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P.2.015 Chronic effects of ‘biased’ 5-HT1A receptor agonists on object pattern separation performance and hippocampal plasticity
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Behavioural and systems
Conclusions: Based on our systems biological approach, we propose that manic-like behavior in Ank3+/ − mice is connected to a disturbance of the kinesin cargo system, resulting in a dysfunction of ion channel and AMPA receptor transport and scaffolding in hippocampal neurons. Lithium reverses this molecular signature, suggesting the promotion of anterograde microtubular transport as part of its mechanism of action in Ank3+/ − mice. Reference(s) [1] Gottschalk, M.G., Wesseling, H., Guest, P.C., Bahn, S., 2015. Proteomic enrichment analysis of psychotic and affective disorders reveals common signatures in presynaptic glutamatergic signaling and energy metabolism. Int J Neuropsychopharmacol 18(2). [2] Leussis, M.P., Berry-Scott, E.M., Saito, M., Jhuang, H., de Haan, G., Alkan, O., Luce, C.J., Madison, J.M., Sklar, P., Serre, T., Root, D.E., Petryshen, T.L., 2013. The ANK3 bipolar disorder gene regulates psychiatric-related behaviors that are modulated by lithium and stress. Biol Psychiatry 73(7) 683−90. [3] Cristino, A.S., Williams, S.M., Hawi, Z., An, J.Y., Bellgrove, M.A., Schwartz, C.E., Costa Lda, F., Claudianos, C., 2014. Neurodevelopmental and neuropsychiatric disorders represent an interconnected molecular system. Mol Psychiatry 19(3) 294–301. [4] Wesseling, H., Gottschalk, M.G., Bahn, S., 2015. Targeted multiplexed selected reaction monitoring analysis evaluates protein expression changes of molecular risk factors for major psychiatric disorders. Int J Neuropsychopharmacol 18(1). [5] Barry, J., Gu, Y., Jukkola, P., O’Neill, B., Gu, H., Mohler, P.J., Rajamani, K.T., Gu, C., 2014. Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons. Dev Cell 28(2) 117−31. P.2.015 Chronic effects of ‘biased’ 5-HT1A receptor agonists on object pattern separation performance and hippocampal plasticity B. Van Hagen1 ° , N.P. Van Goethem1 , R. Schreiber2 , A. Newman-Tancredi3 , M. Varney3 , J. Prickaerts1 . 1 Maastricht University, Dept of Psychiatry & Neuropsychology, Maastricht, The Netherlands; 2 Suadeo Drug Discovery, Consulting LLC, Watertown, MA, USA; 3 Neurolixis, Inc, Dana Point, CA, USA Introduction: Pattern separation, the formation of distinct representations out of similar inputs, is an important
hippocampal process implicated in memory formation. Impairments in pattern separation could underlie cognitive symptoms of disorders like PTSD and schizophrenia. Our previous research showed that acute treatment with the ‘biased’ 5-HT1A R agonist F15599, which preferentially activates postsynaptic cortical heteroreceptors, improved rat performance in a spatial object pattern separation (OPS) task. In contrast, F13714, which preferentially activates raphe-located autoreceptors, impaired performance in this task [1]. Here we investigated the effects of chronic treatment with F15599 and F13714 on OPS performance in rats. We hypothesized that, through desensitization of raphe-located autoreceptors by F13714, OPS performance impairment would ameliorate due to increased activation of postsynaptic receptors. We hypothesize an increase in plasticity and/or newborn neurons to underlie the increase in OPS performance. Methods: 48 three-month-old Wistar rats received a 14-day chronic treatment with either 0.32 mg/kg F15599 (N = 16), 0.02 mg/kg F13714 (N = 16) or vehicle (PBS, N = 16) (daily i.p. injections; injection volume of 2 ml/kg). Daily body temperature measurements were taken to indirectly measure hypothalamic post-synaptic 5-HT1A R activation. On day 1, 7 and 15 OPS task performance was measured [2], the d2 index (the difference in object exploration times relative to the total exploration time) was used as an outcome measure and a difference from zero (chance level) was evaluated with a onesample t-test. After behavioral assessment on day 15 the rats were sacrificed and from N = 8 animals per group the hippocampus was dissected for westernblot analysis of (pro)BDNF, (p)CREB, P11, synaptophysin and PSD95. For immunohistochemical analysis of hippocampal doublecortin (DCX) and 5-HT1A R’s in the Raphe nucleus N = 8 brains per treatment group were fixed in 4% PF. Stereological quantification of DCX is currently being performed. One-way ANOVA’s were used to compare between treatment groups for the biochemical and temperature measurements, if significant a Dunnett’s t-test comparing the treatment groups to vehicle was used. Results: The OPS impairment after acute treatment of F13714 was replicated (d2: 0.02; t-test comparing to zero, NS) and subsequently a gradual increase of performance was found, resembling vehicle treatment on day 15 (d2 F13714: 0.17; d2 vehicle: 0.16; t-tests zero P < 0.05). Body temperature dropped by 1 degree Celsius from treatment day 5 onwards (Dunnett’s t-test from vehicle: P < 0.001; repeated measures ANOVA from day 1: P < 0.1). F15599 showed an enhancement in OPS performance compared to vehicle acutely (d2: 0.33; t-test zero P < 0.001), and this was maintained through day 15 (d2: 0.27; t-test P < 0.001). Westernblot analysis of BDNF/GAPDH ratio
Behavioural and systems showed a significant increase in the dorsal hippocampus of the F15599 treated rats (4.456) compared to vehicle (1.381; Dunnett’s t-test; P < 0.05) but not to F13714. Other protein measurements showed no significant differences [P11, PSD95, synaptophysin, (p)CREB]. Discussion: These results imply desensitization of 5-HT1A autoreceptors by F13714, leading to stimulation of postsynaptic 5-HT1A Rs which could explain the drop in temperature. The increase in hippocampal BDNF does imply some enhanced plasticity in the F15599 treated rats but the DCX quantification results are needed to confirm an increase in adult hippocampal plasticity. Reference(s) [1] van Goethem, N.P., Schreiber, R., NewmanTancredi, A., Varney, M., Prickaerts, J., 2015. Divergent effects of the ’biased’ 5-HT1A receptor agonists F15599 and F13714 in a novel object pattern separation task. Br J Pharmacol 172:10, 1476–5381. [2] van Hagen, B.T.J., van Goethem, N.P., Lagatta, D.C., Prickaerts, J., 2015. The object pattern separation (OPS) task: a behavioral paradigm derived from the object recognition task. Beh Brain Research 285, 44−52. P.2.016 Muscimol effectively reverses impaired spatial memory in Alzheimer’s diseasetype streptozocin icv rat model V. Pilipenko1 ° , J. Pupure1 , J. Rumaks1 , U. Beitnere1 , Z. Dzirkale1 , B. Jansone1 , R. Skumbins1 , V. Klusa1 . 1 University of Latvia, Pharmacology, Riga, Latvia Background: GABAergic inhibitory action regulates learning and memory processes and contributes to neurotransmission [1]. Existing evidence suggests that GABAergic system is involved in pathophysiology of Alzheimer’s disease (AD) via inhibitory interneuron deficits [2] and decrease in functional GABA-A receptors [3]. In vitro, GABA and GABA-A receptor agonist muscimol blocked neuronal death induced by Ab in rat hippocampal and cortical neurons [4]. Intracerebroventricular (icv) streptozocin (STZ)-induced AD non-transgenic rat model is an easy and effective method for the detection of early behavioural disturbances such as loss of spatial memory and learning. Our concept suggests that low doses of muscimol may prevent learning and memory deficits in icv STZ-induced AD nontransgenic rat model. Methods: Male Wistar rats (280±20 g) received pretreatment with ip saline (control) or muscimol (0.01 and 0.05 mg/kg) on days 1−3. On day 4, rats received icv STZ
S37
(100 mg/ml) or aCSF. From days 19 to 22, rats received ip muscimol again, and spatial learning and memory were assessed in the water maze test (4 trials/day) by recording the escape latency, platform zone crossings and time in platform quadrant. A probe trial was carried out 24 h after the trainings. To investigate neuroinflammation, GFAP detection was implemented. All data were calculated using ANOVA with Bonferroni post-test (training days) and Tukey’s post-test (probe trial and GFAP density). Results and Discussion: Rats in STZ group showed statistically longer escape latencies vs. control group starting from day 2 (p < 0.0001). Muscimol at both doses significantly shortened escape latencies in MUSC 0.01 and MUSC 0.05 + STZ group rats vs. STZ from day 2, reversing STZ effect by about 90% on days 3 and 4 (p < 0.0001). In probe trial, STZ group rats showed a significant decrease in the number of platform crossings (p < 0.01) and the time spent in platform quadrant (p < 0.05) compared to control. Muscimol-treated STZ rats showed a significantly longer time spent in platform quadrant (p < 0.01) and a significantly higher number of platform crossings (p < 0.05 in MUSC 0.05 + STZ and p < 0.01 in MUSC 0.01 + STZ) vs. STZ group. Muscimol at both doses per se showed values comparable to control in both training days and probe trial. In the STZ group, a significant increase in GFAP density was observed compared vs. control (p < 0.0001). Muscimol at both doses showed significantly lower levels of GFAP in MUSC + STZ vs. STZ group. Conclusions: Obtained data suggest that icv STZ significantly decreased rat spatial learning and memory abilities in adult rats. Muscimol at both low doses, however, significantly improved learning and memory outcomes impaired by icv STZ, as well as alleviated the neuroinflammation. Therefore, novel therapeutic approaches aimed at the modulation of neuronal excitability through the control of GABAergic inhibitory inputs might be considered viable viable in the search of new anti-AD treatments. Reference(s) [1] Gong, N., Li, Y., Cai, G.Q., Niu, R.F., Fang, Q., Wu, K., Chen, Z., Lin, L.N., Xu, L., Fei, J., Xu, T.L., 2009. GABA transporter-1 activity modulates hippocampal theta oscillation and theta burst stimulation-induced long-term potentiation. J Neurosci 29, 15836–14845. [2] Verret, L., Mann, E.O., Hang, G.B., Barth, A.M., Cobos, I., Ho, K., Devidze, N., Masliah, E., Kreitzer, A.C., Mody, I., Mucke, L., Palop, J.J., 2012. Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell 149, 708–721.
Behavioural and systems
Conclusions: Based on our systems biological approach, we propose that manic-like behavior in Ank3+/ − mice is connected to a disturbance of the kinesin cargo system, resulting in a dysfunction of ion channel and AMPA receptor transport and scaffolding in hippocampal neurons. Lithium reverses this molecular signature, suggesting the promotion of anterograde microtubular transport as part of its mechanism of action in Ank3+/ − mice. Reference(s) [1] Gottschalk, M.G., Wesseling, H., Guest, P.C., Bahn, S., 2015. Proteomic enrichment analysis of psychotic and affective disorders reveals common signatures in presynaptic glutamatergic signaling and energy metabolism. Int J Neuropsychopharmacol 18(2). [2] Leussis, M.P., Berry-Scott, E.M., Saito, M., Jhuang, H., de Haan, G., Alkan, O., Luce, C.J., Madison, J.M., Sklar, P., Serre, T., Root, D.E., Petryshen, T.L., 2013. The ANK3 bipolar disorder gene regulates psychiatric-related behaviors that are modulated by lithium and stress. Biol Psychiatry 73(7) 683−90. [3] Cristino, A.S., Williams, S.M., Hawi, Z., An, J.Y., Bellgrove, M.A., Schwartz, C.E., Costa Lda, F., Claudianos, C., 2014. Neurodevelopmental and neuropsychiatric disorders represent an interconnected molecular system. Mol Psychiatry 19(3) 294–301. [4] Wesseling, H., Gottschalk, M.G., Bahn, S., 2015. Targeted multiplexed selected reaction monitoring analysis evaluates protein expression changes of molecular risk factors for major psychiatric disorders. Int J Neuropsychopharmacol 18(1). [5] Barry, J., Gu, Y., Jukkola, P., O’Neill, B., Gu, H., Mohler, P.J., Rajamani, K.T., Gu, C., 2014. Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons. Dev Cell 28(2) 117−31. P.2.015 Chronic effects of ‘biased’ 5-HT1A receptor agonists on object pattern separation performance and hippocampal plasticity B. Van Hagen1 ° , N.P. Van Goethem1 , R. Schreiber2 , A. Newman-Tancredi3 , M. Varney3 , J. Prickaerts1 . 1 Maastricht University, Dept of Psychiatry & Neuropsychology, Maastricht, The Netherlands; 2 Suadeo Drug Discovery, Consulting LLC, Watertown, MA, USA; 3 Neurolixis, Inc, Dana Point, CA, USA Introduction: Pattern separation, the formation of distinct representations out of similar inputs, is an important
hippocampal process implicated in memory formation. Impairments in pattern separation could underlie cognitive symptoms of disorders like PTSD and schizophrenia. Our previous research showed that acute treatment with the ‘biased’ 5-HT1A R agonist F15599, which preferentially activates postsynaptic cortical heteroreceptors, improved rat performance in a spatial object pattern separation (OPS) task. In contrast, F13714, which preferentially activates raphe-located autoreceptors, impaired performance in this task [1]. Here we investigated the effects of chronic treatment with F15599 and F13714 on OPS performance in rats. We hypothesized that, through desensitization of raphe-located autoreceptors by F13714, OPS performance impairment would ameliorate due to increased activation of postsynaptic receptors. We hypothesize an increase in plasticity and/or newborn neurons to underlie the increase in OPS performance. Methods: 48 three-month-old Wistar rats received a 14-day chronic treatment with either 0.32 mg/kg F15599 (N = 16), 0.02 mg/kg F13714 (N = 16) or vehicle (PBS, N = 16) (daily i.p. injections; injection volume of 2 ml/kg). Daily body temperature measurements were taken to indirectly measure hypothalamic post-synaptic 5-HT1A R activation. On day 1, 7 and 15 OPS task performance was measured [2], the d2 index (the difference in object exploration times relative to the total exploration time) was used as an outcome measure and a difference from zero (chance level) was evaluated with a onesample t-test. After behavioral assessment on day 15 the rats were sacrificed and from N = 8 animals per group the hippocampus was dissected for westernblot analysis of (pro)BDNF, (p)CREB, P11, synaptophysin and PSD95. For immunohistochemical analysis of hippocampal doublecortin (DCX) and 5-HT1A R’s in the Raphe nucleus N = 8 brains per treatment group were fixed in 4% PF. Stereological quantification of DCX is currently being performed. One-way ANOVA’s were used to compare between treatment groups for the biochemical and temperature measurements, if significant a Dunnett’s t-test comparing the treatment groups to vehicle was used. Results: The OPS impairment after acute treatment of F13714 was replicated (d2: 0.02; t-test comparing to zero, NS) and subsequently a gradual increase of performance was found, resembling vehicle treatment on day 15 (d2 F13714: 0.17; d2 vehicle: 0.16; t-tests zero P < 0.05). Body temperature dropped by 1 degree Celsius from treatment day 5 onwards (Dunnett’s t-test from vehicle: P < 0.001; repeated measures ANOVA from day 1: P < 0.1). F15599 showed an enhancement in OPS performance compared to vehicle acutely (d2: 0.33; t-test zero P < 0.001), and this was maintained through day 15 (d2: 0.27; t-test P < 0.001). Westernblot analysis of BDNF/GAPDH ratio
Behavioural and systems showed a significant increase in the dorsal hippocampus of the F15599 treated rats (4.456) compared to vehicle (1.381; Dunnett’s t-test; P < 0.05) but not to F13714. Other protein measurements showed no significant differences [P11, PSD95, synaptophysin, (p)CREB]. Discussion: These results imply desensitization of 5-HT1A autoreceptors by F13714, leading to stimulation of postsynaptic 5-HT1A Rs which could explain the drop in temperature. The increase in hippocampal BDNF does imply some enhanced plasticity in the F15599 treated rats but the DCX quantification results are needed to confirm an increase in adult hippocampal plasticity. Reference(s) [1] van Goethem, N.P., Schreiber, R., NewmanTancredi, A., Varney, M., Prickaerts, J., 2015. Divergent effects of the ’biased’ 5-HT1A receptor agonists F15599 and F13714 in a novel object pattern separation task. Br J Pharmacol 172:10, 1476–5381. [2] van Hagen, B.T.J., van Goethem, N.P., Lagatta, D.C., Prickaerts, J., 2015. The object pattern separation (OPS) task: a behavioral paradigm derived from the object recognition task. Beh Brain Research 285, 44−52. P.2.016 Muscimol effectively reverses impaired spatial memory in Alzheimer’s diseasetype streptozocin icv rat model V. Pilipenko1 ° , J. Pupure1 , J. Rumaks1 , U. Beitnere1 , Z. Dzirkale1 , B. Jansone1 , R. Skumbins1 , V. Klusa1 . 1 University of Latvia, Pharmacology, Riga, Latvia Background: GABAergic inhibitory action regulates learning and memory processes and contributes to neurotransmission [1]. Existing evidence suggests that GABAergic system is involved in pathophysiology of Alzheimer’s disease (AD) via inhibitory interneuron deficits [2] and decrease in functional GABA-A receptors [3]. In vitro, GABA and GABA-A receptor agonist muscimol blocked neuronal death induced by Ab in rat hippocampal and cortical neurons [4]. Intracerebroventricular (icv) streptozocin (STZ)-induced AD non-transgenic rat model is an easy and effective method for the detection of early behavioural disturbances such as loss of spatial memory and learning. Our concept suggests that low doses of muscimol may prevent learning and memory deficits in icv STZ-induced AD nontransgenic rat model. Methods: Male Wistar rats (280±20 g) received pretreatment with ip saline (control) or muscimol (0.01 and 0.05 mg/kg) on days 1−3. On day 4, rats received icv STZ
S37
(100 mg/ml) or aCSF. From days 19 to 22, rats received ip muscimol again, and spatial learning and memory were assessed in the water maze test (4 trials/day) by recording the escape latency, platform zone crossings and time in platform quadrant. A probe trial was carried out 24 h after the trainings. To investigate neuroinflammation, GFAP detection was implemented. All data were calculated using ANOVA with Bonferroni post-test (training days) and Tukey’s post-test (probe trial and GFAP density). Results and Discussion: Rats in STZ group showed statistically longer escape latencies vs. control group starting from day 2 (p < 0.0001). Muscimol at both doses significantly shortened escape latencies in MUSC 0.01 and MUSC 0.05 + STZ group rats vs. STZ from day 2, reversing STZ effect by about 90% on days 3 and 4 (p < 0.0001). In probe trial, STZ group rats showed a significant decrease in the number of platform crossings (p < 0.01) and the time spent in platform quadrant (p < 0.05) compared to control. Muscimol-treated STZ rats showed a significantly longer time spent in platform quadrant (p < 0.01) and a significantly higher number of platform crossings (p < 0.05 in MUSC 0.05 + STZ and p < 0.01 in MUSC 0.01 + STZ) vs. STZ group. Muscimol at both doses per se showed values comparable to control in both training days and probe trial. In the STZ group, a significant increase in GFAP density was observed compared vs. control (p < 0.0001). Muscimol at both doses showed significantly lower levels of GFAP in MUSC + STZ vs. STZ group. Conclusions: Obtained data suggest that icv STZ significantly decreased rat spatial learning and memory abilities in adult rats. Muscimol at both low doses, however, significantly improved learning and memory outcomes impaired by icv STZ, as well as alleviated the neuroinflammation. Therefore, novel therapeutic approaches aimed at the modulation of neuronal excitability through the control of GABAergic inhibitory inputs might be considered viable viable in the search of new anti-AD treatments. Reference(s) [1] Gong, N., Li, Y., Cai, G.Q., Niu, R.F., Fang, Q., Wu, K., Chen, Z., Lin, L.N., Xu, L., Fei, J., Xu, T.L., 2009. GABA transporter-1 activity modulates hippocampal theta oscillation and theta burst stimulation-induced long-term potentiation. J Neurosci 29, 15836–14845. [2] Verret, L., Mann, E.O., Hang, G.B., Barth, A.M., Cobos, I., Ho, K., Devidze, N., Masliah, E., Kreitzer, A.C., Mody, I., Mucke, L., Palop, J.J., 2012. Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell 149, 708–721.