| In the senile process, testosterone (T) level decreased gradually and the motor behavior and exploratory behavior were impaired in aged male organisms. Aged men or aged male rats often displayed the disturbances of the body balance and motor coordination. Testosterone is involved in the neuropathophysiology of some neuropsychiatric disorders. The resting tremor and fine motor control were significantly improved after testosterone administration in a parkinsonian with testosterone deficiency. The improvement in motor symptoms correlated with serum testosterone levels. Nonmotor symptoms of Parkinson disease, which are virtually identical to the testosterone deficiency symptoms, were ameliorated by transdermal testosterone gel of a daily dose.Currently, testosterone replacement therapies include oral therapy, intramuscular injections and subcutaneous testosterone implants. The administration routes above have been shown some disadvantages. Orally administered testosterone has lower bioavailability because of first-pass metabolism. Intramuscular injections are painful and inappropriate to be used, especially in long-term testosterone replacement. The insertion of T implants requires minor surgery and can be painful, and extrusion of the pellets is common. Transdermal testosterone non-scrotal patches often result in skin irritation in patients.The ideal testosterone replacement therapy should offer safety, efficacy, cost-effectiveness, convenience, a good release profile, dosing flexibility, and effective normalization of T levels. Targeting testosterone to the central nervous system should allow the administration of lower doses of testosterone and fewer peripheral side effects.Nasal cavity was a part of respiratory system. The lining mucosa of nasal cavity includes respiratory region and olfactory region. The olfactory cells of olfactory region belong to nerve cells, which contact with rhinencephalon through fila olfactoria. Based on the mucous membrane characteristics, intranasal administration of drugs has become preferred method in recent year.Therefore, the intact male rats, gonadectomized (GDX) rats and aged male rats were used as experimental animal models in present study. The influence of intranasal administration of T upon the central nervous system was studied by analyzing the open field behavior of rats after long-term intranasal administration of T and the effects of intranasal delivery of T on mensencephalic dopaminergic and serotoninergic neurons were analyzed by immunocytochemistry, immunoblotting and HPLC. It is hoped that intranasal administration of T/TP could be used in adjutant therapy for some CNS diseases.Prat 1: Effects of intranasal administration of T/TP on c-Fos in rat brainObjective: To study whether the intranasal administration of T/TP could induce the expression of c-Fos in rat brain and activate different brain regions.Methods: Radioimmunoassay (RIA) was used to detect testosterone concentration in cerebrospinal fluid and in serum after intranasal administration of TP. Immunocytochemistry was used to detect the expression of c-Fos protein in different brain regions.Results: 1. RIA: Testosterone in cerebrospinal fluid and in serum was decreased by 45% (P<0.01) and 69% (P<0.01) respectively in GDX rats compared to intact rats. Subcutaneous injection of TP in GDX rats (GDX-sc.TP) only increased testosterone in serum. Testosterone in cerebrospinal fluid and in serum was increased after intranasal administration of TP in GDX rats (GDX-TP). Testosterone in cerebrospinal fluid of GDX-TP rats was higher than that in GDX-sc.TP rats (P<0.01), however testosterone in serum of GDX-TP rats was lower than that in GDX-sc.TP rats (P<0.05). Intranasal administration of TP in intact rats also increased testosterone in cerebrospinal fluid and in serum compared to intact rats. 2. Immunocytochemistry: After intranasal administration of TP or testosterone, the number of c-Fos-positive neurons was increased in Tu, PrL, M1,M2, Cg, CPu-DM, CPu-DL, Acb, PH, SNL and DR brain regions and the c-Fos immunoreactive intensity was enhanced in OB, Tu, PrL, M1,M2, Cg, CPu-DM, CPu-DL, Acb, HIP, PH, SNL and DR brain regions. However, after subcutaneous injection of TP, the number of c-Fos-positive neurons was increased only in Tu, PrL, CPu-DL and Acb brain regions and the c-Fos immunoreactive intensity was enhanced only in OB and Tu.Conclusions: 1. Testosterone in cerebrospinal fluid and in serum was significantly decreased after GDX. Subcutaneous injection of TP in GDX rats only increased the testosterone in serum. Testosterone in cerebrospinal fluid and in serum was increased after intranasal administration of TP in intact rats and GDX rats. 2. The expression of c-Fos was incresead only in a few brain regions after subcutaneous injection of TP, however, more brain regions were activated after intranasal administration of TP or testosterone.Prat 2: Behavior in open field test was affected after intranasal administration of testosterone to ratsObjective: To study the effects of long-term intranasal administration of testosterone on the central nervous system by analyzing the open field behavior of rats.Methods: The open field test was used to analyze five types of behavioral patterns in present study: immobile-sniffing, exploratory behavior, thigmotaxic behavior, motor behavior and grooming behavior.Results: 1. Immobile-sniffing: The intranasal administration of testosterone in intact rats significantly increased the number of immobile-sniffing events by 46% (P<0.01). Gonadectomy decreased the immobile-sniffing events, with a 44% (P<0.01) reduction and the intranasal administration of testosterone in GDX rats significantly increased the number of immobile-sniffing events (P<0.01), which was even more than in sham rats by 88% (P<0.01). 2. Exploratory behavior: The intranasal administration of testosterone significantly increased the amount of walking, climbing, rearing and sniffing in intact rats by 82% (P<0.01), 54% (P<0.05), 85% (P<0.01) and 65% (P<0.01) respectively. Gonadectomy decreased the amount of above four behaviors by 70% (P<0.01), 57% (P<0.01), 68% (P<0.01) and 62% (P<0.01) respectively and that the amount of above behaviors in GDX rats was restored to the level of sham rats after intranasal administration of testosterone, except for the rearing. The amount of rearing in GDX-T rats was still lower than in the sham rats by 47% (P<0.05), even though the amount was increased 68% (P>0.05) compared to GDX rats. 3. Motor behavior: The intranasal administration of T significantly increased the amount of vertical activity and horizontal activity as well as the total path length in intact rats by 61% (P<0.01), 69% (P<0.05) and 75% (P<0.01) respectively. The amount of vertical activity and horizontal activity in GDX rats was significantly lower than in the sham rats by 60% (P<0.01) and 60% (P<0.01) respectively, the total path length in GDX rats was significantly less than in the sham rats by 59% (P<0.01) and the amount of vertical activity and horizontal activity as well as the total path length in GDX rats were restored to the level of sham rats after intranasal administration of testosterone. 4. Grooming behavior: The intranasal administration of T significantly increased the number of grooming events and the duration of grooming in intact rats by 72% (P<0.01) and 61% (P<0.01) respectively. The number of grooming events in GDX rats was significantly less than in the sham rats by 54% (P<0.01), the duration of grooming in GDX rats was significantly shorter than in the sham rats by 55% (P<0.01) and the number of grooming events as well as the duration of grooming in GDX rats was restored to the level of sham rats after intranasal administration of testosterone. 5. Subcutaneous injection results: The amount of walking and horizontal activity, the total path length was only restored to the level of sham-sc rats after subcutaneous injection of testosterone. Conclusions: 1. Significant decreases in open field activity were observed in GDX rats. 2. The open field activity scores was significantly increased in GDX rats and in intact rats compared with the corresponding GDX rats and intact rats after intranasal administration of testosterone. 3. Intranasal route improved the impaired behaviors better than subcutaneous injection.Prat 3: Intranasal administration of testosterone increased the dopaminergic activity in SN-CPu and VTA-Acb neurons of ratsObjective: To sduty the effects of long-term intranasal administration of testosterone on dopaminergic neurons in SN-CPu and VTA-Acb of rats.Methods: Immunohistochemistry and immunoblotting were used to detect the expression of TH and DAT. HPLC was used to detect DA and its metabolites DOPAC and HVA.Results: 1. Immunocytochemistry: 1) The intranasal administration of testosterone significantly increased the expression of TH in intact rats by 7% (P<0.05) in SN, increased by 27% (P<0.01), 30% (P<0.01), 17% (P<0.01) and 18% (P<0.01) in dorsomedial CPu (CPu-DM), ventromedial CPu (CPu-VM), ventrolateral CPu (CPu-VL) and dorsolateral CPu (CPu-DL) respectively, increased by 5% (P<0.05) in VTA, increased by 18% (P<0.01) and 15% (P<0.01) in core and shell of Acb respectively. Gonadectomy decreased the expression of TH above eight brain regions by 13% (P<0.05), 18% (P<0.05), 16% (P<0.05), 11% (P<0.05), 14% (P<0.05), 26% (P<0.05), 17% (P<0.01) and 16% (P<0.05) and that the expression of TH above eight brain regions in GDX rats was restored to the level of sham rats after intranasal administration of testosterone, even though the expression of TH was higher than sham rats (P<0.01). 2) The intranasal administration of testosterone significantly increased the expression of DAT in intact rats by 7% (P<0.01) in SN, increased by 27% (P<0.01), 23% P<0.05), 43% (P<0.01) and 22% (P<0.01) in CPu-DM, CPu-VM, CPu-VL and CPu-DL respectively, increased by 10% (P<0.01) in VTA, increased by 21% (P<0.01) and 13% (P<0.05) in core and shell of Acb respectively. Gonadectomy decreased the expression of DAT above eight brain regions by 13% (P<0.01), 15% (P<0.05), 16% (P<0.05), 15% (P<0.05), 15% (P<0.01), 15% (P<0.05), 18% (P<0.01) and 17% (P<0.05) and that the expression of DAT above eight brain regions in GDX rats was restored to the level of sham rats after intranasal administration of testosterone. 3) The expression of DAT in SN and VTA was only restored to the level of sham-sc rats after subcutaneous injection of testosterone. The expression of TH in CPu-DL and VTA and the expression of DAT in CPu-DM, CPu-DL and shell of Acb were improved after subcutaneous injection of testosterone. 2. Immunoblotting: 1) The intranasal administration of testosterone significantly increased the expression of TH in intact rats by 123% (P<0.01), 8% (P<0.01), 27% (P<0.01) and 51% (P<0.01) in SN, CPu, VTA and Acb respectively. Gonadectomy decreased the expression of TH above four brain regions by 39% (P<0.01), 11% (P<0.01), 35% (P<0.01) and 43% (P<0.01) and that the expression of TH above four brain regions in GDX rats was improved after intranasal administration of testosterone. 2) DAT bands included Glycosylated-DAT (80KDa) and Non-Glycosylated-DAT (50KDa). The intranasal administration of testosterone significantly increased the expression of two kinds of DAT in intact rats by 14% (P<0.01) and 10% (P<0.01) in SN respectively, increased by 40% (P<0.01) and 11% (P<0.01) in CPu respectively, increased by 22% (P<0.01) and 10% (P<0.05) in Acb respectively, the expression of Glycosylated-DAT increased by 53% in VTA (P<0.01). Gonadectomy decreased the expression of two kinds of DAT by 20% (P<0.01) and 23% (P<0.01) in SN respectively, decreased by 58% (P<0.01) and 23% (P<0.01) in VTA respectively, decreased by 51% (P<0.01) and 17% (P<0.01) in Acb respectively, the expression of Non-Glycosylated-DAT decreased by 12% (P<0.05) in CPu. The expression of DAT above four brain regions in GDX rats was improved after intranasal administration of testosterone. 3) The expression of TH in SN and the expression of two kinds of DAT in CPu were only improved after subcutaneous injection of testosterone. 3. HPLC: There are no significant differences on DA concentration in SN and CPu after intranasal administration of testosterone (P>0.05). The intranasal administration of testosterone significantly increased the concentration of DOPAC and HVA by 38% (P<0.01) and 60% (P<0.01) in CPu respectively, increased the ratio of DOPAC+HVA/DA by 35% (P<0.05) in CPu. There are no significant differences on DA concentration in SN and CPu, and the metabolites and metabolic rate in CPu after gonadectomy (P>0.05). The intranasal administration of testosterone in GDX rat increased the concentration of HVA by 23% (P<0.05) in CPu, the concentration of DOPAC and HVA increased by 18% (P<0.01) and 51% (P<0.01) compared to sham group respectively.Conclusions: 1. The expression of TH and DAT was significantly decreased in SN, CPu, VTA and Acb in GDX rats. 2. Long-term intranasal administration of testosterone significantly increased both the expression of TH and DAT in SN, CPu, VTA and Acb and the metabolites of dopaminergic neurons in GDX rats and Intact rats. 3. Intranasal route improved the reduced dopaminergic neurons activity in SN-CPu and VTA-Acb better than subcutaneous injection.Prat 4: Intranasal administration of testosterone increased the serotoninergic activity in dorsal raphe nucleus of rat.Objective: To study the effects of long-term intranasal administration of testosterone on the serotoninergic neurons in DR of rats.Methods: Immunohistochemistry was used to detect the expression of TPH, 5-HT and SERT in DR. HPLC was used to detect 5-HT and its metabolites 5-HIAA in DR, CPu and Acb.Results: 1. Immunocytochemistry: 1) The intranasal administration of testosterone significantly increased the expression of TPH, 5-HT and SERT in intact rats by 3% (P<0.05), 9% (P<0.05) and 15% (P<0.01) in DR respectively. Gonadectomy decreased the expression of TPH, 5-HT and SERT by 21% (P<0.05), 22% (P<0.01) and 29% (P<0.05) compared to sham rats and that the expression of TPH, 5-HT and SERT in GDX rats was restored to the level of sham rats after intranasal administration of testosterone, and the expression of SERT was even more than in sham rats (P<0.05). 2) The expression of TPH, 5-HT and SERT were improved, however, which were lower compared to sham-sc rats after subcutaneous injection of testosterone. 2. HPLC: The intranasal administration of testosterone significantly increased the concentration of 5-HT by 32% (P<0.01) in DR, increased the concentration of 5-HIAA and the ratio of 5-HIAA/5-HT by 27% (P<0.05) and 26% (P<0.05) in CPu respectively, increased the concentration of 5-HIAA and the ratio of 5-HIAA/5-HT by 29% (P<0.05) and 28% (P<0.05) in Acb respectively. Gonadectomy decreased the concentration of 5-HT by 26% (P<0.05), 26% (P<0.05) and 12% (P<0.05) in DR, CPu and Acb respectively, decreased the concentration of 5-HIAA by 13% (P<0.05) and 23% (P<0.05) in CPu and Acb respectively compared to sham rats and that were restored to the level of sham rats after intranasal administration of testosterone.Conclusions: 1. The expression of TPH, 5-HT and SERT was significantly decreased in DR of GDX rats. 2. Long-term intranasal administration of testosterone significantly increased the expression of TPH, 5-HT and SERT in DR of GDX rats and intact rats. Intranasal route improved the decreased serotoninergic neurons activity better than subcutaneous injection. 3. The concentration of 5-HT and 5-HIAA was significantly reduced in DR, CPu and Acb in GDX rats, and long-term intranasal administration of testosterone could restore them to sham level. Prat 5: Influence of intranasal administration of TP upon the behaviors, dopaminergic and serotoninergic neurons in aged ratsObjective: To study the influence of long-term intranasal administration of TP on behaviors and dopaminergic and serotoninergic neurons in aged rats.Methods: Open field test, tilting-plane test, horizontal-wire test and adhesive removal test were used to detect behaviors. Immunohistochemistry and immunoblotting were used to detect the expression of TH, DAT, TPH, 5-HT and SERT.Results: 1. Open field test: The amount of immobile-sniffing events, walking,climbing,rearing,sniffing, vertical activity, horizontal activity, total path length, number of grooming events and the duration of grooming were decreased in 24Mon rats by 39% (P<0.01), 55% (P<0.01), 39% (P<0.01), 74% (P<0.01), 53% (P<0.01), 53% (P<0.05), 58% (P<0.01), 58% (P<0.01), 56%(P<0.01) and 57% (P<0.01) respectively, and the latency of grooming were increased by 506% (P<0.01) compared to 6Mon rats. All behaviors above were significantly improved after intranasal administration of TP compared to 24Mon rats. 2. Tilting-plane test: The angle of sliding off decreased by 27% (P<0.05) and the number of sliding off at 50°angle increased by 660% (P<0.01) in 24Mon rats compared to 6Mon rats. Intranasal administration of TP increased the angle of sliding off and decreased the number of sliding off at 50°angle. 3. Horizontal-wire test: The duration time of hanging was shorter by 69% (P<0.01) in 24Mon rats compared to 6Mon rats and intranasal administration of TP improved the duration time of hanging compared to 24Mon rats. 4. Adhesive removal test: The latency to remove adhesive paper on left nose and right nose increased by 435% (P<0.05) and 656% (P<0.05) in 24Mon rats compared to 6Mon rats respectively and the latency to remove adhesive paper were restored to the level of 6Mon rats after intranasal administration of TP. 5. Immunocytochemistry: 1) The expression of TH was decreased by 22% (P<0.05) in SN, 21% (P<0.05), 25% (P<0.05), 20% (P<0.05) and 24% (P<0.01) in CPu-DM, CPu-VM, CPu-VL and CPu-DL respectively, 12% (P<0.05) in VTA, 20% (P<0.05) and 15% (P<0.01) in core and shell of Acb respectively in 24Mon rats compared to 6Mon rats. Intranasal administration of TP improved the expression of TH in brain regions above. 2) The expression of DAT was decreased by 10% (P<0.05) in SN, 17% (P<0.05), 16% (P<0.05), 15% (P<0.05) and 17% (P<0.05) in CPu-DM, CPu-VM, CPu-VL and CPu-DL respectively, 9% (P<0.05) in VTA, 19% (P<0.05) and 17% (P<0.01) in core and shell of Acb respectively in 24Mon rats compared to 6Mon rats. Intranasal administration of TP improved the expression of DAT in brain regions above except for CPu-VM. 3) The expression of TPH, 5-HT and SERT was decreased by 10% (P<0.05), 14% (P<0.01) and 12% (P<0.01) in 24Mon rats compared to 6Mon rats respectively. Intranasal administration of TP improved the expression of TPH, 5-HT and SERT. 6. Immunoblotting: 1) The expression of TH was decreased by 25% (P<0.01), 18% (P<0.01) 41% (P<0.01) and 24% (P<0.01) in SN, CPu, VTA and Acb in 24Mon rats compared to 6Mon rats respectively and intranasal administration of TP improved the expression of TH four brain regions above. 2) The expression of two kinds of DAT were decreased by 10% (P<0.05) and 40% (P<0.01) in SN respectively, 36% (P<0.01) and 16% (P<0.05) in CPu respectively, 70% (P<0.01) and 10% (P<0.05) in VTA respectively, 12% (P<0.05) and 14% (P<0.01) in Acb respectively. The expression of Non-Glycosylated-DAT in SN, CPu and VTA were restored after long-term intranasal administration of TP.Conclusions: 1. Open field behavior, balancing reactions, muscular strength performance and motor coordination ability were significantly reduced in 24Mon rats compared to 6Mon rats, which were improved after long-term intranasal administration of TP. 2. The expression of TH, DAT, TPH, 5-HT and SERT were significantly reduced in 24Mon rats compared to 6Mon rats, which were improved after long-term intranasal administration of TP.
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