RESUMEN
Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.
Asunto(s)
Transporte Axonal/fisiología , Encéfalo/citología , Virus Defectuosos/fisiología , Dependovirus/fisiología , Proteínas Fluorescentes Verdes/análisis , Virus Helper/fisiología , Herpesvirus Humano 1/fisiología , Vías Nerviosas/ultraestructura , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Trazadores del Tracto Neuronal/análisis , Neuronas/ultraestructura , Virus Reordenados/fisiología , Animales , Línea Celular , Núcleo Celular/virología , Virus Defectuosos/genética , Dependovirus/genética , Genes Reporteros , Genes Sintéticos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Virus Helper/genética , Herpesvirus Humano 1/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/virología , Virus Reordenados/genética , Proteinas del Complejo de Replicasa Viral/genética , Replicación ViralRESUMEN
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.
Asunto(s)
Drogas de Diseño , Radioisótopos de Flúor/análisis , Trazadores del Tracto Neuronal/análisis , Animales , Encéfalo , Clozapina/análogos & derivados , Clozapina/química , Células HEK293 , Haplorrinos , Humanos , Ligandos , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Trazadores del Tracto Neuronal/química , Tomografía de Emisión de Positrones/métodos , RoedoresRESUMEN
Lateral hypothalamus (LH) orexin neurons are essential for the expression of a cocaine place preference. However, the afferents that regulate the activity of these orexin neurons during reward behaviors are not completely understood. Using tract tracing combined with Fos staining, we examined LH afferents for Fos induction during cocaine preference in rats. We found that the ventral bed nucleus of the stria terminalis (vBNST) was a major input to the LH orexin cell field that was significantly Fos-activated during cocaine conditioned place preference (CPP). Inactivation of the vBNST with baclofen plus muscimol blocked expression of cocaine CPP. Surprisingly, such inactivation of the vBNST also increased Fos induction in LH orexin neurons; as activity in these cells is normally associated with increased preference, this result indicates that a vBNST-orexin connection is unlikely to be responsible for CPP that is dependent on vBNST activity. Because previous studies have revealed that vBNST regulates dopamine cells in the ventral tegmental area (VTA), which is known to be involved in CPP and other reward functions, we tested whether vBNST afferents to the VTA are necessary for cocaine CPP. We found that disconnection of the vBNST and VTA (using local microinjections of baclofen plus muscimol unilaterally into the vBNST and contralateral VTA) significantly attenuated expression of cocaine preference. However, blocking ionotropic glutamatergic afferents to the VTA from the vBNST did not significantly reduce cocaine preference. These results indicate that a non-glutamatergic vBNST-VTA projection is involved in expression of cocaine preference.
Asunto(s)
Cocaína/farmacología , Condicionamiento Operante/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Núcleos Septales/fisiología , Área Tegmental Ventral/fisiología , Animales , Baclofeno/farmacología , Neuronas Dopaminérgicas/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Agonistas de Receptores GABA-B/farmacología , Masculino , Muscimol/farmacología , Trazadores del Tracto Neuronal/análisis , Neuronas Aferentes/fisiología , Proteínas Proto-Oncogénicas c-fos/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Neuronal injury is a key feature of neonatal hypoxic-ischemic (HI) brain injury. However, the mechanisms underpinning neuronal losses, such as in the brainstem, are poorly understood. One possibility is that disrupted neural connections between the cortex and brainstem may compromise the survival of neuronal cell bodies in the brainstem. We investigated whether brainstem raphé serotonergic neurons that project to the cortex are lost after HI. We also tested if neuroinflammation has a role in disrupting brainstem raphé projections. Postnatal day 3 (P3) rats underwent unilateral carotid artery ligation followed by hypoxia (6% oxygen for 30 min). A retrograde tracer, choleratoxin b, was deposited in the motor cortex on P38. On P45 we found that retrogradely labelled neurons in the dorsal raphé dorsal, ventrolateral, interfascicular, caudal and ventral nuclei were lost after P3 HI. All retrogradely labelled neurons in the raphé nuclei were serotonergic. Numbers of retrogradely labelled neurons were also reduced in the ventromedial thalamus and basolateral amygdala. Minocycline treatment (45 mg/kg 2 h post-HI, 22.5 mg/kg daily P4-P9) attenuated losses of retrogradely labelled neurons in the dorsal raphé ventrolateral, interfascicular and ventral raphé nuclei, and the ventromedial thalamus. These results indicate that raphé neurons projecting to the cortex constitute a population of serotonergic neurons that are lost after P3 HI. Furthermore, neuroinflammation has a role in the disruption of raphé and thalamic neural projections. Future studies investigating the cellular mechanisms of axonal degeneration may reveal new targets for interventions to prevent neuronal losses after neonatal HI.