**3. Results**

Figure 1 shows CBD plasma levels following single (Figure 1a) or repeated injections (Figure 1b) with 5, 15 or 30 mg/kg of CBD. The concentration of CBD in the plasma increased dose-dependently under both experimental conditions (Figure 1a: KW = 18.24, *p* = 0.0001; CBD 5 mg/kg = +32.53 ng/mL; CBD 15 mg/kg = +79.78 ng/mL vs. 5 mg/kg, *p* = 0.0327; CBD 30 mg/kg = +181.998 ng/mL vs. 5 mg/kg, *p* < 0.0001; Figure 1b: F(2,21) = 102.4, *p* < 0.0001; CBD 5 mg/kg = +2.04 ng/mL; CBD 15 mg/kg = +10.51 ng/mL vs. 5 mg/kg *p* = 0.0010, CBD 30 mg/kg + 23,96 ng/mL vs. 15 mg/kg, *p* < 0.0001).

**Figure 1.** Concentration of CBD in the plasma of rats treated with a single (**a**) or repeated (**b**) CBD administration at the dose of 5 mg/kg, 15 mg/kg, or 30 mg/kg and killed respectively one (**a**) and twenty-four hours (**b**) after the last treatment. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Red dashed line in panel a indicates the y axis limit (100) of the bar chart represented in panel b. Ordinary one-way ANOVA followed by Tukey's multiple comparisons test, or Kruskal−Wallis one-way ANOVA for ranks followed by Dunn's multiple comparisons test for non-normally distributed data. \* *p* < 0.05, \*\*\* *p* < 0.001 vs. 5 mg/kg, ### *p* < 0.001 vs. 15 mg/kg. CBD 5 mg/kg *n* = 8 CBD 15 mg/kg *n* = 8, CBD 30 mg/kg *n* = 8.

Figure 2 shows CBD cortical levels following single (Figure 2a) or repeated injections (Figure 2b) with 5, 15, or 30 mg/kg of CBD. At variance from plasma CBD levels, it appears that cortical CBD concentration rises significantly over untreated rats only at the highest dose employed (i.e., 30 mg/kg) under both experimental conditions (Figure 2a: KW = 12.89, *p* = 0.0016; CBD 30 mg/kg = +0.31 ng CBD/ng protein vs. 5 mg/kg, *p* = 0.0010; Figure 2b: KW = 17.90, *p* = 0.0001; CBD 30 mg/kg + 0.10 ng CBD/ng protein vs. 5 mg/kg, *p* = 0.0001; CBD 30 mg/kg + 0.09 ng CBD/ng protein vs. 15 mg/kg, *p* = 0.0070). Of note, CBD was undetectable at the 5 mg/kg dose following both single and repeated administrations.

**Figure 2.** Concentration of CBD in the medial prefrontal cortex (mPFC) of rats treated with a single (**a**) or repeated (**b**) CBD administration at the dose of 5 mg/kg, 15 mg/kg, or 30 mg/kg and killed respectively one (**a**) and twenty-four hours (**b**) after the last treatment. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Red dashed line in panel a indicates the y axis limit (0.35) of the bar chart represented in panel b. Ordinary one-way ANOVA followed by Tukey's multiple comparisons test, or Kruskal−Wallis one-way ANOVA for ranks followed by Dunn's multiple comparisons test for non-normally distributed data. \* *p* < 0.05, \*\*\* *p* < 0.001 vs. 5 mg/kg, ### *p* < 0.001 vs. 15 mg/kg. CBD 5 mg/kg *n* = 8 CBD 15 mg/kg *n* = 8, CBD 30 mg/kg *n* = 8.

We next evaluated the dose-dependent effects of acute CBD exposure on the gene expression levels of *total Bdnf* and related exons in the mPFC. In particular, we analyzed

the main *Bdnf* exons, i.e., *exon IV*, the most abundant exon of somatic origin, and *exon VI*, which is expressed at dendrite level. Figure 3 illustrates the effects of a single injection of 5, 15, and 30 mg/kg of CBD on the gene expression levels of *total Bdnf* and its exons. While *total Bdnf* as well as *exon VI* gene expression levels are unchanged (Figure 3a: F(3,27) = 1.553, *p* = 0.224; Figure 3c: F(3,27) = 1.980, *p* = 0.1407), the transcription of *exon IV* is significantly upregulated in the mPFC, but only at the highest dose employed (Figure 3b: KW= 10.24, *p* = 0.0166, + 21.14% vs. saline *p* = 0.0173).

**Figure 3.** Effects of acute CBD exposure on Bdnf gene expression levels in the mPFC. Rats were treated with a single injection of CBD 5 mg/kg, 15 mg/kg, or 30 mg/kg, and killed one hour after the treatment. *Total Bdnf* (**a**), *Bdnf exon IV* (**b**), and *Bdnf exon VI* (**c**) mRNA levels in mPFC are expressed as percentages of saline-treated rats. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Ordinary one-way ANOVA followed by Tukey's multiple comparisons test, or Kruskal−Wallis one-way ANOVA for ranks followed by Dunn's multiple comparisons test for non-normally distributed data. \* *p* < 0.01 vs. saline-treated rats. Saline *n* = 8 CBD 5 mg/kg *n* = 8 CBD 15 mg/kg *n* = 8, CBD 30 mg/kg *n* = *7*.

We then analyzed protein levels of the mature form of the neurotrophin BDNF (mBDNF). As shown in Figure 4, acute CBD treatment up-regulates mBDNF levels in the whole homogenate of the mPFC at 30 mg/kg (Figure 4a: F(3,27) = 3.361, *p* = 0.0333, + 30.00% vs. saline *p* = 0.0230), but not at the lower doses used. Similarly, we found increased phosphorylation of the BDNF high-affinity receptor TrkB in Tyr(Y)706 (Figure 4b: F(3,28) = 7.893, *p* = 0.0006, + 43.63% vs. saline *p* = 0.0035), with no changes in its total expression (Figure 4c: KW 4.440, *p* = 0.2177). Of note, the levels of TrkB receptor activation, expressed as the ratio between the phosphorylated and non-phosphorylated (pTrkB/TrkB), are increased only following the single injection of CBD 30 mg/kg (Figure 4d: KW= 17.82, *p* = 0.0005, + 15.25% vs. saline *p* = 0.0220), with no effect at the lower doses.

**Figure 4.** Effects of acute CBD exposure on mBDNF and TrkB receptor protein levels in the mPFC. Rats were treated with a single injection of CBD 5 mg/kg, 15 mg/kg, or 30 mg/kg and killed one hour after treatment. Protein levels of BDNF (**a**), phospho(p)-TrkBY706 (**b**), TrkB (**c**), and of the ratio pTrkB/TrkB (**d**) measured in the homogenate of mPFC are expressed as percentages of saline-treated

rats. Below the graphs representative immunoblots are shown for mBDNF (14 kDa), pTrkB Y706 (145 kDa), TrkB (145 kDa), and β-Actin (43 kDa) proteins. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Ordinary one-way ANOVA followed by Tukey's multiple comparisons test, or Kruskal-Wallis one-way ANOVA for ranks followed by Dunn's multiple comparisons test for non-normally distributed data. \* *p* < 0.05 vs. saline-treated rats. Saline *n* = 8, CBD 5 mg/kg *n* = 8 CBD 15 mg/kg *n* = 8, CBD 30 mg/kg *n* = 8 (mBDNF *n* = 7).

Then, to investigate whether alterations in the BDNF-TrkB system induced by a single CBD exposure would engage the recruitment of BDNF downstream pathways, we analyzed the expression and phosphorylation of Akt and ERK2 effectors (Figure 5). As shown in Figure 5, while no significant changes are observed either in the phosphorylated (Ser473) (Figure 5a: F(3.28) = 2.907, *p* = 0.0521) or in the total form of Akt (Figure 5b: KW = 6.804, *p* = 0.0784), their ratio expressed as pAkt/Akt is significantly increased following the acute CBD treatment at 30 mg/kg (Figure 5c: F(3.28) = 2.959, *p* = 0.0494, + 33.38% vs. saline *p* = 0.0328). Conversely, the analysis of ERK2 phosphorylation (Thr185/Tyr187) does not show any relevant changes (Figure 5d: KW= 8.619, *p* = 0.0348; Figure 5f: KW= 1.054, *p* = 0.7882), whereas, despite that the ANOVA analysis of ERK2 expression is statistically significant, the multiple comparisons test does not show any relevant change (Figure 5e: KW = 4.662, *p* = 0.1983).

**Figure 5.** Effects of acute CBD exposure on BDNF-downstream signaling in the mPFC. Rats were treated with a single injection of CBD 5 mg/kg, 15 mg/kg, or 30 mg/kg and killed one hour after treatment. Protein levels of phospho(p)-AktS473 (**a**), Akt (**b**), pAkt/Akt (**c**), pERK2T185-Y187 (**d**), ERK2 (**e**), and pERK2/ERK2 (**f**) measured in the homogenate of mPFC are expressed as percentages of saline-treated rats. Below the graphs, representative immunoblots are shown for pAktS473 (60 kDa), Akt (60 kDa), pERK2T185-Y187 (42 kDa), ERK2 (42 kDa), and β-Actin (43 kDa) proteins. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Ordinary one-way ANOVA followed by Tukey's multiple comparisons test, or Kruskal-Wallis oneway ANOVA for ranks followed by Dunn's multiple comparisons test for non-normally distributed data. \* *p* < 0.05 vs. saline-treated rats. Saline *n* = 8, CBD 5 mg/kg *n* = 8 CBD 15 mg/kg *n* = 8, CBD 30 mg/kg *n* = 8.

Based on the results observed following the single exposure of CBD, showing a significant effect on BDNF and its downstream signaling only at the highest dose employed, we decided to further analyze the neuroplastic effect of a repeated treatment with the phytocannabinoid employing only the highest dose. We found that repeated exposure to CBD (30 mg/kg) did not cause any change in the gene expression levels of *total Bdnf*, and *exon IV* and *VI* in the mPFC (Figure 6a: −10.75% vs. saline, t(14) = 1.224, *p* = 0.2410; Figure 6b: −9% vs. saline, t(14) = 0.8836, *p* = 0.3918; Figure 6c: −9.75% vs. saline, t(14) = 1.027, *p* = 0.3219). Of note, despite that no alterations are present in the mRNA levels of Bdnf and related exons, repeated CBD exposure reduces slightly, but significantly, mBDNF protein levels (Figure 7a: −11.75% vs. saline, t(14) = 2.443, *p* = 0.0284) and pTrkB/TrkB ratio (Figure 7d: −14.25% vs. saline, t(14) = 2.708, *p* = 0.0170).

**Figure 6.** Effects of repeated CBD exposure on Bdnf gene expression levels in the mPFC. Rats were treated with repeated injections of CBD 30 mg/kg for seven days and killed twenty-four hours after the last treatment. *Total Bdnf* (**a**), *exon IV* (**b**), and *exon VI* (**c**) mRNA levels in mPFC are expressed as percentages of saline-treated rats. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Unpaired Student's *t*-test. Saline *n* = 8, CBD 30 mg/kg *n* = 8.

**Figure 7.** Effects of repeated CBD exposure on mBDNF and TrkB receptor protein levels in the mPFC. Rats were treated with repeated injections of 30 mg/kg for seven days and killed twenty-four hours after the last treatment. Protein levels of BDNF (**a**), phospho(p)-TrkBY706 (**b**), TrkB (**c**), and of the ratio pTrkB/TrkB (**d**) measured in the homogenate of mPFC are expressed as percentages of saline-treated rats. Below the graphs, representative immunoblots are shown for mBDNF (14 kDa), pTrkBY706 (145 kDa), TrkB (145 kDa), and β-Actin (43 kDa) proteins. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Unpaired Student's *t*-test or Wilcoxon−Mann−Whitney test. \* *p* < 0.05 vs. saline-treated rats. Saline *n* = 8, CBD 30 mg/kg *n* = 8.

Interestingly, we found that the pAkt/Akt ratio is reduced (Figure 8c: −33.13% vs. saline, t(14) = 2.959, *p* = 0.0104), in line with reduced levels of Akt phosphorylation in Ser473 (Figure 8a), whereas no changes are observed in total Akt levels (Figure 8b: +9% vs. saline, t(14) = 0.6370, *p* = 0.5344). As previously shown after a single injection, the analysis of ERK2 does not show any alteration following repeated CBD exposure when compared to saline-treated animals (Figure 8d: −0.88% vs. saline, t(14) = 0.0594, *p* = 0.9535; Figure 8e: −7% vs. saline, U = 17, *p* = 0.1304; Figure 8f: +7% vs. saline, t(14) = 0.5305, *p* = 0.6041).

**Figure 8.** Effects of repeated CBD exposure on BDNF-downstream signaling in the mPFC. Rats were treated with repeated injections of 30 mg/kg for seven days and killed twenty-four hours after the last treatment. Protein levels of phospho(p)-AktS473 (**a**), Akt (**b**), pAkt/Akt (**c**), pERK2T185-Y187 (**d**), ERK2 (**e**), and pERK2/ERK2 (**f**) measured in the homogenate of mPFC are expressed as percentages of saline-treated rats. Below the graphs, representative immunoblots are shown for pAktS473 (60 kDa), Akt (60 kDa), pERK2T185-Y187 (42 kDa), ERK2 (42 kDa), and β-Actin (43 kDa) proteins. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Unpaired Student's *t*-test or Wilcoxon−Mann−Whitney test. \* *p* < 0.05, \*\* *p* < 0.01 vs. saline-treated rats. Saline *n* = 8, CBD 30 mg/kg *n* = 8.

BDNF protein is known to undergo anterograde transport from the mPFC toward striatum [21]. In line with the reduction observed in the mPFC, we analyzed the BDNF-TrkB system in the striatum. Of note, accordingly, mBDNF protein levels are increased following repeated exposure to 30 mg/kg of CBD (Figure 9a: +36.5% vs. saline, U= 0, *p* = 0.0002). No changes are observed in the TrkB receptor levels (Figure 9b–d), either in the phosphorylated (Figure 9b: −12.75% vs. saline, U= 20, *p* = 0.3969) or in the total form of TrkB receptor (Figure 9c: −8.37% vs. saline, t(14) = 1.454, *p* = 0.1680) as well as in the pTrkB/TrkB ratio (Figure 9d: −7.5% vs. saline, U = 19, *p* = 0.3357). The evaluation of BDNF downstream effectors Akt and ERK2 revealed a significant increase in pAkt (Ser473) (+29.13% vs. saline, t(14) = 4.599, *p* = 0.0004) and total Akt (+24.88% vs. saline, t(14) = 5.550, *p* < 0.0001) as shown in Figure 10a,b, respectively, with no changes in the pAkt/Akt ratio (Figure 10c: (+3.75% vs. saline, t(14) = 0.7222, *p* = 0.4821). In line with our previous observations, no changes are detected for ERK2 (Figure 10d: −12.38% vs. saline, t(14) = 1.579, *p* = 0.1366; Figure 10e: +1.38% vs. saline, t(14) = 0.1393, *p* = 0.8912; Figure 10f: −8.13% vs. saline, U= 20, *p* = 0.2345).

**Figure 9.** Effects of repeated CBD exposure on mBDNF and TrkB receptor protein levels in the striatum. Rats were treated with repeated injections of 30 mg/kg for seven days and killed twentyfour hours after the last treatment. Protein levels of mBDNF (**a**), phospho(p)-TrkBY706 (**b**), TrkB (**c**), and of the ratio pTrkB/TrkB (**d**) measured in the homogenate of the striatum are expressed as percentages of saline-treated rats. Below the graphs, representative immunoblots are shown for mBDNF (14 kDa), pTrkBY706 (145 kDa), TrkB (145 kDa), and β-Actin (43 kDa) proteins. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Unpaired Student's *t*-test. \*\*\* *p* < 0.001 vs. saline-treated rats. Saline *n* = 8 (pTrkB *n* = 7), CBD 30 mg/kg *n* = 8.

**Figure 10.** Effects of repeated CBD exposure on BDNF-downstream signaling in the striatum. Rats were treated with repeated injections of 30 mg/kg for seven days and killed twenty-four hours after the last treatment. Protein levels of phospho(p)-AktS473 (**a**), Akt (**b**), pAkt/Akt (**c**), pERK2T185-Y187 (**d**), ERK2 (**e**), and pERK2/ERK2 (**f**) measured in the homogenate of the striatum are expressed as percentages of saline-treated rats. Below the graphs, representative immunoblots are shown for pAktS473 (60 kDa), Akt (60 kDa), pERK2T185-Y187 (42 kDa), ERK2 (42 kDa), and β-Actin (43 kDa) proteins. Bar graphs represent the mean ± SEM from eight independent determinations for each experimental group. Unpaired Student's *t*-test. \*\*\* *p* < 0.001 vs. saline-treated rats. Saline *n* = 8, CBD 30 mg/kg *n* = 8.
