Lack of CB1 receptor activity impairs serotonergic negative feedback

Serotonergic and endocannabinoid systems are important substrates for the control of emotional behaviour and growing evidence show an involvement in the pathophysiology of mood disorders. In the present study, the absence of the activity of the CB₁ cannabinoid receptor impaired serotonergic negative feedback in mice. Thus, in vivo microdialysis experiments revealed increased basal 5-HT extracellular levels and attenuated fluoxetine-induced increase of 5-HT extracellular levels in the prefrontal cortex of CB₁ knockout compared with wild-type mice. These observations could be related to the significant reduction in the 5-HT transporter binding site density detected in frontal cortex and hippocampus of CB₁ knockout mice. The lack of CB₁ receptor also altered some 5-HT receptors related to the 5-HT feedback. Extracellular recordings in the dorsal raphe nucleus (DRN) revealed that the genetic and pharmacological blockade of CB₁ receptor induced a 5-HT_1A autoreceptor functional desensitization. In situ hybridization studies showed a reduction in the expression of the 5-HT_2C receptor within several brain areas related to the control of the emotional responses, such as the DRN, the nucleus accumbens and the paraventricular nucleus of the hypothalamus, whereas an over-expression was observed in the CA3 area of the ventral hippocampus. These results reveal that the lack of CB₁ receptor induces a facilitation of the activity of serotonergic neurons in the DRN by altering different components of the 5-HT feedback as well as an increase in 5-HT extracellular levels in the prefrontal cortex in mice.     

CB1 knockout mice display impaired functionality of 5-HT1A and 5-HT2A/C receptors

Interaction between brain endocannabinoid (EC) and serotonin (5-HT) systems was investigated by examining 5-HT-dependent behavioral and biochemical responses in CB₁ receptor knockout mice. CB₁ knockout animals exhibited a significant reduction in the induction of head twitches and paw tremor by the 5-HT_2A/C receptor selective agonist (±) DOI, as well as a reduced hypothermic response following administration of the 5-HT_1A receptor agonist (±)-8-OH-DPAT. Additionally, exposure to the tail suspension test induced enhanced despair responses in CB₁ knockout mice. However, the tricyclic antidepressant imipramine and the 5-HT selective reuptake inhibitor fluoxetine induced similar decreases in the time of immobility in the tail suspension test in CB₁ receptor knockout and wild-type mice. No differences were found between both genotypes with regard to 5-HT_2A receptor and 5-HT_1A receptors levels, measured by autoradiography in different brain areas. However, a significant decrease in the ability of both, the 5-HT_1A receptor agonist (±)-8-OH-DPAT and the 5-HT_2A/C receptor agonist (−)DOI, to stimulate [³⁵S]GTPγS binding was detected in the hippocampal CA₁ area and fronto-parietal cortex of CB₁ receptor knockout mice, respectively. This study provides evidence that CB₁ receptors are involved in the regulation of serotonergic responses mediated by 5-HT_2A/C and 5-HT_1A receptors, and suggests that a reduced coupling of 5-HT_1A and 5-HT_2A receptors to G proteins might be involved in these effects.     

Modulation of neuropeptide Y and Y1 receptor expression in the amygdala by fluctuations in the brain content of neuroactive steroids during ethanol drinking discontinuation in Y1R/LacZ transgenic mice

Previous studies have shown that GABAergic neuroactive steroids increase Y₁ receptor (Y₁R) gene expression in the amygdala of Y ₁ R / LacZ transgenic mice, harbouring the murine Y₁R gene promoter linked to a LacZ reporter gene. As ethanol is known to increase GABAergic neuroactive steroids, we investigated the relationship between fluctuations in the brain content of neuroactive steroids induced by chronic voluntary ethanol consumption or ethanol discontinuation and both the level of neuropeptide Y (NPY) immunoreactivity and Y₁R gene expression in the amygdala of Y ₁ R / LacZ transgenic mice. Ethanol discontinuation (48 h) after voluntary consumption of consecutive solutions of 3%, 6%, 10% and 20% (v/v) ethanol over 4 weeks produced an anxiety-like behaviour as measured by elevated plus maze. Voluntary ethanol intake increased the cerebrocortical concentration of the progesterone metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) that returned to control level 48 h after discontinuation of ethanol intake. Ethanol discontinuation significantly decreased NPY immunoreactivity and concomitantly increased Y ₁ R / LacZ transgene expression in the amygdala, whereas chronic ethanol intake failed to affect these parameters. The 5α-reductase inhibitor finasteride prevented both the increase in the cerebrocortical concentration of 3α,5α-TH PROG apparent after 4 weeks of ethanol intake and the changes in NPY immunoreactivity and transgene expression induced by ethanol discontinuation. Data suggest that 3α,5α-TH PROG plays an important role in the changes in NPY–Y₁R signalling in the amygdala during ethanol discontinuation.     

磁场对小鼠两种迷宫学习记忆的影响

据发现,磁场对生物体有一定作用,但是磁场对于人类或实验动物的学习记忆是否有影响,目前的报道结果很不一致。本实验采用实验小白鼠,给予不同强度（65高斯／50Hz,35高斯/25Hz）的低频磁场照射（每天1小时,持续25天）。磁场照射后,采用旷场行为测试、Y-迷宫和Morris水迷宫,检测小鼠的活动性、空间辨别、空间学习记忆和非空间学习记忆能力。结果表明：65高斯／50Hz磁场显著增高小鼠的活动性,并损伤小鼠Y-迷宫的空间辨别能力,但对Morris水迷宫的空间、非空间学习记忆无明显影响。35高斯／25Hz磁场处理动物行为在三个指标上均接近对照组。提示：长期的磁场照射可能会给动物,甚至人类造成一些影响。     

Abnormal anxiety-related behavior in serotonin transporter null mutant mice: the influence of genetic background

Serotonin transporter (5-HTT) null mutant mice provide a model system to study the role genetic variation in the 5-HTT plays in the regulation of emotion. Anxiety-like behaviors were assessed in 5-HTT null mutants with the mutation placed on either a B6 congenic or a 129S6 congenic background. Replicating previous findings, B6 congenic 5-HTT null mutants exhibited increased anxiety-like behavior and reduced exploratory locomotion on the light ↔ dark exploration and elevated plus-maze tests. In contrast, 129S6 congenic 5-HTT null mutant mice showed no phenotypic abnormalities on either test. 5-HTT null mutants on the 129S6 background showed reduced 5-HT_1A receptor binding (as measured by quantitative autoradiography) and reduced 5-HT_1A receptor function (as measured by 8-OH-DPAT-indcued hypothermia). These data confirm that the 5-HTT null mutation produced alterations in brain 5-HT function in mice on the 129S6 background, thereby discounting the possibility that the absence of an abnormal anxiety-like phenotype in these mice was due to a suppression of the mutation by 129 modifier genes. Anxiety-like behaviors in the light ↔ dark exploration and elevated plus-maze tests were significantly higher in 129S6 congenic +/+ mice as compared to B6 congenic +/+ mice. This suggests that high baseline anxiety-like behavior in the 129S6 strain might have precluded detection of the anxiety-like effects of the 5-HTT null mutation on this background. Present findings provide further evidence linking genetic variation in the 5-HTT to abnormalities in mood and anxiety. Furthermore, these data highlight the utility of conducting behavioral phenotyping of mutant mice on multiple genetic backgrounds.     

Working memory impairment in a transgenic amyloid precursor protein TgCRND8 mouse model of Alzheimer's disease

The most profound deficits observed in Alzheimer's disease (AD) are in domains of episodic and working memory systems. Transgenic (Tg) mice expressing mutated human amyloid precursor protein (APP) genes offer a model to study the effect of AD pathology on cognition. We reported previously that APP TgCRND8 mice showed deficits in a reference and working memory evaluated in a Morris water-maze test. In this study, we evaluated the working memory of TgCRND8 mice comparing two training paradigms in a six-arm radial water maze. In the first paradigm, the exploration of the maze was constrained, forcing the mice to use a spatial mapping strategy. In the second paradigm, mice were unconstrained in their exploration of the maze. TgCRND8 mice proved to be significantly impaired in spatial working memory in both paradigms as compared with their non-transgenic littermates. The analysis of data revealed that forcing mice to use a spatial strategy during training caused only a moderate improvement in the performance of all mice. However, unconstrained exploration of the maze not only resulted in a fast learning in control mice, but also facilitated the development of a chaining strategy in spatially impaired TgCRND8 mice. In conclusion, TgCRND8 mice showed impairment in spatial working memory but retained a plasticity to choose alternative search strategies.     

Behavioral profiles of inbred strains on novel olfactory,spatial and emotional tests for reference memory in mice

Studying the behavior of genetic background strains provides important information for the design and interpretation of cognitive phenotypes in mutant mice. Our experiments examined the performance of three commonly used strains (C57BL/6J, 129S6, DBA/2J) on three behavioral tests for learning and memory that measure very different forms of memory, and for which there is a lack of data on strain differences. In the social transmission of food preference test (STFP) all three strains demonstrated intact memory for an odor-cued food that had been sampled on the breath of a cagemate 24 hours previously. While C57BL/6J and 129S6 mice showed good trace fear conditioning, DBA/2J mice showed a profound deficit on trace fear conditioning. In the Barnes maze test for spatial memory, the 129S6 strain showed poor probe trial performance, relative to C57BL/6J mice. Comparison of strains for open field exploratory activity and anxiety-like behavior suggests that poor Barnes maze performance reflects low exploratory behavior, rather than a true spatial memory deficit, in 129S6 mice. This interpretation is supported by good Morris water maze performance in 129S6 mice. These data support the use of a C57BL/6J background for studying memory deficits in mutant mice using any of these tasks, and the use of a 129S6 background in all but the Barnes maze. A DBA/2J background may be particularly useful for investigating the genetic basis of emotional memory using fear conditioning.     

Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABA_A receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABA_A receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α₁ and α₅ subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α₁ and α₅ subunit immunoreactivity was reduced. In the VTA, levels of α₁ subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α₁ subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α₅ mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABA_A receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.     

Early life stress effects on adult stress-induced corticosterone secretion and anxiety-like behavior in the C57BL/6 mouse are not as robust as initially thought

Understanding environmental effects on mouse brain development would allow us to take advantage of powerful genetic tools to determine the interaction between genetic and epigenetic factors governing brain development in C57BL/6 mice. Experiment 1 examined whether time of day for neonatal manipulations affects adult stress-induced hormone secretion. Three rearing groups were examined: early handled (EH; dam removed 10 min/day); maternal separated (MS; dam removed 180 min/day); and an animal facility raised (AFR) control. Separations occurred during either the first or last 3 h of the light phase. Corticosterone (CORT) secretion in response to 100 dB white noise was assessed in adulthood. Both EH and MS males separated during the last 3 h of the light phase exhibited blunted stress-induced CORT compared to all other groups. Experiment 2 varied time of behavior testing. A fourth group was also added: maternal isolated (MI; separated from dam and littermates 180 min/day). Adult male behavior was assessed in three different tests. EH males tested in the elevated zero maze (EZM) during the light phase and MS males tested in the EZM during the dark phase exhibited diminished anxiety-like behavior compared to the other groups. We conclude that the EH protocol is marginally effective in blunting stress-induced CORT secretion and anxiety-like behavior in C57BL/6 mice, and these early handling effects are influenced by time of day. We also conclude that the 3 h MS or MI protocol is not effective in exacerbating future adult stress-induced CORT secretion or anxiety-like behavior in C57BL/6 mice.     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

Comprehensive behavioural study of GluR4 knockout mice: implication in cognitive function

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA‐type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1–4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long‐term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T‐maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.     

Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mouse

Studies were performed to determine if cyclooxygenase (COX)-2 regulates muscarinic receptor-initiated signaling involving brain phospholipase A₂ (PLA₂) activation and arachidonic acid (AA; 20 : 4n-6) release. AA incorporation coefficients, k* (brain [1–¹⁴C]AA radioactivity/integrated plasma radioactivity), representing this signaling, were measured following the intravenous injection of [1–¹⁴C]AA using quantitative autoradiography, in each of 81 brain regions in unanesthetized COX-2 knockout (COX-2^–/–) and wild-type (COX-2^+/+) mice. Mice were administered arecoline (30 mg/kg i.p.), a non-specific muscarinic receptor agonist, or saline i.p. (baseline control). At baseline, COX-2^–/– compared with COX-2^+/+ mice had widespread and significant elevations of k*. Arecoline increased k* significantly in COX-2^+/+ mice compared with saline controls in 72 of 81 brain regions, but had no significant effect on k* in any region in COX-2^–/– mice. These findings, when related to net incorporation rates of AA from brain into plasma, demonstrate enhanced baseline brain metabolic loss of AA in COX-2^–/– compared with COX-2^+/+ mice, and an absence of a normal k* response to muscarinic receptor activation. This response likely reflects selective COX-2-mediated conversion of PLA₂-released AA to prostanoids.     

Transmembrane 36CI− Flux Measurements and Desensitization of the γ-Aminobutyric AcidA Receptor

Abstract: Some data on the concentration range of response and the concentration for half-response (EC₅₀) of γ-aminobutyric acid (GABA) for the GABA_A receptor are reviewed and compared. An analysis of the ³⁶CI⁻ flux assay demonstrates that both the EC₅₀ and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the ³⁶CI⁻ concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC₅₀ can be decreased by orders of magnitude by increasing the assay time. The EC₅₀ measured in a finite time is less than the half-response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC₅₀ depends on the rate of receptor desensitization. The computer simulations showed that a GABA_A receptor with a half-response concentration of 100 μ M GABA can give ³⁶CI⁻ flux measurements with an EC₅₀ value 100-fold lower.     

Guinea pig hippocampal 5-HT1E receptors: a tool for selective drug development

Recent studies have indicated that the serotonin [5-hydroxytryptamine (5-HT)] 1E receptor, originally discovered in human brain tissue, is not expressed in rat or mouse brain. Thus, there have been few reports on 5-HT_1E receptor drug development. However, expression of 5-HT_1E receptor mRNA has been shown in guinea pig brain. To establish this species as an animal model for 5-HT_1E drug development, we identified brain regions that exhibit 5-carboxyamidotryptamine, ritanserin, and LY344864 – insensitive [³H]5-HT binding (characteristic of the 5-HT_1E receptor). In hippocampal homogenates, where 5-HT_1E receptor density was sufficiently high for radioligand binding analysis, 100 nM 5-carboxyamidotryptamine, 30 nM ritanserin, and 100 nM LY344864 were used to mask [³H]5-HT binding at non-5-HT_1E receptors. The K _d of [³H]5-HT was 5.7 ± 0.7 nM and is indistinguishable from the cloned receptor K _d of 6.5 ± 0.6 nM. The affinities of 16 drugs for the cloned and hippocampal-expressed guinea pig 5-HT_1E receptors are essentially identical ( R ² = 0.97). These findings indicate that using these conditions autoradiographical distribution and signal transduction studies of the 5-HT_1E receptor in guinea pig brain are feasible. Using the guinea pig as an animal model should provide important insights into possible functions of this receptor and the therapeutic potential of selective human 5-HT_1E drugs.     

Inhibition of Adenylyl Cyclase Activity by a Homogeneous Population of Dopamine Receptors: Selective Blockade by Antisera Directed Against Gi1 and/or Gi2

Abstract: The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D₂ receptors. [³H]-Sulpiride binding to 7315c cell homogenates was specific and saturable, and K _i values for compounds to compete for these sites were highly correlated with values for the same compounds at D₂ receptors in brain. Dopamine maximally inhibited ∼65% of forskolin-stimulated cyclase activity in cell membranes. Some D₂ agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known Gα subunits revealed that G_i1, G_i2, G_i3, G_o, G_q, and G_s are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C-terminal regions of Gα_i1 and Gα_i2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C-terminal regions of the other Gα subunits had no effect. These findings suggest that the dopamine D₂ receptor regulates cyclase inhibition predominantly via G_i1 and/or G_i2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D₂ receptors in the absence of other dopamine receptor subtypes.     

The effect of mGlu8 deficiency in animal models of psychiatric diseases

The metabotropic glutamate receptor subtype 8 (mGlu₈) is presynaptically located and regulates the release of the transmitter. Dysfunctions of this mechanism are involved in the pathophysiology of different psychiatric disorders. mGlu₈ deficient mice have been previously investigated in a range of studies, but the results are contradictory and there are still many open questions. Therefore, we tested mGlu₈-deficient animals in different behavioral tasks that are commonly used in neuropsychiatric research. Our results show a robust contextual fear deficit in mGlu₈-deficient mice. Furthermore, novel object recognition, chlordiazepoxide-facilitated extinction of operant conditioning and the acoustic startle response were attenuated by mGlu₈ deficiency. We found no changes in sensory processing, locomotor activity, prepulse inhibition, phencyclidine-induced changes in locomotion or prepulse inhibition, operant conditioning, conditioned fear to a discrete cue or in animal models of innate fear and post-traumatic stress disorder. We conclude that mGlu₈ might be a potential target for disorders with pathophysiological changes in brain areas where mGlu₈ modulates glutamate and gamma-amino butyric acid (GABA) transmission. Our data especially point to anxiety disorders involving exaggerated contextual fear, such as generalized anxiety disorders, and to conditions with disturbed declarative memory.     

The effect of intrahippocampal insulin microinjection on spatial learning and memory

Insulin is best known for its action on peripheral target tissues such as the adipocyte, muscle and liver to regulate glucose homeostasis. Insulin and its receptor are found in specific area of CNS with a variety of region-specific functions different from its direct glucose regulation in the periphery. The hippocampus and cerebral cortex distributed insulin/insulin receptor has been shown to be involved in brain cognitive functions. Previous studies about the effect of insulin on memory are controversial. In the present study, the effect of insulin microinjection into CA1 region of rat hippocampus on water maze performance has been investigated. Insulin had a discrepant effect dose dependently. The spatial learning and memory were impaired with lower dose of insulin, had not changed with intermediate doses, while they improved with higher doses. These results suggest that insulin may have a dose-dependent effect on spatial learning and memory.     

Immunolabeling of Central Serotonin 5-HT1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti-Peptide Antiserum

Abstract: A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT_1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT_1B/1Dβ receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin- O -carboxymethylglycyl[¹²⁵I]iodotyrosinamide ([¹²⁵I]GTI; a selective 5-HT_1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT_1B receptors. The remaining 40% of [¹²⁵I]GTI binding sites were shown to be 5-HT_1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT_1B/1Dβ, but not the 5-HT_1D/1Dα, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT_1Dβ receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [¹²⁵I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT_1Dβ component of [¹²⁵I]GTI specific receptor binding sites.