RESUMO
Mice lacking functional neurokinin-1 receptors (NK1R-/-) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: 'Hom') wildtype and NK1R-/- mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R-/- progeny of heterozygous parents ('Het', derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R-/- mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5-Choice Serial Reaction-Time Task (5-CSRTT). During training, NK1R-/- mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R-/- mice from the Hom colony were more impulsive than their wildtypes, but NK1R-/- mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R-/- mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R-/- mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors.
Assuntos
Comportamento Animal/fisiologia , Comportamento de Escolha/fisiologia , Comportamento Impulsivo/fisiologia , Receptores da Neurocinina-1/genética , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Camundongos Knockout , Fenótipo , Tempo de Reação/genética , Receptores da Neurocinina-1/deficiênciaRESUMO
BACKGROUND: Fear-conditioned analgesia (FCA) is the profound suppression of pain during exposure to conditioned aversive stimuli and is mediated at spinal and supraspinal levels. The endocannabinoid system plays a key role in FCA. This study investigated brain and spinal cord expression of genes implicated in pain- and fear-related plasticity (Zif268 and Sgk1), following expression of formalin-evoked nociception, contextual fear or endocannabinoid-mediated FCA. METHODS: Adult male Lister-Hooded rats received intra-plantar injection of formalin or saline, with or without administration of the CB1 receptor antagonist AM251 (3 mg/kg, i.p.) or vehicle, 30 min prior to re-exposure to an arena paired 24 h previously with footshock. Real time quantitative polymerase chain reaction was used to measure expression of Zif268 and Sgk1 mRNA in the dorsal horn of the spinal cord (DHSC) and rostral ventromedial medulla (RVM) 30 min following arena re-exposure. RESULTS: Intra-plantar injection of formalin resulted in an increase in Zif268 and Sgk1 mRNA expression in the ipsilateral DHSC of non-fear-conditioned rats, effects not observed in rats expressing FCA. Systemic administration of the CB1 receptor antagonist/inverse agonist AM251 attenuated both FCA and the FCA-associated suppression of Zif268 expression in the ipsilateral DHSC without affecting expression of Sgk1. Conditioned fear was associated with an increase in Zif268 mRNA expression in the RVM of saline-, but not formalin-treated rats. CONCLUSIONS: The present findings suggest that Zif268 in the DHSC is an important molecular correlate of endocannabinoid-mediated FCA, and that fear-related expression of Zif268 in the RVM is influenced by the presence of nociceptive tone.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Endocanabinoides/metabolismo , Medo/efeitos dos fármacos , Receptor CB1 de Canabinoide/antagonistas & inibidores , Analgesia , Animais , Comportamento Animal/efeitos dos fármacos , Condicionamento Psicológico , Modelos Animais de Doenças , Masculino , Manejo da Dor , Ratos , Receptor CB1 de Canabinoide/metabolismo , Medula Espinal/metabolismoRESUMO
Genetically-altered mice, lacking functional NK1 receptors (NK1R-/-), express abnormal behaviours that are prominent in Attention Deficit Hyperactivity Disorder: namely, inattentiveness and impulsivity (indicated by their greater % omissions and premature responses in the 5-Choice Serial Reaction-Time Task (5-CSRTT) and locomotor hyperactivity. We investigated how behaviour in the 5-CSRTT is affected by repeated testing and whether the abnormalities expressed by NK1R-/- mice are mimicked by treating wild type mice with a NK1R antagonist (L 733060 or RP 67580; 5 or 10 mg/kg). Repeated testing with a variable (VITI) or fixed, prolonged (LITI) intertrial interval reduced % omissions. Premature responses also declined, but only in NK1R-/- mice, in the VITI test. By contrast, perseveration increased in both genotypes. RP 67580 (10 mg/kg) increased the % omissions in both genotypes in the VITI, an action which cannot be attributed to NK1R antagonism. Neither drug affected perseveration. However, for premature responses, the response profile suggested that the low and high doses of RP 67580 (VITI) and L 733060 (LITI) had opposing effects on this behaviour. We infer that the effect of NK1R antagonists in the 5-CSRTT is confounded by animals' test experience and non-specific drug effects at sites other than NK1R, possibly L-type Ca²âº(v) channels.
Assuntos
Comportamento de Escolha/efeitos dos fármacos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Tempo de Reação/efeitos dos fármacos , Receptores da Neurocinina-1/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Comportamento de Escolha/fisiologia , Masculino , Camundongos , Camundongos Knockout , Tempo de Reação/genética , Receptores da Neurocinina-1/genéticaRESUMO
The substance P/NK1 receptor system plays an important role in the regulation of stress and emotional responding and as such had been implicated in the pathophysiology of anxiety and depression. The present study investigated whether alterations in the substance P/NK1 receptor system in brain areas which regulate emotional responding accompany the depressive behavioural phenotype observed in the olfactory bulbectomised (OB) mouse. The effect of NK1 receptor deletion on behavioural responding and monoamine levels in discrete brain regions of the OB model, were also examined. Substance P levels in the frontal cortex and NK1 receptor expression in the amygdala and hippocampus were enhanced following olfactory bulbectomy. Although NK1 receptor knockout (NK1-/-) mice did not exhibit altered behavioural responding in the open field test, noradrenaline levels were enhanced in the frontal cortex, amygdala and hippocampus, as were serotonin levels in the frontal cortex. Locomotor activity and exploratory behaviour were enhanced in wild type OB mice, indicative of a depressive-like phenotype, an effect attenuated in NK1-/- mice. Bulbectomy induced a decrease in noradrenaline and 5-HIAA in the frontal cortex and an increase in serotonin in the amygdala, effects attenuated in OB NK1-/- mice. The present studies indicate that alterations in substance P/NK1 receptor system underlie, at least in part, the behavioural and monoaminergic changes in this animal model of depression.
Assuntos
Monoaminas Biogênicas/metabolismo , Depressão/metabolismo , Depressão/fisiopatologia , Receptores da Neurocinina-1/fisiologia , Substância P/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Depressão/genética , Depressão/psicologia , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Bulbo Olfatório/cirurgia , Receptores da Neurocinina-1/biossíntese , Receptores da Neurocinina-1/genéticaRESUMO
Mice with functional genetic ablation of the Tacr1 (substance P-preferring receptor) gene (NK1R-/-) are hyperactive. Here, we investigated whether this is mimicked by NK1R antagonism and whether dopaminergic transmission is disrupted in brain regions that govern motor performance. The locomotor activity of NK1R-/- and wild-type mice was compared after treatment with an NK1R antagonist and/or psychostimulant (d-amphetamine or methylphenidate). The inactivation of NK1R (by gene mutation or receptor antagonism) induced hyperactivity in mice, which was prevented by both psychostimulants. Using in vivo microdialysis, we then compared the regulation of extracellular dopamine in the prefrontal cortex (PFC) and striatum in the two genotypes. A lack of functional NK1R reduced (>50%) spontaneous dopamine efflux in the prefrontal cortex and abolished the striatal dopamine response to d-amphetamine. These behavioural and neurochemical abnormalities in NK1R-/- mice, together with their atypical response to psychostimulants, echo attention deficit hyperactivity disorder (ADHD) in humans. These findings prompted genetic studies on the TACR1 gene (the human equivalent of NK1R) in ADHD patients in a case-control study of 450 ADHD patients and 600 screened supernormal controls. Four single-nucleotide polymorphisms (rs3771829, rs3771833, rs3771856, and rs1701137) at the TACR1 gene, previously known to be associated with bipolar disorder or alcoholism, were strongly associated with ADHD. In conclusion, our proposal that NK1R-/- mice offer a mouse model of ADHD was borne out by our human studies, which suggest that DNA sequence changes in and around the TACR1 gene increase susceptibility to this disorder.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Polimorfismo de Nucleotídeo Único , Receptores da Neurocinina-1/genética , Animais , Sequência de Bases , Estudos de Casos e Controles , Corpo Estriado/metabolismo , Dextroanfetamina/farmacologia , Dopamina/metabolismo , Predisposição Genética para Doença , Humanos , Masculino , Metilfenidato/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Atividade Motora/genética , Fenótipo , Córtex Pré-Frontal/metabolismo , Receptores da Neurocinina-1/efeitos dos fármacosRESUMO
A large number of cytokines and growth factors support the development and subsequent maintenance of postnatal motor neurons. RegIIIbeta, also known as Reg2 in rat and HIP/PAP1 in humans, is a member of a family of growth factors found in many areas of the body and previously shown to play an important role in both the development and regeneration of subsets of motor neurons. It has been suggested that RegIIIbeta expressed by motor neurons is both an obligatory intermediate in the downstream signaling of the leukemia inhibitory factor/ciliary neurotrophic factor (CNTF) family of cytokines, maintaining the integrity of motor neurons during development, as well as a powerful influence on Schwann cell growth during regeneration of the peripheral nerve. Here we report that in mice with a deletion of the RegIIIbeta gene, motor neuron survival was unaffected up to 28 weeks after birth. However, there was no CNTF-mediated rescue of neonatal facial motor neurons after axotomy in KO animals when compared with wild-type. In mice, RegIIIbeta positive motor neurons are concentrated in cranial motor nuclei that are involved in the patterning of swallowing and suckling. We found that suckling was impaired in RegIIIbeta KO mice and correlated this with a significant delay in myelination of the hypoglossal nerve. In summary, we propose that RegIIIbeta has an important role to play in the developmental fine-tuning of neonatal motor behaviors mediating the response to peripherally derived cytokines and growth factors and regulating the myelination of motor axons.
Assuntos
Fator Neurotrófico Ciliar/metabolismo , Nervo Hipoglosso/metabolismo , Neurônios Motores/metabolismo , Bainha de Mielina/metabolismo , Proteínas/metabolismo , Animais , Fator Neurotrófico Ciliar/genética , Deglutição/fisiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Proteínas Associadas a Pancreatite , Proteínas/genética , Comportamento de Sucção/fisiologiaRESUMO
Reg-2 is a secreted protein that is expressed de novo in motoneurons, sympathetic neurons, and dorsal root ganglion (DRG) neurons after nerve injury and which can act as a Schwann cell mitogen. We now show that Reg-2 is also upregulated by DRG neurons in inflammation with a very unusual expression pattern. In a rat model of monoarthritis, Reg-2 immunoreactivity was detected in DRG neurons at 1 day, peaked at 3 days (in 11.6% of DRG neurons), and was still present at 10 days (in 5%). Expression was almost exclusively in the population of DRG neurons that expresses the purinoceptor P2X(3) and binding sites for the lectin Griffonia simplicifolia IB4, and which is known to respond to glial cell line-derived neurotrophic factor (GDNF). Immunoreactivity was present in DRG cell bodies and central terminals in the dorsal horn of the spinal cord. In contrast, very little expression was seen in the nerve growth factor (NGF) responsive and substance P expressing population. However intrathecal delivery of GDNF did not induce Reg-2 expression, but leukemia inhibitory factor (LIF) had a dramatic effect, inducing Reg-2 immunoreactivity in 39% of DRG neurons and 62% of P2X(3) cells. Changes in inflammation have previously been observed predominantly in the neuropeptide expressing, NGF responsive, DRG neurons. Our results show that changes also take place in the IB4 population, possibly driven by members of the LIF family of neuropoietic cytokines. In addition, the presence of Reg-2 in central axon terminals implicates Reg-2 as a possible modulator of second order dorsal horn cells.
Assuntos
Artrite Experimental/patologia , Gânglios Espinais/patologia , Expressão Gênica/fisiologia , Litostatina/metabolismo , Neurônios/metabolismo , Animais , Expressão Gênica/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Indóis , Lectinas/metabolismo , Fator Inibidor de Leucemia/farmacologia , Masculino , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos , Ratos Wistar , Receptor trkA/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X3 , Substância P/metabolismo , Fatores de TempoRESUMO
Genetic background affects animal phenotype and therefore is of particular relevance to studies using genetically manipulated mice. Strain differences in hypothalamic-pituitary-adrenocortical (HPA) axis activity may contribute to background-specificity of some mutations. Here, we analysed components of the HPA axis in mice lacking a functional neurokinin-1 receptor (NK1-/-) on two backgrounds: backcrossed C57BL/6 (B6) and mixed C57BL/6 x 129/sv (129B6). We hypothesized that HPA axis activity would vary between these strains, leading to differences in the NK1-/- phenotype. We compared levels of plasma corticosterone between the groups, and found 129B6 mice exhibited elevated levels of stress-induced corticosterone compared with B6 mice, regardless of genotype. Although the level of basal corticotrophin-releasing factor and stress-induced c-fos mRNAs did not differ between the genotypes of either strain, examination of glucocorticoid receptor immunoreactivity within the hippocampus revealed that NK1-/- mice on the 129B6 background had elevated expression compared with wild-type, whilst there was no difference between genotypes in the B6 strain. Similarly, hippocampal neurogenesis in NK1-/- mice was greater than in wild-type on the 129B6 strain, and did not differ between genotypes on the B6 background. Finally, novelty- and morphine-induced locomotion were assessed. NK1-/- mice on the 129B6 background exhibited hyperlocomotion in response to novelty and greater sensitivity to the locomotor-stimulating properties of morphine than wild-type. In contrast, in B6 mice, no differences were observed between genotypes for either locomotor behaviour. In summary, we find that HPA axis activity differs between the strains and that there are profoundly background-specific effects of the NK1 receptor mutation.
Assuntos
Encéfalo/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Receptores da Neurocinina-1/genética , Estresse Fisiológico/metabolismo , Animais , Encéfalo/citologia , Proliferação de Células , Corticosterona/sangue , Corticosterona/metabolismo , Resistência a Medicamentos/genética , Comportamento Exploratório/fisiologia , Predisposição Genética para Doença/genética , Genótipo , Hipocampo/citologia , Hipocampo/metabolismo , Sistema Hipotálamo-Hipofisário/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Mutação/genética , Fenótipo , Receptores de Glucocorticoides/metabolismo , Especificidade da Espécie , Estresse Fisiológico/genética , Estresse Fisiológico/fisiopatologia , Taquicininas/metabolismo , Regulação para Cima/genéticaRESUMO
We used a molecular screen to identify genes upregulated in regenerating adult rat dorsal root ganglion cells. FLRT3 mRNA and protein characterized by a fibronectin type III domain and a leucine-rich repeat motif was upregulated in damaged sensory neurons. The protein was then transported into their peripheral and central processes where the FLRT3 protein was localized to presynaptic axon terminals. In vitro, the FLRT3 protein was expressed at the cell surface, regulated neurite outgrowth in sensory neurons, but did not exhibit homophilic binding. FLRT3 was widely expressed in the developing embryo, particularly in the central nervous system and somites. However, in the adult, we found no evidence for accumulation or reexpression of the FLRT3 protein in damaged axons of the central nervous system. We conclude that FLRT3 codes for a putative cell surface receptor implicated in both the development of the nervous system and in the regeneration of the peripheral nervous system (PNS).
Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/fisiologia , Neuritos/metabolismo , Neurônios Aferentes/metabolismo , Neuropatia Ciática/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/genéticaRESUMO
The use of viral vectors for gene delivery to motor neurons in vivo has been hampered by the need to perform invasive surgery to inject directly the vector into the anterior horn of the spinal cord. Here, we have characterized the features of herpes simplex virus-1 (HSV)-derived vectors, in terms of gene mutations and promoter constructs, that are required to allow efficient transduction of motor neurons following a relatively noninvasive peripheral administration via sciatic nerve or footpad injection. Owing to the wide variety of animal models used to study neurodegenerative diseases of motor neurons, we analysed the effectiveness of these vectors in adult mice and adult and neonatal rats. We tested viruses with differing degrees of disablement based on the 1764 backbone (deleted for ICP34.5 and an insertional inactivation in VP16) rendered completely replication incompetent by the deletion of the essential immediate-early genes ICP27 and/or ICP4. In the adult mouse, prolonged gene expression in motor neurons was obtained after sciatic nerve inoculation with a vector defective in ICP4 and ICP27. In the adult rat, both the vector defective in ICP4 and the vector defective in ICP4 and ICP27 were capable of transducing motor neurons for extended periods of time during viral latency. This study demonstrates the feasibility of using HSV vectors for persistent transgene expression in motor neurons in a safe and nontoxic manner following peripheral administration. These vectors are potentially useful tools to investigate the functions of genes involved in motor neuronal survival and regeneration in models of motor neuron diseases in vivo.
Assuntos
Gânglios Espinais/metabolismo , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Herpesvirus Humano 1/genética , Neurônios Motores/metabolismo , Doenças Neurodegenerativas/terapia , Animais , Animais Recém-Nascidos , Deleção de Genes , Expressão Gênica , Vetores Genéticos/genética , Humanos , Proteínas Imediatamente Precoces/genética , Injeções , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Modelos Animais , Regeneração Nervosa , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático , Fatores de Tempo , Transdução Genética/métodos , Transgenes , Latência ViralRESUMO
We have used differential display to profile and compare the mRNAs expressed in the hippocampus of freely moving animals after the induction of long-term potentiation (LTP) at the perforant path-dentate gyrus synapse with control rats receiving low-frequency stimulation. We have combined this with in situ hybridization and have identified A-kinase anchoring protein of 150 kDa (AKAP-150) as a gene selectively up-regulated during the maintenance phase of LTP. AKAP-150 mRNA has a biphasic modulation in the dentate gyrus following the induction of LTP. The expression of AKAP-150 was 29% lower than stimulated controls 1 h after the induction of LTP. Its expression was enhanced 3 (50%), 6 (239%) and 12 h (210%) after induction, returning to control levels by 24 h postinduction. The NMDA receptor antagonist CPP blocked the tetanus-induced modulation of AKAP-150 expression. Interestingly, strong generalized stimulation produced by electroconvulsive shock did not increase the expression of AKAP-150. This implies that the AKAP-150 harbours a novel property of selective responsiveness to the stimulation patterns that trigger NMDA-dependent LTP in vivo. Its selective up-regulation during LTP and its identified functions as a scaffold for protein kinase A, protein kinase C, calmodulin, calcineurin and ionotropic glutamate receptors suggest that AKAP-150 encodes is an important effector protein in the expression of late LTP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Proteínas de Ancoragem à Quinase A , Animais , Anticonvulsivantes/farmacologia , Estimulação Elétrica , Eletrochoque , Hibridização In Situ , Masculino , Plasticidade Neuronal/fisiologia , Piperazinas/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/metabolismo , Regulação para CimaRESUMO
The primary epineural repair of human peripheral nerves is most often achieved using non-absorbable microsutures, which can elicit a foreign-body reaction. We describe a new system for neural-tissue approximation, consisting of non-penetrating vascular closure staples (VCS) applied to the epineurium. These clips were initially developed for use in microvascular anastomosis, with no knowledge of their effectiveness in neural-tissue approximation. We compare the efficacies of VCS clips and monofilament nylon microsutures in the repair of transected sciatic nerves in 36 adult Sprague Dawley rats (18 treated with 9/0 sutures and 18 treated with VCS clips). In the rat, regeneration starts by day 5, and is well advanced by 4 weeks. To assess the overall repair success, the site of injury, after perfusion fixation, was harvested at 5, 7 and 30 days. The two methods were compared in terms of operative time, the degree of self-mutilation (autotomy), the macroscopic specimens in vivo and the microanatomical continuity through the repair site. Continuity was studied by using PGP-9.5-labelled cryosections and fluorescent secondary antibodies to visualise axonal regeneration. Clip repair was significantly faster in the VCS group (mean +/- s.e.m. = 7.09 +/- 0.36 min versus mean +/- s.e.m. 11.56 +/- 0.51 min in the sutured group) and an equal and minimal degree of autotomy was observed. Macroscopically, all 36 nerves were in continuity and free from neuroma. The use of VCS clips resulted in equivalent visualised regeneration across the repair site at each time point. We believe the use of VCS clips to be a faster and comparable alternative to non-absorbable sutures in primary nerve repair in this experimental model.
Assuntos
Procedimentos de Cirurgia Plástica/métodos , Nervo Isquiático/cirurgia , Suturas , Titânio , Animais , Feminino , Imuno-Histoquímica , Masculino , Regeneração Nervosa , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/anatomia & histologia , Automutilação , Fatores de TempoRESUMO
The phenotype of NK1R-/- mice was compared with that of acute pharmacological blockade of the tachykinin NK1 receptor on sensorimotor function and in assays relevant to depressive illness and anxiety. The dose range for L-760735 and GR205171 that was associated with functional blockade of central NK1 receptors in the target species was established by antagonism of the behavioural effects of intracerebroventricular NK1 agonist challenge in gerbils, mice and rats. The caudal grooming and scratching response to GR73632 was absent in NK1R-/- mice, confirming that the receptor had been genetically ablated. There was no evidence of sedation or motor impairment in NK1R-/- mice or following administration of L-760735 to gerbils, even at doses in excess of those required for central NK1 receptor occupancy. In the resident-intruder and forced swim test, the behaviour of NK1R-/- mice, or animals treated acutely with L-760735 or GR205171, resembled that seen with the clinically used antidepressant drug fluoxetine. However, the effects of GR205171 were not clearly enantioselective in mice. In contrast, although NK1R-/- mice also exhibited an increase in the duration of struggle behaviour in the tail suspension test, this was not observed following pharmacological blockade with L-760735 in gerbils or GR205171 in mice, suggesting that this may reflect a developmental alteration in the knockout mouse. There was no effect of NK1 receptor blockade with L-760735 in guinea-pigs or GR205171 in rats, or deletion of the NK1 receptor in mice, on behaviour in the elevated plus-maze test for anxiolytic activity. These findings extend previous observations on the phenotype of the NK1R-/- mouse and establish a broadly similar profile following acute pharmacological blockade of the receptor. These studies also serve to underscore the limitations of currently available antagonists that are suitable for use in rat and mouse behavioural assays.
Assuntos
Ansiolíticos/farmacologia , Antidepressivos/farmacologia , Ansiedade/genética , Nível de Alerta/genética , Depressão/genética , Mutação/genética , Fenótipo , Receptores da Neurocinina-1/genética , Animais , Nível de Alerta/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Gerbillinae , Cobaias , Desamparo Aprendido , Masculino , Camundongos , Morfolinas/farmacologia , Motivação , Antagonistas dos Receptores de Neurocinina-1 , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Tetrazóis/farmacologiaRESUMO
Antagonists at substance P receptors of the neurokinin 1 (NK1) type have been shown to represent a novel class of antidepressant drugs, with comparable clinical efficacy to the selective serotonin (5-HT) reuptake inhibitors (SSRIs). Because 5-HT(1A) receptors may be critically involved in the mechanisms of action of SSRIs, we examined whether these receptors could also be affected in a model of whole-life blockade of NK1 receptors, i.e. knock-out mice lacking the latter receptors (NK1-/-). 5-HT(1A) receptor labeling by the selective antagonist radioligand [(3)H]N-[2-[4-(2-methoxyphenyl)1-piperazinyl]-ethyl]-N-(2-pyridinyl)-cyclohexanecarboxamide (WAY 100635) and 5-HT(1A)-dependent [(35)S]GTP-gamma-S binding at the level of the dorsal raphe nucleus (DRN) in brain sections, as well as the concentration of 5-HT(1A) mRNA in the anterior raphe area were significantly reduced (-19 to -46%) in NK1-/- compared with NK1+/+ mice. Furthermore, a approximately 10-fold decrease in the potency of the 5-HT(1A) receptor agonist ipsapirone to inhibit the discharge of serotoninergic neurons in the dorsal raphe nucleus within brainstem slices, and reduced hypothermic response to 8-OH-DPAT, were noted in NK1-/- versus NK1+/+ mice. On the other hand, cortical 5-HT overflow caused by systemic injection of the SSRI paroxetine was four- to sixfold higher in freely moving NK1-/- mutants than in wild-type NK1+/+ mice. Accordingly, the constitutive lack of NK1 receptors appears to be associated with a downregulation/functional desensitization of 5-HT(1A) autoreceptors resembling that induced by chronic treatment with SSRI antidepressants. Double immunocytochemical labeling experiments suggest that such a heteroregulation of 5-HT(1A) autoreceptors in NK1-/- mutants does not reflect the existence of direct NK1-5-HT(1A) receptor interactions in normal mice.
Assuntos
Antidepressivos/farmacologia , Autorreceptores/metabolismo , Receptores da Neurocinina-1/deficiência , Receptores de Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Resistência a Medicamentos/fisiologia , Eletrofisiologia , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paroxetina/farmacologia , Piperazinas/farmacocinética , Piridinas/farmacocinética , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Núcleos da Rafe/citologia , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/metabolismo , Receptores da Neurocinina-1/genética , Receptores de Serotonina/genética , Receptores 5-HT1 de Serotonina , Antagonistas da Serotonina/farmacocinética , Agonistas do Receptor de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Substância P/metabolismoRESUMO
Pain is necessary for survival, but persistent pain can result in anxiety, depression and a reduction in the quality of life. The discriminative and affective qualities of pain are both thought to be regulated in an activity-dependent fashion. Recent studies have identified cells and molecules that regulate pain sensitivity and the parallel pathways that distribute nociceptive information to limbic or sensory areas of the forebrain. Here, we emphasize the cellular and neurobiological consequences of pain, especially those that are involved in the generation and maintenance of chronic pain. These new insights into pain processing will significantly alter our approach to pain control and the development of new analgesics.
Assuntos
Córtex Cerebral/metabolismo , Fibras Nervosas/metabolismo , Neurônios Aferentes/metabolismo , Dor/metabolismo , Receptores da Neurocinina-1/metabolismo , Medula Espinal/metabolismo , Animais , Doença Crônica , Discriminação Psicológica , Humanos , Camundongos , Camundongos Knockout , Neurônios Aferentes/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/fisiopatologia , Tratos Piramidais/metabolismo , Tratos Espinotalâmicos/metabolismoRESUMO
Substance P and its receptor (NK1) are thought to play an important role in pain and hyperalgesia. Here we have further examined this role by comparing the behavioural responses to intradermal capsaicin of mutant mice with a disruption of the NK1 receptor (NK1 KO) and wild-type (WT) mice. We have also evaluated the contribution of peripheral NK1 receptors to capsaicin-evoked behaviour by selective blockade of peripheral NK1 receptors in WT mice using a non-brain penetrant NK1 receptor antagonist. Injection of 6 microg capsaicin into the heel evoked paw licking with the same latency in WT and KO mice, but a significantly longer duration in WT mice. A higher dose (30 microg) evoked a similar duration of licking in both groups. There were no differences in mechanical sensitivity tested with von Frey hairs between WT and KO mice before capsaicin. Both capsaicin doses resulted in pronounced increases in responses to von Frey hairs (hyperalgesia) and novel responses to cotton wisps (allodynia) applied to the digits of the injected paw in WT mice, but no significant changes from baseline in KO mice. Selective blockade of peripheral NK1 receptors in WT mice resulted in a complete inhibition of capsaicin-evoked plasma extravasation, but the mechanical hyperalgesia induced by 30 microg capsaicin intraplantar was still significantly greater than that seen in KO mice. We conclude that the response to intradermal capsaicin is still present but abbreviated in mice lacking NK1 receptors, such that secondary hyperalgesia is not observed even after a high dose. Further, the lack of secondary hyperalgesia in NK1 KO mice is largely due to the loss of central rather than peripheral NK1 receptors. The phenotype of the NK1 KO mice is consistent with a loss of function of mechanically-insensitive nociceptors, and thus we propose that substance P may be expressed by this group of primary sensory neurones and required for their function.
Assuntos
Hiperalgesia , Tempo de Reação/fisiologia , Receptores da Neurocinina-1/fisiologia , Animais , Capsaicina , Hiperalgesia/induzido quimicamente , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medição da Dor/efeitos dos fármacos , Tempo de Reação/efeitos dos fármacos , Receptores da Neurocinina-1/efeitos dos fármacosRESUMO
Behavioral analysis of the NK1 receptor gene knock-out (NK1-/-) mouse indicated that substance P was closely involved in orchestrating the physiological and behavioral response of the animal to major environmental stressors. In particular, endogenous pain control mechanisms, such as stress-induced analgesia were substantially impaired in mutant mice, suggesting a reduction in descending inhibitory controls to the spinal cord from the brainstem. To directly test the integrity of descending controls in NK1-/- mice, we have analyzed c-Fos expression in laminae I-II of the lumbar and cervical cord and in the rostral ventromedial medulla in an experimental paradigm known to require recruitment of descending inhibitory controls. Anesthetized mice were stimulated with water at 50 degrees C either on their forepaw, hindpaw, or on both the hindpaw plus forepaw concurrently. Wild-type mice, naive or treated with an NK1 antagonist (RP67580) or its inactive isomer (RP68651), were compared with NK1-/- mice. C-Fos expression at the lumbar laminae I-II level was significantly reduced, whereas it was significantly greater in the raphe magnus and pallidus nuclei in the double stimulation situation in wild-type compared with NK1-/- mice. Blocking the NK1 receptor pharmacologically reproduced, in an enantiomere-selective manner, the data from NK1-/- mice, with no evidence for recruitment of descending inhibition at the lumbar cord level after forepaw stimulation. The present study demonstrates that the NK1 receptor is essential for the full development of noxiously evoked descending inhibition.
Assuntos
Inibição Neural , Dor/metabolismo , Receptores da Neurocinina-1/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Contagem de Células , Condicionamento Psicológico , Cruzamentos Genéticos , Membro Anterior , Membro Posterior , Temperatura Alta , Imuno-Histoquímica , Região Lombossacral , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Pescoço , Antagonistas dos Receptores de Neurocinina-1 , Dor/fisiopatologia , Medição da Dor , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores da Neurocinina-1/deficiência , Receptores da Neurocinina-1/genética , Medula Espinal/citologia , Medula Espinal/metabolismo , Estresse Fisiológico/metabolismo , Estresse Fisiológico/fisiopatologia , Substância P/metabolismoRESUMO
Synaptic plasticity in the hippocampus requires activity-dependent gene expression. We have therefore profiled gene expression in area CA1 following the induction of an electroshock-evoked maximal seizure. Using cDNA microarrays, the differential expression of approximately 9000 cDNAs was examined. In situ hybridization on 14 transcripts that showed strongest modulation in the microarray screen (1.8-2-fold) confirmed the differential expression of a single gene that encodes for the nuclear hormone receptor NGFI-B (Nur77, N10). Although this gene is only modestly up-regulated (approximately 2-fold) in area CA1, in situ hybridization revealed that maximal seizures induce a marked (approximately 12-fold) up-regulation of NGFI-B in the dentate gyrus. These data support the notion [French et al. (2001) Eur. J. Neurosci., 13, 968-976] that CA1 pyramidal neurons are more refractory than granule cells of the dentate gyrus with respect to activity-dependent gene transcription. Furthermore, our results argue against a large cohort of activity-dependent genes in area CA1.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Convulsões/metabolismo , Transmissão Sináptica/fisiologia , Fatores de Transcrição/genética , Regulação para Cima/genética , Animais , Giro Denteado/metabolismo , Estimulação Elétrica , Hipocampo/citologia , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Células Piramidais/metabolismo , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Convulsões/fisiopatologiaRESUMO
This unit outlines the polymerase chain reaction (PCR)-based technique of mRNA differential display, which identifies genes that are differentially expressed between cells or tissues. The basic protocol describes the actual differential display PCR reaction along with details of the identification, reamplification, and cloning of candidate differentially expressed genes. A support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.