Phototoxicities Caused by Continuous Light Exposure Were Not Induced in Retinal Ganglion Cells Transduced by an Optogenetic Gene.

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.

Hyperactivation of proprioceptors induces microglia-mediated long-lasting pain in a rat model of chronic fatigue syndrome.

BACKGROUND: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model. METHODS: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity. RESULTS: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior. CONCLUSION: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS.

Behavioural and neural sequelae of stressor exposure are not modulated by controllability in females.

The degree of behavioural control that an organism has over a stressor is a potent modulator of the stressor's impact; controllable stressors produce none of the neurochemical and behavioural sequelae that occur if the stressor is uncontrollable. Research demonstrating the importance of control and the neural mechanisms responsible has been conducted almost entirely with male rats. It is unknown if behavioural control is stress blunting in females, and whether or not a similar resilience circuitry is engaged. Female rats were exposed to controllable, yoked uncontrollable or no tailshock. In separate experiments, behavioural (juvenile social exploration, fear and shuttle box escape) and neurochemical (activation of dorsal raphe serotonin and dorsal raphe-projecting prelimbic neurons) outcomes, which are sensitive to the dimension of control in males, were assessed. Despite successful acquisition of the controlling response, behavioural control did not mitigate dorsal raphe serotonergic activation and behavioural outcomes induced by tailshock, as it does in males. Moreover, behavioural control failed to selectively engage prelimbic cells that project to the dorsal raphe as in males. Pharmacological activation of the prelimbic cortex restored the stress-buffering effects of control. Collectively, the data demonstrate stressor controllability phenomena are absent in females and that the protective prelimbic circuitry is present but not engaged. Reduced benefit from coping responses may represent a novel approach for understanding differential sex prevalence in stress-related psychiatric disorders.

The Cerebello-Hypothalamic and Hypothalamo-Cerebellar Pathways via Superior and Middle Cerebellar Peduncle in the Rat.

The connections between the cerebellum and the hypothalamus have been well documented. However, the specific cerebellar peduncle through which the hypothalamo-cerebellar and cerebello-hypothalamic connections pass has not been demonstrated. The present study aims to define the specific cerebellar peduncle through which connects the cerebellum to specific hypothalamic nuclei. Seventeen male albino rats received 20-50-nl pressure injections of either Fluoro-Gold (FG) or biotinylated dextran amine (BDA) tracer into the superior (SCP), middle (MCP), and inferior (ICP) cerebellar peduncle. Following 7-10 days of survival period, the animals were processed according to the appropriate protocol for the two tracers used. Labeled cells and axons were documented using light or fluorescence microscopy. The present study showed connections between the hypothalamus and the cerebellum via both the SCP and the MCP but not the ICP. The hypothalamo-cerebellar connections via the SCP were from the lateral, dorsomedial, paraventricular, and posterior hypothalamic nuclei, and cerebello-hypothalamic connections were to the preoptic and lateral hypothalamic nuclei. The hypothalamo-cerebellar connections via the MCP were from the lateral, dorsomedial, ventromedial, and mammillary hypothalamic nuclei; and cerebello-hypothalamic connections were to the posterior, arcuate, and ventromedial hypothalamic nuclei. The hypothlamo-cerebellar connections were denser compared to the cerebello-hypothlamic connections via both the SCP and the MCP. The connection between the cerebellum and the hypothalamus was more prominent via the SCP than MCP. Both the hypothlamo-cerebellar and cerebello-hypothalamic connections were bilateral, with ipsilateral preponderance. Reciprocal connections were with the lateral hypothalamic nucleus via the SCP and the ventromedial nucleus via the MCP were observed. Cerebellum takes part in the higher order brain functions via its extensive connections. The knowledge of hypothalamo-cerebellar and cerebello-hypothalamic connections conveyed within the SCP and MCP can be important for the lesions involving the MCP and SCP. These connections can also change the conceptual architecture of the cerebellar circuitry and deepen current understanding.

No latency to dentate granule cell epileptogenesis in experimental temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: To determine when spontaneous granule cell epileptiform discharges first occur after hippocampal injury, and to identify the postinjury "latent" period as either a "silent" gestational state of epileptogenesis or a subtle epileptic state in gradual transition to a more obvious epileptic state. METHODS: Nonconvulsive status epilepticus evoked by perforant path stimulation in urethane-sedated rats produced selective and extensive hippocampal injury and a "latent" period that preceded the onset of the first clinically obvious epileptic seizures. Continuous granule cell layer depth recording and video monitoring assessed the time course of granule cell hyperexcitability and the onset/offset times of spontaneous epileptiform discharges and behavioral seizures. RESULTS: One day postinjury, granule cells in awake rats were hyperexcitable to afferent input, and continuously generated spontaneous population spikes. During the ~2-4 week "latent" period, granule cell epileptiform discharges lasting ~30 seconds caused subtle focal seizures characterized by immobilization and facial automatisms that were undetected by behavioral assessment alone but identified post hoc. Granule cell layer epileptiform discharge duration eventually tripled, which caused the first clinically obvious seizure, ending the "latent" period. Behavioral seizure duration was linked tightly to spontaneous granule cell layer events. Granule cell epileptiform discharges preceded all behavioral seizure onsets, and clonic behaviors ended abruptly within seconds of the termination of each granule cell epileptiform discharge. Noninjurious hippocampal excitation produced no evidence of granule cell hyperexcitability or epileptogenesis. SIGNIFICANCE: The latent period in this model is a subtle epileptic state in transition to a more clinically obvious epileptic state, not a seizure-free "gestational" state when an unidentified epileptogenic mechanism gradually develops. Based on the onset/offset times of electrographic and behavioral events, granule cell behavior may be the prime determinant of seizure onset, phenotype, duration, and offset in this model of hippocampal-onset epilepsy. Extensive hippocampal neuron loss could be the primary epileptogenic mechanism.

Quantitative Analysis of Mouse Dural Afferent Neurons Expressing TRPM8, VGLUT3, and NF200.

OBJECTIVE: To quantify the abundance of dural afferent neurons expressing transient receptor potential channel melastatin 8 (TRPM8), vesicular glutamate transporter 3 (VGLUT3), and neurofilament 200 (NF200) in adult mice. BACKGROUND: With the increasing use of mice as a model system to study headache mechanisms, it is important to understand the composition of dural afferent neurons in mice. In a previous study, we have measured the abundance of mouse dural afferent neurons that express neuropeptide calcitonin gene-related peptide as well as two TRP channels TRPV1 and TRPA1, respectively. Here, we conducted quantitative analysis of three other dural afferent subpopulations in adult mice. METHODS: We used the fluorescent tracer Fluoro-Gold to retrogradely label dural afferent neurons in adult mice expressing enhanced green fluorescent protein in discrete subpopulations of trigeminal ganglion (TG) neurons. Mechanoreceptors with myelinated fibers were identified by NF200 immunoreactivity. We also conducted Ca2+ -imaging experiments to test the overlap between TRPM8 and VGLUT3 expression in mouse primary afferent neurons (PANs). RESULTS: The abundance of TRPM8-expressing neurons in dural afferent neurons was significantly lower than that in total TG neurons. The percentages of dural afferent neurons expressing VGLUT3 and NF200 were comparable to those of total TG neurons, respectively. TRPM8 agonist menthol evoked Ca2+ influx in less than 7% VGLUT3-expressing PANs in adult mice. CONCLUSIONS: TG neurons expressing TRPM8, VGLUT3, and NF200 all innervate adult mouse dura. TRPM8 and VGLUT3 are expressed in distinct subpopulations of PANs in adult mice. These results provide an anatomical basis to investigate headache mechanisms in mouse models.

Genetic inactivation of glutamate neurons in the rat sublaterodorsal tegmental nucleus recapitulates REM sleep behaviour disorder.

SEE SCHENCK AND MAHOWALD DOI101093/AWW329 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Idiopathic REM sleep behaviour disorder is characterized by the enactment of violent dreams during paradoxical (REM) sleep in the absence of normal muscle atonia. Accumulating clinical and experimental data suggest that REM sleep behaviour disorder might be due to the neurodegeneration of glutamate neurons involved in paradoxical sleep and located within the pontine sublaterodorsal tegmental nucleus. The purpose of the present work was thus to functionally determine first, the role of glutamate sublaterodorsal tegmental nucleus neurons in paradoxical sleep and second, whether their genetic inactivation is sufficient for recapitulating REM sleep behaviour disorder in rats. For this goal, we first injected two retrograde tracers in the intralaminar thalamus and ventral medulla to disentangle neuronal circuits in which sublaterodorsal tegmental nucleus is involved; second we infused bilaterally in sublaterodorsal tegmental nucleus adeno-associated viruses carrying short hairpin RNAs targeting Slc17a6 mRNA [which encodes vesicular glutamate transporter 2 (vGluT2)] to chronically impair glutamate synaptic transmission in sublaterodorsal tegmental nucleus neurons. At the neuroanatomical level, sublaterodorsal tegmental nucleus neurons specifically activated during paradoxical sleep hypersomnia send descending efferents to glycine/GABA neurons within the ventral medulla, but not ascending projections to the intralaminar thalamus. These data suggest a crucial role of sublaterodorsal tegmental nucleus neurons rather in muscle atonia than in paradoxical sleep generation. In line with this hypothesis, 30 days after adeno-associated virus injections into sublaterodorsal tegmental nucleus rats display a decrease of 30% of paradoxical sleep daily quantities, and a significant increase of muscle tone during paradoxical sleep concomitant to a tremendous increase of abnormal motor dream-enacting behaviours. These animals display symptoms and behaviours during paradoxical sleep that closely mimic human REM sleep behaviour disorder. Altogether, our data demonstrate that glutamate sublaterodorsal tegmental nucleus neurons generate muscle atonia during paradoxical sleep likely through descending projections to glycine/GABA premotor neurons in the ventral medulla. Although playing a role in paradoxical sleep regulation, they are, however, not necessary for inducing the state itself. The present work further validates a potent new preclinical REM sleep behaviour disorder model that opens avenues for studying and treating this disabling sleep disorder, and advances potential regions implicated in prodromal stages of synucleinopathies such as Parkinson's disease.

Cortical, subcortical and brain stem connections of the cerebellum via the superior and middle cerebellar peduncle in the rat.

The role of cerebellum in coordination of somatic motor activity has been studied in detailed in various species. However, experimental and clinical studies have shown the involvement of the cerebellum with various visceral and cognitive functions via its vast connections with the central nervous system. The present study aims to define the cortical and subcortical and brain stem connections of the cerebellum via the superior (SCP) and middle (MCP) cerebellar peduncle using biotinylated dextran amine (BDA) and Fluoro-Gold (FG) tracer in Wistar albino rats. 14 male albino rats received 20-50-nl pressure injections of either FG or BDA tracer into the SCP and MCP. Following 7-10 days of survival period, the animals were processed according to the related protocol for two tracers. Labelled cells and axons were documented using light and fluorescence microscope. The SCP connects cerebellum to the insular and infralimbic cortices whereas, MCP addition to the insular cortex, it also connects cerebellum to the rhinal, primary sensory, piriform and auditory cortices. Both SCP and MCP connected the cerebellum to the ventral, lateral, posterior and central, thalamic nuclei. Additionally, SCP also connects parafasicular thalamic nucleus to the cerebellum. The SCP connects cerebellum to basal ganglia (ventral pallidum and clastrum) and limbic structures (amygdaloidal nuclei and bed nucleus of stria terminalis), however, the MCP have no connections with basal ganglia or limbic structures. Both the SCP and MCP densely connects cerebellum to various brainstem structures. Attaining the knowledge of the connections of the SCP and MCP is important for the diagnosis of lesions in the MCP and SCP and would deepen current understanding of the neuronal circuit of various diseases or lesions involving the SCP and MCP.

Thalamic and Entorhinal Network Activity Differently Modulates the Functional Development of Prefrontal-Hippocampal Interactions.

Precise information flow during mnemonic and executive tasks requires the coactivation of adult prefrontal and hippocampal networks in oscillatory rhythms. This interplay emerges early in life, most likely as an anticipatory template of later cognitive performance. At neonatal age, hippocampal theta bursts drive the generation of prefrontal theta-gamma oscillations. In the absence of direct reciprocal interactions, the question arises of which feedback mechanisms control the early entrainment of prefrontal-hippocampal networks. Here, we demonstrate that prefrontal-hippocampal activity couples with discontinuous theta oscillations and neuronal firing in both lateral entorhinal cortex and ventral midline thalamic nuclei of neonatal rats. However, these two brain areas have different contributions to the neonatal long-range communication. The entorhinal cortex mainly modulates the hippocampal activity via direct axonal projections. In contrast, thalamic theta bursts are controlled by the prefrontal cortex via mutual projections and contribute to hippocampal activity. Thus, the neonatal prefrontal cortex modulates the level of hippocampal activation by directed interactions with the ventral midline thalamus. Similar to the adult task-related communication, theta-band activity ensures the feedback control of long-range coupling in the developing brain. SIGNIFICANCE STATEMENT: Memories are encoded by finely tuned interactions within large-scale neuronal networks. This cognitive performance is not inherited, but progressively matures in relationship with the establishment of long-range coupling in the immature brain. The hippocampus initiates and unidirectionally drives the oscillatory entrainment of neonatal prefrontal cortex, yet feedback interactions that precisely control this early communication are still unresolved. Here, we identified distinct roles of entorhinal cortex and ventral midline thalamus for the functional development of prefrontal-hippocampal interactions. While entorhinal oscillations modulate the hippocampal activity by timing the neuronal firing via monosynaptic afferents, thalamic nuclei act as a relay station routing prefrontal activation back to hippocampus. Understanding the mechanisms of network maturation represents the prerequisite for assessing circuit dysfunction in neurodevelopmental disorders.

Generating level-dependent models of cervical and thoracic spinal cord injury: Exploring the interplay of neuroanatomy, physiology, and function.

The majority of spinal cord injuries (SCI) occur at the cervical level, which results in significant impairment. Neurologic level and severity of injury are primary endpoints in clinical trials; however, how level-specific damages relate to behavioural performance in cervical injury is incompletely understood. We hypothesized that ascending level of injury leads to worsening forelimb performance, and correlates with loss of neural tissue and muscle-specific neuron pools. A direct comparison of multiple models was made with injury realized at the C5, C6, C7 and T7 vertebral levels using clip compression with sham-operated controls. Animals were assessed for 10weeks post-injury with numerous (40) outcome measures, including: classic behavioural tests, CatWalk, non-invasive MRI, electrophysiology, histologic lesion morphometry, neuron counts, and motor compartment quantification, and multivariate statistics on the total dataset. Histologic staining and T1-weighted MR imaging revealed similar structural changes and distinct tissue loss with cystic cavitation across all injuries. Forelimb tests, including grip strength, F-WARP motor scale, Inclined Plane, and forelimb ladder walk, exhibited stratification between all groups and marked impairment with C5 and C6 injuries. Classic hindlimb tests including BBB, hindlimb ladder walk, bladder recovery, and mortality were not different between cervical and thoracic injuries. CatWalk multivariate gait analysis showed reciprocal and progressive changes forelimb and hindlimb function with ascending level of injury. Electrophysiology revealed poor forelimb axonal conduction in cervical C5 and C6 groups alone. The cervical enlargement (C5-T2) showed progressive ventral horn atrophy and loss of specific motor neuron populations with ascending injury. Multivariate statistics revealed a robust dataset, rank-order contribution of outcomes, and allowed prediction of injury level with single-level discrimination using forelimb performance and neuron counts. Level-dependent models were generated using clip-compression SCI, with marked and reliable differences in forelimb performance and specific neuron pool loss.