Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission.

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.

Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.

Effects of SC58236, a selective COX-2 inhibitor, on epileptogenesis and spontaneous seizures in a rat model for temporal lobe epilepsy.

Inflammation is an important biological process that is activated after status epilepticus and could be implicated in the development of epilepsy. Here we tested whether an anti-inflammatory treatment with a selective cox-2 inhibitor (SC58236) could prevent the development of epilepsy or modify seizure activity during the chronic epileptic phase. SC58236 was orally administered (10mg/kg) during the latent period for 7 days, starting 4h after electrically induced SE. Seizures were monitored using EEG/video monitoring until 35 days after SE. Cell death and inflammation were investigated using immunocytochemistry (NeuN and Ox-42). Sprouting was studied using Timm's staining after 1 week and after 4-5 months when rats were chronic epileptic. SC58236 was also administered during 5 days in chronic epileptic rats. Hippocampal EEG seizures were continuously monitored before, during and after treatment. SC58236 effectively reduced PGE(2) production but did not modify seizure development or the extent of cell death or microglia activation in the hippocampus. SC58236 treatment in chronic epileptic rats did not show any significant change in seizure duration or frequency of daily seizures. The fact that cox-2 inhibition, which effectively reduced prostaglandin levels, did not modify epileptogenesis or chronic seizure activity suggests that this type of treatment (starting after SE) will not provide an effective anti-epileptogenic or anti-epileptic therapy.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death.

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 +/- 8 min in saline controls (N = 4) which increased to 369 +/- 71 and 322 +/- 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90% of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90 percent of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Acute buspirone abolishes the expression of behavioral dopaminergic supersensitivity in mice.

Previous studies have shown that rats withdrawn from long-term treatment with dopamine receptor blockers exhibit dopaminergic supersensitivity, which can be behaviorally evaluated by enhanced general activity observed in an open-field. Recently, it has been reported that co-treatment with the non-benzodiazepine anxiolytic buspirone attenuates the development of haloperidol-induced dopaminergic supersensitivity measured by open-field behavior of rats. The aims of the present study were: 1) to determine, as previously reported for rats, if mice withdrawn from long-term neuroleptic treatment would also develop dopaminergic supersensitivity using open-field behavior as an experimental paradigm, and 2) to examine if acute buspirone administration would attenuate the expression of this behavioral dopaminergic supersensitivity. Withdrawal from long-term haloperidol treatment (2.5 mg/kg, once daily, for 20 days) induced a significant (30%) increase in ambulation frequency (i.e., number of squares crossed in 5-min observation sessions) but did not modify rearing frequency or immobility duration in 3-month-old EPM-M1 male mice observed in the open-field apparatus. Acute intraperitoneal injection of buspirone (3.0 and 10 but not 1.0 mg/kg, 12-13 animals per group) 30 min before open-field exposure abolished the increase in locomotion frequency induced by haloperidol withdrawal. These data suggest that the open-field behavior of mice can be used to detect dopaminergic supersensitivity, whose expression is abolished by acute buspirone administration.

Acute buspirone abolishes the expression of behavioral dopaminergic supersensitivity in mice

Previous studies have shown that rats withdrawn from long-term treatment with dopamine receptor blockers exhibit dopaminergic supersensitivity, which can be behaviorally evaluated by enhanced general activity observed in an open-field. Recently, it has been reported that co-treatment with the non-benzodiazepine anxiolytic buspirone attenuates the development of haloperidol-induced dopaminergic supersensitivity measured by open-field behavior of rats. The aims of the present study were: 1) to determine, as previously reported for rats, if mice withdrawn from long-term neuroleptic treatment would also develop dopaminergic supersensitivity using open-field behavior as an experimental paradigm, and 2) to examine if acute buspirone administration would attenuate the expression of this behavioral dopaminergic supersensitivity. Withdrawal from long-term haloperidol treatment (2.5 mg/kg, once daily, for 20 days) induced a significant (30 percent) increase in ambulation frequency (i.e., number of squares crossed in 5-min observation sessions) but did not modify rearing frequency or immobility duration in 3-month-old EPM-M1 male mice observed in the open-field apparatus. Acute intraperitoneal injection of buspirone (3.0 and 10 but not 1.0 mg/kg, 12-13 animals per group) 30 min before open-field exposure abolished the increase in locomotion frequency induced by haloperidol withdrawal. These data suggest that the open-field behavior of mice can be used to detect dopaminergic supersensitivity, whose expression is abolished by acute buspirone administration

Anxiolytic-like effects of 4-phenyl-2-trichloromethyl-3H-1, 5-benzodiazepine hydrogen sulfate in mice.

The pharmacological effects of 4-phenyl-2-trichloromethyl-3H-1, 5-benzodiazepine hydrogen sulfate (PTMB), a novel synthetic benzodiazepine, were examined in mice. In the elevated plus-maze test of anxiety, 0.3-1 mg/kg diazepam ip (F(3,53) = 3.78; P<0.05) and 1-10 mg/kg PTMB ip increased (F(5,98) = 3.26; P<0.01), whereas 2 mg/kg picrotoxin ip decreased (F(3,59) = 8.32; P<0.001) the proportion of time spent in the open arms, consistent with an anxiolytic action of both benzodiazepines, and an anxiogenic role for picrotoxin. In the holeboard, 1.0 mg/kg diazepam ip increased (F(3,54) = 2.78; P<0.05) and 2 mg/kg picrotoxin ip decreased (F(3, 59) = 4.69; P<0.01) locomotor activity. Rotarod assessment revealed that 1 mg/kg diazepam ip and 3, 10 and 30 mg/kg PTMB ip produced significant motor incoordination compared to vehicle control (F(4, 70) = 7.6; P<0.001). These data suggest that the recently synthesized PTMB compound possesses anxiolytic activity and produces motor incoordination similar to those observed with diazepam.

Anxiolytic-like effects of 4-phenyl-2-trichloromethyl-3H-1, 5-benzodiazepine hydrogen sulfate in mice

The pharmacological effects of 4-phenyl-2-trichloromethyl-3H-1,5-benzodiazepine hydrogen sulfate (PTMB), a novel synthetic benzodiazepine, were examined in mice. In the elevated plus-maze test of anxiety, 0.3-1 mg/kg diazepam ip (F(3,53) = 3.78; P<0.05) and 1-10 mg/kg PTMB ip increased (F(5,98) = 3.26; P<0.01), whereas 2 mg/kg picrotoxin ip decreased (F(3,59) = 8.32; P<0.001) the proportion of time spent in the open arms, consistent with an anxiolytic action of both benzodiazepines, and an anxiogenic role for picrotoxin. In the holeboard, 1.0 mg/kg diazepam ip increased (F(3,54) = 2.78; P<0.05) and 2 mg/kg picrotoxin ip decreased (F(3,59) = 4.69; P<0.01) locomotor activity. Rotarod assessment revealed that 1 mg/kg diazepam ip and 3, 10 and 30 mg/kg PTMB ip produced significant motor incoordination compared to vehicle control (F(4,70) = 7.6; P<0.001). These data suggest that the recently synthesized PTMB compound possesses anxiolytic activity and produces motor incoordination similar to those observed with diazepam

Role of uptake inhibition and autoreceptor activation in the control of 5-HT release in the frontal cortex and dorsal hippocampus of the rat.

1. Using brain microdialysis, we compared the relative role of 5-hydroxytryptamine (5-HT; serotonin) blockade and somatodendritic 5-HT(1A) and/or terminal 5-HT(1B) autoreceptor activation in the control of 5-HT output. 2. Fluoxetine (10 mg kg(-1) i.p.) doubled the 5-HT output in frontal cortex and dorsal hippocampus. The 5-HT(1A) receptor antagonist WAY 100635, (0.3 mg kg(-1) s.c.) potentiated the effect of fluoxetine only in frontal cortex (to approximately 500 % of baseline). 3. Methiothepin (10 mg kg(-1) s.c.) further enhanced the 5-HT rise induced by fluoxetine+WAY 100635, to 835+/-179% in frontal cortex and 456+/-24% in dorsal hippocampus. Locally applied, methiothepin potentiated the fluoxetine-induced 5-HT rise more in the former area. 4. The selective 5-HT(1B) receptor antagonist SB-224289 (4 mg kg(-1) i.p.) enhanced the effect of fluoxetine (10 mg kg(-1) i.p.) in both areas. As with methiothepin, SB-224289 (4 mg kg(-1) i.p.) further enhanced the 5-HT increase produced by fluoxetine+WAY 100635 more in frontal cortex (613+/-134%) than in dorsal hippocampus (353+/-59%). 5. Locally applied, fluoxetine (10 - 300 microM; EC(50)=28 - 29 microM) and citalopram (1 - 30 microM; EC(50)=1.0 - 1.4 microM) increased the 5-HT output two to three times more in frontal cortex than in dorsal hippocampus. These data suggest that the comparable 5-HT increase produced by systemic fluoxetine in frontal cortex and dorsal hippocampus results from a greater effect of reuptake blockade in frontal cortex that is offset by a greater autoreceptor-mediated inhibition of 5-HT release. As a result, 5-HT autoreceptor antagonists preferentially potentiate the effect of fluoxetine in frontal cortex.

Ascorbic acid and alpha-tocopherol attenuate methylmalonic acid-induced convulsions.

The effects of chronic administration of alpha-tocopherol or melatonin, or acute ascorbic acid administration on the convulsant action of methylmalonic acid (MMA) were investigated in adult male rats. Animals were chronically injected with alpha-tocopherol (40 mg kg(-1), i.p.), melatonin (5 mg kg(-1), i.p.) or vehicle for 7 days. Buffered MMA (6 micromol/2 microl) or NaCl (9 micromol/2 microl) was injected intrastriatally and the animals were observed for the appearance of clonic or tonic-clonic convulsions and rotational behavior. Ascorbic acid (100 mg kg(-1), s.c.) was administered 30 min before MMA injection. Alpha-tocopherol and ascorbic acid pretreatment decreased the duration of the convulsive episodes and the rotational behavior elicited by MMA. This study provides evidence that free radical generation may participate in the convulsant effects of methylmalonic acid.

Effects of buspirone on an animal model of tardive dyskinesia.

1. The effects of buspirone were studied on an animal model of tardive dyskinesia, i.e., the quantification of orofacial dyskinesia in rats repeatedly treated with reserpine. 2. Rats were co-treated with saline [SAL] or buspirone [BUS] (3.0 mg/kg, i.p., twice daily) and vehicle [VEH] or reserpine [RES] (0.1 mg/kg, s.c., once every other day) for 19 days. On the day 20, the animals were observed for quantification of the behavioral parameters of orofacial dyskinesia: tongue protrusion and vacuous chewing movements frequencies and duration of twitching of the facial musculature. 3. Rats of the SAL + RES group exhibited a significant increase in the three behavioral parameters of orofacial dyskinesia relative to the rats of the SAL + VEH group. However, animals of the BUS + RES group showed only an increased frequency of vacuous chewing movements when compared to animals of the SAL + VEH group. In addition, the duration of the facial twitching was significantly decreased in the BUS + RES group in relation to rats of the SAL + RES group. There were no significant differences in the orofacial parameters between the BUS + VEH and the SAL + VEH groups. 4. Because it was also verified that chronic buspirone treatment was able to increase apomorphine-induced yawning behavior, the possibility is raised that buspirone attenuates reserpine-induced orofacial dyskinesia through the development of dopamine autoreceptor supersensitivity.

Reserpine does not induce orofacial dyskinesia in spontaneously hypertensive rats.

The nigrostriatal dopaminergic system seems to be involved in both reserpine-induced orofacial dyskinesia in normal rats and in the pathogenesis of hypertension in spontaneously hypertensive rats. In the present study, repeated reserpine administration (1.0 mg/kg, s.c., every other day, for 3 days) increased tongue protrusion and vacuous chewing frequencies as well as the duration of facial twitching in Wistar normotensive but not in spontaneously hypertensive rats. These results suggest that genetic hypertension and drug-induced orofacial movements may be inversely modulated by similar mechanisms in the nigrostriatal dopaminergic system.

Effects of buspirone on dopaminergic supersensitivity.

The effects of buspirone treatment on dopaminergic supersensitivity induced by long-term haloperidol administration were studied; both spontaneous activity (locomotion and rearing frequencies) of rats observed in an open-field and apomorphine-induced stereotypy were used as experimental parameters. Buspirone per se (3.0 mg/kg, twice daily, for 30 days) did not produce dopaminergic supersensitivity. When buspirone was given in combination to haloperidol (2.0 mg/kg, once daily, for 30 days), it decreased the neuroleptic withdrawal symptoms as detected in open-field behavior but not in apomorphine-induced stereotypy. Although single administration of buspirone per se decreased both open-field and apomorphine-induced stereotypy behavior, buspirone single administration did not modify the acute effects of haloperidol on these two behavioral models. Taken together with previous behavioral results showing that buspirone reverses haloperidol-induced catalepsy, the present data suggest that buspirone co-administration may lead to important clinical advantages concerning different extrapyramidal side effects of neuroleptic treatment.

Effects of age on a new animal model of tardive dyskinesia.

The effects of age were studied on a new animal model of tardive dyskinesia, i.e., the quantification of oral dyskinesia in rats repeatedly treated with reserpine. Adult and old rats received two injections of reserpine (0.5 or 1.0 mg/kg s.c.) or vehicle, separated by 48 h. One, 10, 25 and 40 days after the second injection of reserpine or vehicle, the animals were observed for quantification of the behavioral parameters of oral dyskinesia: tongue protrusion and vacuous chewing movement frequencies and duration of twitching of the facial musculature. Phenomenologically, control old rats and reserpine-treated adult animals showed very similar oral dyskinesia. When compared to control adult rats, the significant increase in tongue protrusion frequency induced by reserpine treatment was more persistent in the old rats than in the adult animals. Because it is well known that age increases the persistence of tardive dyskinesia, our data provide further support for the validation of reserpine-induced oral dyskinesia as an animal model of tardive dyskinesia. In addition, the possibility is raised that a common pathophysiological mechanism may underlie tardive dyskinesia and age- and reserpine-induced oral dyskinesia.