a range of human diseases.39 B


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Figure 2. Changes in density of CB1+ puncta in the hollows of row A−E barrels in all groups in experimental (“trained”) and control hemispheres. Values represent the mean numerical density (Nv) of the CB1+ puncta × 105/mm3, ± SEM; ***p < 0.0001. (A) First panel shows Nv intra- and interhemispheral CB1+ puncta networks exist in experimental and control hemispheres in CS+UCS, PSEUDO, CS only, UCS only, and NAIVE a groups of animals. Note that there is a significant difference between experimental (“trained”) and control hemispheres in the group of animals receiving whisker-shock training (CS+UCS); ***p < 0.0001. The asterisk (*) denotes where the experimental hemisphere differs significantly from the control hemisphere of CS+UCS group. (B) Second panel shows Nv interhemispheral differences in CB1+ puncta expression exist in experimental and control hemispheres in hollows of row A−E barrels in all groups of animals; ***p < 0.0001. In the second panel, the triple asterisk denotes where the experimental hemisphere CS+UCS and PSEUDO group differs significantly from the experimental and control hemispheres of control groups. (C) Interhemispherical analysis of Nv of CB1+ labeled puncta in all row barrel hollow areas Ah−Eh (Ah, Bh, Ch, Dh, Eh) CS+UCS, PSEUDO, CS only, UCS only, and NAIVE “trained” hemisphere (exp) and control hemisphere (ctrl); **p < 0.001. Double asterisk denotes where the experimental hemisphere differs significantly from the control hemisphere of the CS+UCS group.

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RESULTS AND DISCUSSION

and PV+ interneurons show strong immunostaining (Figure S3). In tangential sections taken from layer IV of the S1 cortex, nuclear staining with Hoechst 33258 dyes the outline of the barrel cortex (Figure 1A). CB1+ and PV+ puncta in the hollow of the barrel hollow area row barrels A−E (Figure 1B) were subjected to confocal microscopy analysis. The average CB1+ puncta size was 0.96 ± 0.1 μm3 and showed a range of of fluorescence intensity of 1.62 × 103 ± 0.48 ADU (arbitrary densitometry units). The average PV+ puncta size was 1.15 ± 0.03 μm3 and showed a range of fluorescence intensity of 8.40 × 103 ± 1.29 ADU (Figure 1C, D). Size and intensity staining detected for CB1+ and PV+ puncta were similar in all groups of animals. For intra- and interhemispheric comparisons, we counted CB1+ and PV+ puncta in the row B hollow (Bh) barrels and

Distribution of CB1+/PV+ Immunofluorescent Puncta, in Adult Naive Mouse S1 Cortex. Our results obtained for adult naive mice were similar to previously described results for naive rats.28 In layers II/III, CB1+ and PV+ puncta occurred with similar incidence. The highest levels of the PV+ puncta and lowest levels of CB1+ puncta were detected in layers IV and Vb. In layer Va, the opposite was found: higher CB1+ puncta expression was coupled to lower levels of PV+ puncta (Figure S1). The pattern of staining of CB1+ and PV+ puncta was observed in neuronal cell bodies, in their proximal processes and neuropil (Figure S2). The CB1+ neurons are sparse. CB1+ and PV+ puncta occurred around unlabeled cell somata. A number of CB1+ puncta surround the CB1− and PV− neurons in a basketlike manner. PV+ puncta, PV+ axons, C


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Table 1. Intra- and Interhemispheric Changes in the Numerical Density (Nv) of CB1 Puncta × 105/mm3 Expressed by Mean and ±SEM for Each Group: CS+UCS, PSEUDO, CS Only, UCS Only, and NAIVE in Barrel A−E Hollow (Ah−Eh) Regions CS+UCS group exp ctrl PSEUDO group exp ctrl CS only group exp ctrl UCS only group exp ctrl NAIVE group exp ctrl

region Ah

region Bh

region Ch

region Dh

region Eh

4.59 (±0.48) 3.74 (±0.69)

5.45 (±0.20) 3.21 (±0.33)

4.72 (±0.34) 4.13 (±0.13)

4.66 (±0.49) 4.18 (±0.11)

3.54 (±0.20) 4.14 (±0.36)

0.37 (±0.02) 0.46 (±0.06)

0.49 (±0.09) 0.34 (±0.03)

0.68 (±0.11) 0.41 (±0.02)

0.59 (±0.09) 0.40 (±0.02)

0.55 (±0.07) 0.44 (±0.06)

2.98 (±0.25) 3.33 (±0.32)

2.40 (±0.23) 3.04 (±0.27)

2.87 (±0.30) 3.48 (±0.28)

3.25 (±0.37) 3.34 (±0.33)

2.52 (±0.38) 3.32 (±0.45)

3.24 (±1.01) 3.37 (±0.16)

3.11 (±0.56) 2.74 (±0.26)

3.25 (±0.62) 3.68 (±0.60)

3.69 (±0.36) 3.50 (±0.53)

3.18 (±0.35) 3.32 (±0.37)

3.44 (±0.44) 3.25 (±0.83)

3.17 (±0.44) 3.11 (±0.78)

3.31 (±0.41) 3.00 (±0.47)

3.36 (±0.36) 3.23 (±0.47)

3.50 (±0.61) 3.28 (±0.87)

neighboring rows A (Ah), C (Ch), D (Dh), and E (Eh) in both hemispheres (experimental, i.e., right and control, i.e., left hemisphere). Data such as those shown in Figure 2A NAIVE did not show any significant intra- or interhemispheric changes in the numerical density (Nv) of CB1+ puncta experimental vs control barrel hollows in the barrel cortex (Table 1, NAIVE; p > 0.05). Data such as those shown in Figure 3 NAIVE (first panel) did not show any significant intra- or interhemispheric changes in Nv of PV+ puncta exp, i.e., right vs control, i.e., left barrel hollows in the barrel cortex (Table 2, NAIVE; p > 0.05). The Specificity of the Whisker-Shock Conditioning (CS+UCS). The objective is to fill the gaps of knowledge in our understanding of the specificity of the whisker-shock training by investigating the “trained” and nontrained barrels in the “trained” hemisphere compared to that of controls. It seems possible that the entire “trained” hemisphere could have shown increase/decrease in CB1+ and/or PV+ puncta. Indeed, if interand intrahemispheral CB1+ and PV+ puncta networks exist, we would presume the whisker-shock group to have vulnerability in the “trained” cortex compared to that of controls. To examine the specificity of the CS+UCS training, we have compared intra- and interhemispheral effects of training concerning CB1+ and PV+ puncta and their control group littermates. We counted CB1+ and PV+ puncta within “trained hollow” rows B (Bh) and nontrained neighboring rows Ah, Ch, Dh, and Eh in “trained” and nontrained hemisphere. The numerical density (Nv) of CB1+ and PV+ puncta was compared between 5 measured regions (Figure 1A,B) in the same hemisphere (intrahemispheric differences) and between the hemispheres receiving input from the stimulated vibrissae and the contralateral, unstimulated side of the brain (interhemispheral effect). The results obtained from supporting pilot experiments show that most of CB1+ puncta are also immunopositive for VGAT-GABA transporter in the membrane of synaptic vesicles localized to the same axon terminals (see Figures S4 and S5). Behavioral Responses. In the first session of habituation (H), animals reacted to the head holder by turning their head in all directions. In the course of habituation, the number of head turnings decreased significantly from 32.87 ± 0.90 to 5.0 ± 0.37 in the 21 session (paired two-tailed Student’s t tests, t = 27.0; p < 0.0001). This shows that the animals were habituated

Figure 3. Changes in density of PV+ puncta in the hollows of row A− E barrels in all groups in experimental (“trained”) and control hemispheres. The values represent the mean numerical density (Nv) of the PV+ puncta × 106/mm3, ± SEM. Comparisons in all groups in experimental (“trained”) and control hemispheres. First and second panels show Nv intra- and interhemispheral PV+ puncta networks exist in experimental and control hemispheres in CS+UCS, PSEUDO, CS only, UCS only, and NAIVE a groups of animals. Note that there is no significant difference between experimental (“trained”) and control hemispheres in the group of animals receiving whisker-shock training (CS+UCS).

to the neck restraint. In mice from NAIVE group the number of head turnings did not change. D


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Table 2. Intra- and Interhemispheric Changes in the Numerical Density (Nv) of PV Puncta × 106/mm3 Expressed by Mean and ±SEM for Each Group: CS+UCS, PSEUDO, CS Only, UCS Only, and NAIVE in Barrel A−E Hollow (Ah−Eh) Regions CS+UCS group exp ctrl PSEUDO group exp ctrl CS only group exp ctrl UCS only group exp ctrl NAIVE group exp ctrl

region Ah

Region Bh

region Ch

region Dh

region Eh

2.30 (±0.50) 1.99 (±0.07)

2.42 (±0.25) 1.93 (±0.19)

1.84 (±0.32) 1.77 (±0.37)

2.01 (±0.46) 2.37 (±0.42)

1.83 (±0.50) 1.63 (±0.43)

1.64 (±0.25) 1.92 (±0.17)

1.56 (±0.26) 1.96 (±0.20)

1.91 (±0.23) 1.95 (±0.12)

2.07 (±0.24) 1.76 (±0.15)

1.69 (±0.10) 1.82 (±0.23)

1.85 (±0.13) 2.29 (±0.35)

2.18 (±0.50) 1.97 (±0.20)

2.34 (±0.56) 2.92 (±0.23)

2.79 (±0. 34) 2.78 (±0.27)

2.02 (±0.24) 2.58 (±0.30)

1.98 (±0.16) 1.85 (±0.27)

1.57 (±0.22) 1.56 (±0.23)

1.46 (±0.32) 1.25 (±0.37)

1.57 (±0.21) 1.24 (±0.08)

1.72 (±0.28) 1.72 (±0.25)

2.24 (±0.60) 2.56 (±0.85)

2.06 (±0.33) 2.37 (±1.0)

2.63 (±0.28) 3.22 (±0.23)

2.22 (±0.27) 2.07 (±0.65)

2.90 (±0.19) 2.85 (±0.15)

animals subjected to different behavioral manipulations. In experimental and control littermates which were subjected to pseudoconditioned procedure, expression of CB1+ was less pronounced than that in CS+UCS, CS only, UCS only, and NAIVE mice. Analysis of N v CB1+ Puncta in Barrel Cortex in Experimental Groups. Whisker-Shock Conditioned Barrel B Hollows (CS+UCS-Bh) Trained Hemisphere. Interhemispheral comparisons of CB1+ puncta show that Nv of CB1+ labeled puncta in the trained Bh CS+UCS group surpasses the mean Nv of CB1+ labeled puncta in control Bh by 69% (exp: 5.45 ± 0.2 × 105/mm3 vs ctrl: 3.21 ± 0.33 × 105/mm3, F9,89 = 16.16, p < 0.0001; Figure 2A, CS+UCS, Figures 4 and 5, Table 1). ANOVA followed by Bonferroni test revealed that there was also a significant difference in Nv of CB1+ puncta in the hollow trained row B (Bh) and Nv of CB1+ puncta in Bh in all examined groups: PSEUDO, CS only, UCS only, naive (Figure 2B Bh, Table 1). This shows that whisker-shock conditioning had a significant increasing effect on expression of CB1+ puncta in “trained” barrel B hollows, and mechanisms mediating that results are specific to the role of maintaining balance between corresponding barrel hollow regions. No Trained Barrel A Hollow (CS+UCS, Ah) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also a significant difference (F9,50 = 10.42, p < 0.0001) between the mean Nv of CB1+ puncta in the Ah experimental hemisphere CS+UCS group (exp: 4.59 ± 0.48 × 105/mm3) vs the mean Nv of CB1+ puncta in Ah in the pseudoconditioned group: PSEUDO (exp: 0.37 ± 0.02 × 105/ mm3 and ctrl: 0.46 ± 0.06 × 105/mm3) (Figure 2B Ah, Table 1). No Trained Barrel C Hollows (CS+UCS, Ch) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also a significant difference (F9,93 = 14.88, p < 0.0001) between the mean Nv of CB1+ puncta in the Ch trained hemisphere CS+UCS group (exp: 4.72 ± 0.34 × 105/mm3) vs the mean Nv of CB1+ puncta in Ch in the PSEUDO group (exp: 0.68 ± 0.11 × 105/mm3 and ctrl: 0.41 ± 0.02 × 105/mm3) (Figure 2B Ch, Table 1). No Trained Barrel D Hollows (CS+UCS, Dh) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also a significant difference (F9,94 = 18.08, p < 0.0001) between the mean Nv of CB1+ puncta in the

In mice from the UCS only group, the number of head turnings decreased from 10.0 ± 1.90 in the first session to 3.85 ± 0.76 in the third session (paired two-tailed Student’s t test, t = 2.52; p = 0.045). This shows that a tail shock applied alone produced a definite observable response, i.e., reduction of head turnings. The decrease has not been observed in the naive group (NAIVE in the first session 5.2 ± 0.868; in the third session 4.8 ± 0.58; t = 0.78; p = 0.46). During the initial session of whisker-shock conditioning (CS +UCS), the mice often reacted to vibrissal stimulation (CS) by turning their head toward the stimulus. However, in the course of CS+UCS, the number of head turnings decreased from 18.42 ± 1.13 in the first session to 3.14 ± 0.79 in the third session (paired two-tailed Student’s t test, t = 9.55; p < 0.0001). The decrease has not been observed in the case of pseudoconditioning (PSEUDO in the first session 21.28 ± 1.08; in the third session 26.85 ± 4.22; t = 1.70; p = 0.14) and in case of CS only (CS only the first session 21.14 ± 3.29; in the third session 20.42 ± 2.54; t = 0.43; p = 0.67). This shows that only during whisker-shock conditioning (CS+UCS) sessions animals learn freezing (Figure S6). Analysis of Nv CB1+ Puncta in Barrel Cortex (Intrahemispheral Effect). After whisker-shock conditioning (CS +UCS), pseudoconditioning (PSEUDO), whisker stimulation alone (CS only), tail shock alone (UCS only), and in naive animals CB1+ puncta were observed in the hollows of the “trained” row B of barrels (Bh) and untrained rows A, C, D, and E of barrels (Ah, Ch, Dh, Eh) in experimental and control hemispheres. In the hollows of the “trained” row B of barrels (Bh) and untrained rows A, C, D, and E of barrels (Ah, Ch, Dh, Eh) in the experimental hemisphere Nv of CB1+ puncta, labeling was similar (Figure 2A CS+UCS exp, Figure 2A PSEUDO exp, Figure 2A CS only exp, Figure 2A UCS only exp, and Figure 2A NAIVE exp; p > 0.05). In the hollows of the “trained” row B of barrels (Bh) and untrained rows A, C, D, and E of barrels (Ah, Ch, Dh, Eh) in the control hemisphere Nv of CB1+ puncta, labeling was similar (Figure 2A CS+UCS ctrl, Figure 2A PSEUDO ctrl, Figure 2A CS only ctrl, Figure 2A UCS only ctrl, Figure 2A NAIVE ctrl; p > 0.05). Nevertheless, the possibility of differences in Nv CB1+ puncta intrahemispheral expression did not cease to exist for E


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Dh trained hemisphere CS+UCS group (exp: 4.66 ± 0.49 × 105/mm3) vs the mean Nv of CB1+ puncta in Dh in the PSEUDO group (exp: 0.59 ± 0.09 × 105/mm3 and ctrl: 0.40 ± 0.02 × 105/mm3) (Figure 2B Dh, Table 1). No Trained Barrel E Hollows (CS+UCS, Eh) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also a significant difference (F9,86 = 12.64, p < 0.0001) between the mean Nv of CB1+ puncta in the Eh trained hemisphere CS+UCS group (exp: 3.54 ± 0.20 × 105/mm3) vs the mean Nv of CB1+ puncta in Eh in the PSEUDO group (exp: 0.55 ± 0.07 × 105/mm3 and ctrl: 0.44 ± 0.06 × 105/mm3) (Figure 2B Eh, Table 1). Our data indicate also that pseudoconditioning had a large and significant decreasing effect on expression of CB1+ puncta in both the trained and untrained hemispheres as compared to CS+UCS, CS only, UCS only, and NAIVE groups of mice. Pseudoconditioned Barrel B Hollows (PSEUDO, Bh) Trained Hemisphere. Interhemispheral comparisons of CB1+ puncta show that Nv of CB1+ labeled puncta in pseudoconditioned Bh do not differ significantly from the mean Nv of CB1+ labeled puncta in control Bh (exp: 0.49 ± 0.09 × 105/mm3 vs ctrl: 0.34 ± 0.03 × 105/mm3) (Figure 2A PSEUDO). ANOVA followed by Bonferroni test revealed that there was also a significant difference (F9,89 = 16.16, p < 0.0001) in Nv of CB1+ puncta in the hollows of pseudoconditioned row B (Bh) and Nv of CB1+ puncta in Bh in all examined groups: CS+UCS, CS only, UCS only, and naive (Figure 2B Bh, Table 1). No-Trained Barrel A Hollows (PSEUDO, Ah) Trained Hemisphere. The present post pseudoconditioning data demonstrate that there is also a significant difference (F9,50 = 10.42, p < 0.0001) between the mean Nv of CB1+ puncta in the Ah experimental hemisphere PSEUDO group (exp.: 0.37 ± 0.02 × 105/mm3) vs the mean Nv of CB1+ puncta in Ah in the whisker-shock CS+UCS, CS only, UCS only, and naive group (Figure 2B Ah, Table 1).

Figure 4. Photomicrograph showing tangential section of barrel rows A and B of S1 cortex 24 h after whisker-shock training (CS+UCS). Barrel hollow area was detected on each Hoechst 33258 dye image. Only CB1+ (red) puncta inside the barrel hollows were counted. Scale bar = 20 μm.

Figure 5. Three-dimensional (3D) reconstruction of CB1+ and PV+ elements in quadratic prism located in the hollow of row B (Bh). Note the high density of CB1+ puncta located in the CS+UCS trained side compared to control side. Scale bar = 10 μm. F


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control hemisphere (NAIVE exp: 3.31 ± 0.19 × 105/mm3 vs ctrl: 3.16 ± 0.32 × 105/mm3, paired two-tailed Student’s t tests, t = 0.166; p = 0.868, Figure 2C NAIVE). Analysis of N v PV+ Puncta in Barrel Cortex in Experimental Groups. After whisker-shock conditioning (CS +UCS), pseudoconditioning (PSEUDO), whisker stimulation alone (CS only), tail shock alone (UCS only), and in naive animals, PV+ puncta were observed in the hollows of the “trained” row B of barrels (Bh) and untrained rows A, C, D, and E of barrels (Ah, Ch, Dh, Eh) in experimental and control hemispheres. In the hollows of the “trained” row B of barrels (Bh) and untrained rows A, C, D, and E of barrels (Ah, Ch, Dh, Eh) in the experimental hemisphere, Nv of PV+ puncta labeling was similar (Figure 3 CS+UCS exp, Figure 3 PSEUDO exp, Figure 3 CS only exp, Figure 3 UCS only exp, Figure 3 NAIVE exp, first panel; p > 0.05, Table 2). In the hollows of the “trained” row B of barrels (Bh) and untrained rows A, C, D, and E of barrels (Ah, Ch, Dh, Eh) in the control hemisphere, Nv of CB1+ puncta labeling was similar (Figure 3 CS+UCS ctrl, Figure 3 PSEUDO ctrl, Figure 3 CS only ctrl, Figure 3 UCS only ctrl, Figure 3 NAIVE ctrl, first panel; p > 0.05, Table 2). Nevertheless, the possibility of differences in Nv PV+ puncta intrahemispheral expression did not cease to exist for animals subjected to different behavioral manipulations. In experimental and their control littermates which were subjected to CS+UCS procedure, PV+ puncta expression was more pronounced than in PSEUDO, CS only, UCS only, and NAIVE mice. Whisker-Shock Conditioned Barrel B Hollows (CS+UCSBh) Trained Hemisphere. Interhemispheral comparisons of PV + puncta show that Nv of PV+ labeled puncta in trained Bh CS +UCS group is a failure in the mean Nv of PV+ labeled puncta in control Bh (exp: 2.42 ± 0.25 × 106/mm3 vs ctrl: 1.93 ± 0.19 × 106/mm3, F9,86 = 0.94, p = 0.49, considered not significant, post tests were not calculated because the p value was greater than 0.05; Figure 3 CS+UCS, first panel, Figure 5, Figure 6, Table 2). ANOVA revealed that there was no significant difference in Nv of PV+ puncta in hollow trained row B (Bh) and Nv of PV+ puncta in Bh in all examined groups: PSEUDO, CS only, UCS only, and naive (Figure 3 Bh, second panel, Table 2). No-Trained Barrel A Hollows (CS+UCS, Ah) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also no significant difference (F9,44 = 0.62, p = 0.77) between the mean Nv of PV+ puncta in the Ah experimental hemisphere CS+UCS group (exp: 2.30 ± 0.50 × 106/mm3) vs the mean Nv of PV+ puncta in Ah in the pseudoconditioned group PSEUDO, CS only, UCS only, and naive (Figure 3 Ah, second panel, Table 2). No-Trained Barrel C Hollows (CS+UCS, Ch) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also no significant difference (F9,94 = 2.94, p > 0.05) between the mean Nv of PV+ puncta in the Ch trained hemisphere CS+UCS group (exp: 1.84 ± 0.32 × 106/mm3) vs the mean Nv of PV+ puncta in Ch in the PSEUDO group, CS only, UCS only, and naive (Figure 3 Ch, second panel, Table 2). No-Trained Barrel D Hollows (CS+UCS-Dh) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also no significant difference (F9,108 = 1.99, p > 0.05) between the mean Nv of PV+ puncta in the Dh trained hemisphere CS+UCS group (exp: 2.01 ± 0.46 ×

No-Trained Barrel C Hollows (PSEUDO, Ch) Trained Hemisphere. The present post pseudoconditioning data demonstrate that there is also a significant difference (F9,93 = 14.88, p < 0.0001) between the mean Nv of CB1+ puncta in the Ch trained hemisphere PSEUDO group (exp.: 0.68 ± 0.11 × 105/mm3) vs the mean Nv of CB1+ puncta in Ch in the CS +UCS group, CS only, UCS only, and naive (Figure 2B Ch, Table 1). No-Trained Barrel D Hollows (PSEUDO-Dh) Trained Hemisphere. The present post pseudoconditioning data demonstrate that there is also a significant difference (F9,94 = 18.08, p < 0.0001) between the mean Nv of CB1+ puncta in the Dh trained hemisphere PSEUDO group (exp.: 0.59 ± 0.09 × 105/mm3) vs the mean Nv of CB1+ puncta in Dh in the CS +UCS group, CS only, UCS only, and naive (Figure 2B Dh, Table 1). No-Trained Barrel E Hollows (PSEUDO-Eh) Trained Hemisphere. The present post pseudoconditioning data demonstrate that there is also a significant difference (F9,86 = 12.64, p < 0.0001) between the mean Nv of CB1+ puncta in the Eh trained hemisphere PSEUDO group (exp.: 0.55 ± 0.07 × 105/ mm3) vs the mean Nv of CB1+ puncta in Eh in the CS+UCS group, CS only, UCS only, and naive (Figure 2B Eh, Table 1). Comparison of the Interhemispheric Effect of Whisker-Shock Conditioning (CS+UCS) Pseudoconditioning (PSEUDO), CS Only, UCS Only, and Naive Animals upon Nv CB1+ Puncta Labeling in All Five Rows in Barrel Hollows [A (A1h−A4h), B (B1h−B4h), C (C1h−C4h), D (D1h−D4h), and E (E1h−E4h)]. Interhemispheric comparisons of Nv CB1+ puncta in all five rows A−E barrel hollow areas in whisker-shock (CS+UCS) experimental hemisphere surpass significantly Nv CB1+ labeled puncta in control hemisphere by 25% (exp: 4.75 ± 0.18 × 105/mm3 vs ctrl: 3.76 ± 0.2 × 105/mm3, paired two-tailed Student’s t tests, t = 4.302; p < 0.001, Figure 2C CS+UCS). This shows that whisker-shock conditioning had a significant increasing effect on expression of CB1+ puncta in “trained” hemisphere, and mechanisms mediating the results are specific to the integration between barrel hollow regions. Interhemispheric comparisons of Nv CB1+ puncta in all five row A−E barrel hollow areas in pseudoconditioned (PSEUDO) experimental hemisphere did not differ significantly from Nv of CB1+ labeled puncta in control hemisphere (exp: 0.55 ± 0.04 × 105/mm3 vs ctrl: 0.42 ± 0.02 × 105/mm3, paired two-tailed Student’s t tests, t = 0.72; p = 0.476, Figure 2C PSEUDO). This shows that pseudoconditioning has a large and decreasing effect on expression of CB1+ puncta in both trained and untrained hemisphere. Interhemispheric comparisons of Nv CB1+ puncta in all five row A−E barrel hollow areas in CS only, and UCS only experimental hemisphere did not differ significantly from Nv CB1+ labeled puncta in control hemisphere (CS only exp: 2.83 ± 0.14 × 105/mm3 vs ctrl: 3.29 ± 0.14 × 105/mm3, paired twotailed Student’s t tests, t = 1.594; p = 0.118, Figure 2C CS only; UCS only exp: 3.33 ± 0.21 × 105/mm3 vs ctrl: 3.31. ± 0.2 × 105/mm3, paired two-tailed Student’s t tests, t = 0.651; p = 0.518, Figure 2C UCS only). This shows that whisker stimulation alone and tail shock alone have no effect on expression of CB1+ puncta in both trained and untrained hemisphere. Interhemispheric comparisons of Nv CB1+ puncta in all five row A−E barrel hollow areas in naı̈ve experimental hemisphere did not differ significantly from Nv CB1+ labeled puncta in G


Research Article


shock (CS+UCS) conditioning but not during pseudoconditioning sessions animals learn to fear.3,20 The present study suggests that the accompanying learning ability of rational behavior promotes increased expression of CB1+ puncta and is accompanied by plastic changes depending on the learning in the S1 cortex. Pseudoconditioning paradigm generates lack of ability to rationalize the assessment of the situation which led to a reduction in expression of CB1+ puncta in the trained row, trained and control hemisphere compared to CS+UCS, CS only, UCS only, and naive animals. These findings suggest that pseudoconditioning disrupts the pathways and mechanisms mediating expression of CB1+ puncta are specific to the role of maintaining balance, and integration between corresponding somatosensory areas. Here, we investigated the functional plasticity of the barrel hollow area S1 cortex of the adult mice, nonoverlapping populations CB1+ and PV+ puncta, 24 h after whisker-shock (CS+UCS) and control tests. We found that stressed mice exposed to whisker-shock associative training exhibited fear response, acquired the head turning conditioned response at a facilitated rate when stimuli were presented in paired manner. An increase of Nv of CB1+ puncta in trained hemisphere only “trained” row B hollow, suggest learning-dependent active CB1+ interneuron specific connectivity. In contrast, stressed mice exposed to unpaired stimuli (PSEUDO) were not exhibiting a fear response and that induced a massive reduction in CB1+ puncta expression intra and inters barrel cortex hemispheres. In all groups, in barrel cortex both trained and untrained hemisphere PV+ puncta exhibit stable expression. These results suggest that exposure to inescapable stress facilitates PV+ network in associative and control tests in the mouse. On whisker-shock conditioning but not on pseudoconditioning PV+ network prevents CB1+ puncta lost. It is well documented that fear conditioning induces changes in the synaptic connectivity of excitatory and inhibitory markers in the different brain areas.20,40−46 Increased density of CB1+ puncta, to whisker-shock conditioning, seems be relevant for suppressing the input associative related information to “trained” barrel hollow target neurons. Interestingly, in the hippocampal region, it has been found that the interneurons that express the CB1+ receptors are located at the stratum radiatum and stratum lacunosum border and they target the dendritic region of pyramidal cell. They decrease their firing during sharp wave ripple oscillations and were strongly thetamodulated during running periods. It could participate in plastic changes taking place on pyramidal cell dendrites during sharp wave ripple oscillations and in the precise coordination of dendritic inputs during theta rhythm.47 Recently, it has been discovered that in the hippocampal region cannabinoid control of learning and memory through regulation of dendritic excitability has taken place by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that underlie the hcurrent (Ih). Activation of the CB1R-HCN pathway impairs dendritic integration of excitatory inputs, long-term potentiation (LTP), and spatial memory formation.48 If a similar mechanism CB1R-Ih pathway in the trained barrel B hollow accompanies whisker-shock dependent upregulation of CB1+ puncta, then c-Jun-N-terminal kinases (JNKs), nitric oxide synthase, and intracellular cGMP should show an increase in target cells located in regions in which plastic changes were previously detected by 2DG labeling. The increase in the Nv of CB1+ puncta as a specific after-effect of whisker-shock conditioning observed presently may be sensitive to cannabi-

Figure 6. Photomicrograph showing tangential section of barrel rows A and B of S1 cortex 24 h after whisker-shock training (CS+UCS). Barrel hollow area was detected on each Hoechst 33258 dye image. Only PV+ (green) puncta inside the barrel hollows were counted. Scale bar = 20 μm.

106/mm3) vs the mean Nv of CB1+ puncta in Dh in the PSEUDO group, CS only, UCS only, and naive (Figure 3 Dh, second panel, Table 2). No-Trained Barrel E Hollows (CS+UCS-Eh) Trained Hemisphere. The present post fear conditioning data demonstrate that there is also no significant difference (F9,70 = 2.88, p > 0.05) between the mean Nv of PV+ puncta in the Eh trained hemisphere CS+UCS group (exp: 1.83 ± 0.50 × 106/mm3) vs the mean Nv of CB1+ puncta in Eh in the PSEUDO group, CS only, UCS only, and naive (Figure 3 Eh, second panel, Table 2). This shows that whisker-shock conditioning (CS+UCS), PSEUDO, CS only, and UCS only experimental situation had no significant increasing effect on expression of PV+ puncta in “trained” barrel B hollows, or in no trained barrel hollows located in the trained hemisphere. Similar expression of PV+ puncta observed in experimental and naive animals suggests that stable density of its population seems to apply widely. Taken together, we found that whisker-shock conditioning (CS+UCS) led to an increase in expression of CB1+ puncta in the trained row and trained hemisphere compared to pseudoconditioning. Sharp contrast in CB1+ puncta expression detected in the whisker-shock conditioned hemisphere and corresponding cortical fields of pseudoconditioned groups suggests that mechanisms mediating CB1+ puncta expression are specific to the role of maintaining balance and integration between corresponding somatosensory areas. We confirmed also previously described data that show that during whiskerH


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synaptic dynamics in the mouse barrel cortex. Previously we found that associative whisker-shock training induced a large increase in the density of symmetrical synapses in the hollow of “trained” barrel compared to controls. The supernumerary GABAergic synapses were found on double-synapse spines, which showed an increased number after whisker-shock conditioning. Pseudoconditioning led to an increase only in the density of asymmetrical synapses on single-synapse spines, whereas whisker stimulation alone increased the density of excitatory synapses in an unspecified pool.20 Moreover, in the barrel cortex, the density of PV+ interneurons23 and PV+ puncta (present study) is not altered by whisker-shock associative learning and any controls. However, PV+ interneurons and puncta represent the major population of layer IV inhibitory circuit which can indirectly promote previously observed whisker-shock dependent increase in density of double synapse spines. Recently Chen and colleagues 59 reported that selective activation of PV+ interneurons prevents stress-induced synapse loss and stressrelated disorders. Our present data is supporting conception that only whisker-shock associative activation of PV+ interneurons prevents observed previously symmetrical synapse loss in the barrel cortex.20 Our present results suggest this compound to interact/ interfere with cholinergic mechanism and could affect whiskershock to CB1+ puncta, which significantly increases in the hollows of the “trained” row B of barrels but not in controls, is related to the neuronal activity resulting from stimulus presentation. Pseudoconditioning schedule probably does cause cholinergic projection confusion and concomitant plastic changes in S1 cortex losses. However, pseudoconditioned mice seem to learn something, namely, that they cannot escape from an aversive stimulation or that the neck restraining apparatus is related to aversive stimuli. Only during whisker-shock training (CS+UCS) sessions does the mouse learn to fear and have head turning accompanied fear conditioning (Figure S6). Both kinds of acquired experience have been described previously3,20 and were detectable also in our present study using the same behavioral paradigm. Previously, Marsicano and colleagues60 reported that the CB1 signaling controls passive fear responses necessary for extinction of freezing in fear conditioning. In any case, our findings seem to indicate upregulation of CB1+ puncta in a learning-dependent manner. Thus, the endogenous activity of CB1 receptors is necessary for CS+UCS association while destructive integration paradigm of neuronal circuits in pseudoconditioned animals with cellular-level precision decreased CB1+ puncta density. Probably CB1 signaling controls fear responses necessary for freezing in whisker-shock conditioning. In contrast, pseudoconditioned animals behaviorally did not show freezing and the endogenous activity of CB1Rs signaling is not present 24 h after pseudoconditioning. Tail shock applied alone in the UCS only group produces an observable response, i.e., reduction of head turnings and automatic fear responses, but that CB1+ puncta density intra- and interhemispherically did not change significantly. Therefore, the endogenous activity of CB1Rs located in barrel cortex inter- and intrahemispherical regions is not directly responsible for freezing and automating responses to painful stimuli applied to the tail without whisker stimulation. In control groups, naive animals (Student’s t test, p = 0.46) or whisker stimulation applied alone (CS only group; Student’s t test, p = 0.67) did not show any reduction in head

noid control plasticity similar to some forms of hippocampaldependent short-term memory.49 Interestingly, Kirschmann and colleagues50 found that, in adolescent rats, prior small doses of self-administration of the cannabinoid receptor agonist WIN55, 212-2 resulted in improved working memory performance in adulthood. Future CB1R agonist studies in whiskershock fear conditioning trained mice in connection with the present findings should be extremely informative. The fact that pseudoconditioned mice exhibited reduced Nv CB1+ puncta in both intra- and interhemispherical responses to aversive stimulation applied randomly relative to whisker stimulation argues for incorrect suppression of nonassociative information and consequently a disturbed cortical plasticity in pseudoconditioned mice. In fact, pseudoconditioning disrupts correct fear learning acquisition. The mice which received whisker stimulation alone (CS only) and tail-shock alone (UCS only) exhibit inter- and intrahemispherical Nv CB1+ puncta similar to naive animals group (NAIVE). Assuming that whisker-shock activity induces rewiring, what is its relevance? Now, we report that, in mice 24 h after whisker-shock training, the specific increased density of CB1+ might lead to the observed previously synapse remodeling.20 Activity-dependent synaptic plasticity has been detected for both inhibitory and excitatory neurons in the brain.51−54 An increased Nv of CB1+ puncta 24 h after whisker-shock training may reflect selectivity in cholinergic modulation of CCK+ interneurons, which exhibit large, neuronal nicotinic cholinergic receptor (nAChR) mediated depolarization.55 We found previously that whisker-shock training increased the density of CCK+ interneurons.22 However, we must remember that not only CCK+ interneurons, but also 63% of somatostatinexpressing (SST+) and 69% of vasoactive intestinal polypeptide-expressing (VIP+) interneurons and also glutamatergic neurons coexpress CB1Rs.56 Thus, selectively increasing in density of CB1+ puncta in the row B barrel hollow area suggests new functional CB1Rs mediating endocannabinoid effects on neurotransmission to modulate a specific whiskershock learning-dependent barrel cortex network. By contrast, interneurons PV+ are weakly affected by nicotinic receptor agonists.57 The fact that whisker-shock trained mice did not exhibit fear conditioning-mediated increased in Nv of PV+ puncta underscores the importance of cholinergic circuits rewiring for whisker-shock induced S1 plastic changes. Furthermore, the lack of expression CB1+ puncta during pseudoconditioning in all regions of the barrel field, we discovered in the present study, suggests impaired cholinergic cortico-cortical and thalamocortical circuits. Wall and colleagues58 reported that the major classes in the barrel cortex neurons (PV+, SST+, or VIP+) integrate feedforward and feedback information from throughout the brain to modulate the activity of the local cortical circuit. All three classes of interneurons received considerable input from known cortical and thalamic input sources, as well as from probable cholinergic cells in the basal nucleus of Meynert. However, more input to VIP+ than SST+ interneurons suggests that disinhibition of the cortex via VIP+ cells, which inhibit SST + cells, might be a general feature of long-distance corticocortical and thalamocortical circuits. The connectivity of PV+ puncta to barrel hollow principal neurons was unaltered in all regions of the barrel field of all groups. Our present and previous23 findings suggest that PV+ inhibitory circuit in the barrel cortex is crucial for normal I


Research Article


For tail-shock alone (UCS only, n = 7), seven experiment and seven control hemispheres were collected. In this group, whisker stimulation described above was omitted but a single tail-shock was applied for the same duration and the same number of times as in whisker-shock conditioning training. For naive mice (NAIVE, n = 5), five right and five left hemispheres were collected from the nonstimulated control mice. Averaged data from of the barrel hollow area row A barrels (A1h−A4h), from row B barrels (B1h−B4h), from row C barrels (C1h−C4h), from row D barrels (D1h−D4h), and from row E barrels (E1h−E4h) from five right and five left hemispheres were collected from the nonstimulated controls. Behavioral Responses. To evaluate the effects of habituation to a head holder, which requires 21 sessions (10 min per day), we examined head turning during the first and the last session. In the UCS only group, head turnings were counted during 9 s immediately before delivery of the tail shock. In the naive group, head turnings were counted during 9 s similarly to the UCS only group but the tail shock was not delivered. To evaluate the effects of training, we examined head turning in response to CS in all groups. In CS+UCS, PSEUDO, and CS only groups, head turnings were counted in the time during application of row B whisker stroking. Tissue Sampling. At 24 h after the end of the experiments, the animals were euthanized with an overdose of Nembutal (150 mg/kg i.m.) and perfused transcardially with 20 mL of 0.9% saline-heparin (5000 IU/L), followed by 150 mL of cold fixative composed of 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS), pH = 7.4. The brains were then removed and postfixed in PFA for 2 h at 4 °C. The fixed brains were cryoprotected by treatment with 10%, 20%, and 30% sucrose solution sequentially, then frozen (−70 °C) and cryosectioned (−18 °C) paracoronally68 (see Figure S1) and tangentially to the barrel field in sections (100 μm thickness) using a cryostat. The section thickness was confirmed by focusing up and down through the sections, and no significant differences were detected; thickness and block advance BA = 100 μm. The BA (i) determines the hitting probability of the particles within the block, (ii) avoids deformation in the z-axis (the height),62 and (iii) avoids mutability in the barrel area which could be related to differences in the cutting plane. Sections were then collected in 0.1 M PBS. Only one section taken from layer IV of the S1 cortex, where rows A−E were readily visible under low magnification, was used in present CB1+/PV + immunofluorescent study. The barrel hollow regions were defined according to criteria of Woolsey and Van der Loos.63 Double Label Immunofluorescence Staining Procedure CB1+/PV +. Sections were washed in BBS for 10 min before blocking of nonspecific endogenous peroxidase activity with 1% hydrogen peroxide for duration of 30 min at room temperature free floating. A further wash was performed at room temperature with (a) 10% NGS and (b) 10% NHS (Vector Laboratories, Inc., Burlingame, CA) for 2 h to exhaust endogenous peroxidase activity and to block nonspecific reaction, respectively. Sections were then incubated free floating with (a) rabbit polyclonal anti-CB1 (1:500; ab23703, Abcam, Cambridge, UK) raised against the C-terminal amino acid 461−472 of human CB1; (b) mouse antiparvalbumin (1:12000; PARV-19, P3088; Sigma-Aldrich, Gillingham, Dorset, UK) for 48 h at 4 °C under vigorous shaking. Sections were then washed in BBS for 3 × 10 min then incubated free floating with (a) biotinylated anti-rabbit IgG (1:100; Vector Laboratories, Inc., Burlingame, CA) followed by Texas Red or Alexa Red fluorescence (1:100; Vector Laboratories, Inc., Burlingame, CA); (b) biotinylated anti-mouse IgG (1:100; Vector Laboratories, Inc., Burlingame, CA) followed by fluorescein avidin DCS (1:100; Vector Laboratories, Inc., Burlingame, CA, or Alexa 561 1:500 dilution; Molecular Probes, green fluorescence). Nuclear staining with Hoechst 33258 (0.5 μg/mL; Molecular Probes, Carlsbad, CA) delineated the barrel cortex prior to mounting in Vectashield Mounting Medium (Vector Laboratories, Inc., Burlingame, CA). Sections from the “trained” and control hemispheres were processed together. In all cases, the experimenter conducting the analysis was blinded to group identity.

turning, did not show an increase in cortical CB1+ puncta expression, and fear responses were never to be observed. Decreased cholinergic input in APP/PS1 double transgenic mice61 possibly diminishes CB1 present interneuron response to aversive stimulation, should prevents density symmetrical synapses gain observed 24 h after CS+UCS20 and impairs learning acquirement, while treatment with a cholinergic agonist should restore whisker-shock learning dependent changes in APP/PS1 mice. Future experiments are designed to influence the expression of CB1Rs in mouse brain after whisker-shock learning, delay, and extinction as well as to elucidate the additional influences assumed to control the completion of the memory trace and potentially in human learning disorders.

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METHODS

Animals. Thirty-three 2-month-old Swiss-Webster mice (25−30g) were used in the study. The mice were reared in a 12:12 light/dark cycle in standard cages, and they had ad libitum access to water and food. The mice were given a habituation period to become accustomed to a neck restraint by being placed in a restraining apparatus for 10 min a day for 21 days prior to the start of experiments. All experimental procedures were approved by the First Ethical Commission in Warsaw, Poland (Permit Number: No. 652/2014) and were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize the number of animals used and their suffering. After habituation sessions, the mice were divided into the following five groups: whisker-shock conditioning (CS+UCS), pseudoconditioning (PSEUDO), whisker-stimulation alone (CS only), tail shock alone (UCS only), and naive animals. Behavioral Training and Testing. For whisker-shock conditioning (CS+UCS, n = 7), seven experimental and seven control hemispheres were collected. The mice were placed in the restraining apparatus, and row B vibrissae were stimulated manually using a fine paint brush. Tactile stimulation (CS) comprised three strokes on one side of the snout. Each stroke lasted for 3 s and was applied in the posterior− anterior direction along row B of the mystacial vibrissae. Great care was taken not to touch adjacent rows of whiskers or the fur growing between the rows. In the last second of the last stroke, a tail shock representing the UCS was applied (single, square, pulse 0.5 s, 0.5 mA). The electrical stimulation was discontinued simultaneously with the end of stroking. After a 6 s interval, the trial was repeated. The CS +UCS stimuli were repeated four times per minute, for 10 min per day, for 3 consecutive days. Animals received 120 pairings of CS+UCS trials of whisker-shock conditioning.1 Fort pseudoconditioning (PSEUDO, n = 7), seven experiment and seven control hemispheres were collected. In the pseudoconditioning schedule (active control), animals received stimulation of row B vibrissae (CS), which comprised three strokes on one side of the snout. Each stroke lasted for 3 s and was applied in the posterior− anterior direction along row B of the mystacial vibrissae. Pseudoconditioning schedule allows that the CS was presented regularly every 15 s, and UCS was presented at random relative to CS. These mice received the same number of CS and UCS per session as applied for whisker-shock fear conditioning but CS and UCS has never been paired. The pseudoconditioning schedule was applied for 10 min per day, which was maintained for 3 consecutive days.1 For whisker stimulation alone (CS only, n = 7), seven experiment and seven control hemispheres were collected. In this group, animals received stimulation of row B vibrissae (CS only), which was applied for the same duration as in the whisker-shock conditioning group, regularly ever 15 s. Mice received the CS consisting of three strokes to the whiskers of row B lasting a total 9 s, and after a 6 s interval this was repeated. These control mice received only stroking of row B vibrissae on one side of the snout 4 times/min, for 10 min/day, for 3 days (CS only). J


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ACS Chemical Neuroscience Double Label Immunofluorescence Staining Procedure CB1+/ VGAT+. Additional control study investigated whether CB1+ puncta and VGAT+ puncta (vesicular GABA transporter) are coexpressed in the barrel B hollow in the trained side CS+UCS group. Sections were washed in BBS for10 min before blocking of nonspecific endogenous peroxidase activity with 1% hydrogen peroxide for the duration of 30 min at room temperature free floating. A further wash was performed at room temperature with 10% NGS (Vector Laboratories, Inc., Burlingame, CA) for 2 h to exhaust endogenous peroxidase activity and to block nonspecific reaction. Sections were then incubated free floating with (a) rabbit polyclonal anti-CB1 (1:500; ab23703, Abcam, Cambridge, UK) raised against the C-terminal amino acid 461−472 of human CB1; (b) VGAT (1:250; Synaptic System/131 013, 37079 Göttingen, Germany), for 48 h at 4 °C under vigorous shaking. Sections were then washed in BBS for 3 × 10 min then incubated free floating with (a) biotinylated anti-rabbit IgG (1:100; Vector Laboratories, Inc., Burlingame, CA) followed by Texas Red or Alexa Red fluorescence (1:100; Vector Laboratories, Inc., Burlingame, CA); (b) biotinylated anti-guinea pig IgG (1:100; Vector Laboratories, Inc., Burlingame, CA) followed by fluorescein avidin DCS (1:100; Vector Laboratories, Inc., Burlingame, CA, or Alexa 561 1:500 dilution; Molecular Probes, green fluorescence). Nuclear staining with Hoechst 33258 (0.5 μg/mL; Molecular Probes, Carlsbad, CA) delineated the barrel cortex prior to mounting in Vectashield Mounting Medium (Vector Laboratories, Inc., Burlingame, CA). Control Immunofluorescence Staining Procedure. To confirm further the specificity, the primary CB1 antibody was preabsorbed overnight at 4 °C with its immunogenic peptide (20−100 μg/mL; ab50542; Abcam, Cambridge, UK) prior to incubation on the sections. Additionaly, the specifity of the primary CB1 antibody used in this study has been confirmed by the company (Abcam, Cambridge, UK) of origin and has been used previously in other publications.64,65 The specifity of primary PV antibody has been used in studies previously in the laboratory23 and confirmed by the company (Sigma-Aldrich, Gillingham, Dorset, UK). As a control for the specificity of the secondary antibody binding, one section from each animal was processed according to the same protocol but omitting incubation with the anti-CB1 and anti-PV primary antibody. For control purposes, sections were processed with PBS/NGS and PBS/NHS instead of primary antibodies. Controls for secondary antibody cross-reaction in mouse tissues were performed by incubating sections with a nonmatching antispecies antiserum. Specific immunostaining was not observed in any of these control sections. Sections from the trained and control sides of mice S1 cortex were processed together. Image Acquisition and Analysis. Series of optical sections were registered using a spinning disc confocal system (ZEISS/Yokogawa) equipped with a 63× oil immersion objective (NA = 1.4) and EMCCD camera (Photometrics Evolve 512). Fluorescence of Hoechst 33258 dye in neural nuclei was excited with a 405 nm diode laser and detected in the 419−465 nm range. Fluorescence of Alexa 488 (CB1+ puncta) was excited with a 489 nm diode laser and detected in the 500−550 nm range, whereas Alexa 561 (PV+ puncta) was excited with a 561 nm DPSS laser and detected in the 571−639 nm range. The fluorescence signal was digitized with 16 bit precision, and the image acquisition parameters (time, gain) were set so as to fill 65% the dynamic range. A single optical section comprised 512 × 512 pixels (corresponding to 0.21 × 0.21 μm each), and the spacing between the sections was 0.7 μm. The 3D images comprised 7 × 7 tiles (registered with 10% overlap and corresponding to the area of 680 μm × 680 μm). The 3D images (Alexa 488 and 561) were subjected to median filtering (kernel of 5 × 5 × 5 elements). The background was calculated using grayscale opening with elliptical structuring element (radius of 5 × 5 × 2 voxels). Following background subtraction, the 3D images were filtered with a Gaussian filter (standard deviation of 0.35). Regions of high fluorescence intensity (corresponding to CB1+ and PV+ puncta) were segmented using background threshold (SD of the respective distribution multiplied by 5). The identified regions were fitted using 3D ellipsoids. The regions for which the main axis

radius exceeded 20 voxels or the ratio of the axis to the cross section radius exceeded 5 were excluded from further analysis. The remaining regions were split to single puncta using watersheding (the minimum object size of 9 voxels). To define the shape of each barrel, this study uses Hoechst 33258 dye for staining cell nucleic acid that emits blue fluorescence. The same areas bordering the hollows of rows A (A1h−A4h), B (B1h−B4h), C (C1h−C4h), D (D1h−D4h), and E (E1h−E4h) were delineated (semiautomatically) on Hoechst+, CB1+, and PV+ images (Figure 1). These regions and time window were chosen because our previous studies indicated increases in the density of GABAergic markers69 24 h after whisker-shock training.1 A Z-stack series was collected with a spinning disc confocal system (ZEISS/Yokogawa), and 3D animations were assembled using Imaris software. Maximum intensity projections were generated from the 3D images of nuclei Hoechst 33258 dye. Regions corresponding to single barrels of the cortex were delineated manually and used as masks for calculation numbers of the segmented CB1+ and PV+ puncta. This procedure is equivalent to imaging of an optical section in the plane of interest and an optical section above this plane required for the stereological procedures.66,67 Results were expressed as the numerical density (Nv, the number per unit volume) of CB1+ (red) and PV+ (green) puncta (mean values, ± SEM) at the same barrel hollows in which the positive puncta numbers were counted. To quantify the size and intensity of CB1 and PV immunoreactivity, optical density morphometry was performed on 150 individual CB1+ and PV+ puncta. For each condition, at least three independent set of experiments were analyzed. Statistics. To compare the effect of whisker-shock conditioning upon numerical density (Nv) of CB1+ puncta and numerical density (Nv) of PV+ puncta labeling in CS+UCS mice and their controls (PSEUDO, CS only, UCS only, and naive littermates), one-way analysis of variance followed by Bonferroni correction for experimental and control regions was performed. To compare the interhemispheric effect of whisker-shock conditioning (CS+UCS) and controls mice upon Nv CB1+puncta labeling in rows A−E paired, two-tailed Student’s t test was performed. Values of Nv of CB1+ puncta and Nv of PV+ puncta obtained for rows A (A1h−A4h), B (B1h−B4h), C (C1h−C4h), D (D1h−D4h), and E (E1h−E4h) were averaged and expressed as mean ± SEM. In behavioral studies, paired two-tailed Student’s t test has been used. Statistical analysis was performed using the GraphPad Prism 5 software (GraphPad Software, Inc.). The significance was considered at p values < 0.05 for all comparisons.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.7b00500. Hoechst 33258 dye (blue), CB1+ and PV+ immunostaining in paracoronal section through the mouse S1 cortex; double label immunofluorescence staining for CB1+ (red) and PV+ (green) neurons and puncta in the barrel hollow region; 3D high-resolution position (X, Y, Z) of CB1+ puncta and PV+ puncta recognition/ localization in merging data from multiple views spatial profiles in the barrel hollow region; 3D high-resolution position (X, Y, Z) of CB1+ puncta and VGAT+ puncta recognition/localization in merging data from multiple views spatial profiles in the barrel hollow region; micrographs of 15 optical sections of 700 nm thickness laser scanning confocal optical sections illustrating of CB1+ puncta and VGAT+ puncta recognition/colocalization in merging data in the barrel B3 hollow of the CS +UCS experimental side mouse; behavioral response to training (PDF) K


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AUTHOR INFORMATION

Corresponding Author

*Phone: +48225892371. Fax: +48228225342. E-mail: e. [email protected]. ORCID

Ewa Siucinska: 0000-0002-4541-2864 Author Contributions

Conceptualization: E.S. Funding acquisition: E.S. Investigation: E.S. Methodology: E.S. and T.B. Project administration: E.S. Supervision: E.S. Visualization: E.S., W.B., and T.B. Writing original draft: E.S. Review and editing: E.S., T.B., and W.B. Funding

We thank Polish National Science Centre Grant No. 6420/B/ P01/2011/40 to E. S. for funding. Notes

The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Professor Krzysztof Zablocki for helpful discussions. We wish to acknowledge Mr. Artur Wolny for confocal microscopy visualization expert engineering support.

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