Increased Accuracy to c-Fos-Positive Neuron Counting

There is not a described method to count the core label of c-Fos-positive neurons, avoiding false-positive and false-negative results. The aim of this manuscript is to provide guidelines for a secure and accurate method to calculate a threshold to select which core of c-Fos-positive neurons marked by immunofluorescence has to be scored. A background percentage was calculated by dividing the intensity value (0 to 255) of the core of c-Fos-positive neurons by its surrounding background from the 8-bit images obtained in a previous study. Using the background percentage from 20% up to 98%, raising 2% once for each score, as threshold to choose which core has to be counted, a script was written for the R program to count the number of the c-Fos-positive neurons and the comparison between control and experimental groups. The differences of the average number of the core counted c-Fos-positive neurons between control and experimental groups, at all thresholds studied, showed a rising value related to an increase of the background percentage threshold as well as a decrease of its p value related to an increase of the threshold of background percentage. For the smallest thresholds (high intensity of label), the differences between groups are suppressed (false negative). However, for the biggest thresholds (nonspecific label), these differences are always the same (false positive). Therefore, to avoid the false-negative and the false-positive values, it was chosen as the threshold of 62% the inflection point of the linear regression, which is equally different from the biggest and smallest values of the differences between groups.

Neonatal valproic acid exposure produces altered gyrification related to increased parvalbumin-immunopositive neuron density with thickened sulcal floors

Valproic acid (VPA) treatment is associated with autism spectrum disorder in humans, and ferrets can be used as a model to test this; so far, it is not known whether ferrets react to developmental VPA exposure with gyrencephalic abnormalities. The current study characterized gyrification abnormalities in ferrets following VPA exposure during neonatal periods, corresponding to the late stage of cortical neurogenesis as well as the early stage of sulcogyrogenesis. Ferret pups received intraperitoneal VPA injections (200 μg/g of body weight) on postnatal days (PD) 6 and 7. BrdU was administered simultaneously at the last VPA injection. Ex vivo MRI-based morphometry demonstrated significantly lower gyrification index (GI) throughout the cortex in VPA-treated ferrets (1.265 ± 0.027) than in control ferrets (1.327 ± 0.018) on PD 20, when primary sulcogyrogenesis is complete. VPA-treated ferrets showed significantly smaller sulcal-GIs in the rostral suprasylvian sulcus and splenial sulcus but a larger lateral sulcus surface area than control ferrets. The floor cortex of the inner stratum of both the rostral suprasylvian and splenial sulci and the outer stratum of the lateral sulcus showed a relatively prominent expansion. Parvalbumin-positive neuron density was significantly greater in the expanded cortical strata of sulcal floors in VPA-treated ferrets, regardless of the BrdU-labeled status. Thus, VPA exposure during the late stage of cortical neurogenesis may alter gyrification, primarily in the frontal and parietotemporal cortical divisions. Altered gyrification may thicken the outer or inner stratum of the cerebral cortex by increasing parvalbumin-positive neuron density.

Molecular origin of somatostatin-positive neuron vulnerability

Reduced somatostatin (SST) and SST-positive (SST+) neurons are hallmarks of neurological disorders and associated with mood disturbances, but their origin are unknown. Chronic psychosocial stress induces behavioral emotionality deficits and deregulates unfolded protein response (UPR) of the endoplasmic reticulum (ER) preferentially in SST+ neurons. Here we confirm that chronic stress increases ER stress levels in SST+ neurons of mouse prefrontal cortex, and show that genetically suppressing ER stress in SST+ neurons, but not in pyramidal neurons, normalized psychosocial stress-induced behavioral emotionality. Forced expression of SST precursor protein (preproSST), mimicking psychosocial stress-induced early proteomic changes, induces ER stress, whereas mature SST or processing-incompetent preproSST does not. Biochemical analyses further show that psychosocial stress induces SST protein aggregation under elevated ER stress conditions. These results demonstrate that SST processing is a SST+ neuron-intrinsic vulnerability factor under conditions of sustained or over-activated UPR in the ER, hence negatively impacting SST+ neuron functions.

Resistance to noise-induced gap detection impairment in FVB mice is correlated with reduced neuroinflammatory response and parvalbumin-positive neuron loss

Exposure to loud noises results in neuroinflammatory responses in the central auditory pathway. Noise-induced neuroinflammation is implicated in auditory processing deficits such as impairment in gap detection. In this study, we examined whether strain differences between the FVB and C57BL/6 mice in noise-induced impairment in gap detection are correlated with strain differences in neuroinflammatory responses. We found that noise induced more robust TNF-α expression in C57BL/6 than in FVB mice. Noise-induced microglial deramification was observed in C57BL/6 mice, but not in FVB mice. Furthermore, noise exposure resulted in a reduction in parvalbumin-positive (PV+) neuron density in the C57BL/6 mice, but not in FVB mice. These results suggest that neuroinflammatory responses and loss of PV+ neurons may contribute to strain differences in noise-induced impairment in gap detection.

Hypomyelination leads to alterations in inhibitory function and parvalbumin-positive neuron density in auditory cortex

ABSTRACTMyelination increases axonal conduction velocity and contributes to neural circuit function. Consequently, myelin disruption causes behavioral and cognitive defects. Myelin was thought to be restricted to excitatory neurons, and studies on dysmyelination focused primarily on these cells. However, axons of inhibitory neurons are also myelinated (Micheva et al., 2016), but the effects of myelin on inhibitory circuits are unknown. We thus studied mice that have mild hypomyelination due to loss of oligodendrocyte ErbB receptor signaling, focusing on primary auditory cortex (A1). We found that mutant mice have reduced A1 myelin and inhibitory connections to L2/3 neurons without affecting excitatory inputs, thus altering excitatory/inhibitory balance. These effects are not associated with altered expression of GABAergic and glutamatergic synaptic components, but with reduced parvalbumin-positive (PV+) neuron density and PV mRNA levels. These results demonstrate that mild hypomyelination can impact cortical neuronal networks, reducing inhibitory activity, and shifting networks towards excitation.Impact StatementMild hypomyelination alters excitatory/inhibitory balance in the primary auditory cortex by reducing the number of parvalbumin-positive interneurons and reducing inhibition.

Salidroside Protects Dopaminergic Neurons by Preserving Complex I Activity via DJ-1/Nrf2-Mediated Antioxidant Pathway

The pathogenic mechanism of Parkinson’s disease (PD) remains to be elucidated; however, mitochondrial dysfunction at the level of complex I and oxidative stress is suggestively involved in the development of PD. In our previous work, salidroside (Sal), an active component extracted from the medicinal plant Rhodiola rosea L., might protect dopaminergic (DA) neurons through modulating ROS–NO-related pathway. However, the mechanism of Sal-induced neuroprotective effects against PD remains poorly understood. Therefore, we further investigated whether Sal plays neuroprotective effects by activating complex I via DJ-1/Nrf2-mediated antioxidant pathway. The results showed that Sal remarkably attenuated MPP+/MPTP-induced decline in cell viability, accompanied by decreases in reactive oxygen species (ROS), malondialdehyde (MDA), and 8-hydroxy-deoxyguanosine (8-OHdG) contents and increases in the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), as well as glutathione (GSH) levels. Furthermore, Sal greatly improved the behavioral performance and prevented the severe reduction of TH-positive neuron numbers in the substantia nigra (SN). Moreover, in comparison with the MPP+/MPTP group, Sal increased the nuclear translocation of DJ-1 and Nrf2 and the mitochondrial translocation of DJ-1, accompanied by activating complex I. Furthermore, silencing of DJ-1/Nrf2 inhibited the increase of complex I activity and cell viability elicited by Sal. Together, these results support the neuroprotective effect of Sal against MPP+/MPTP-induced DA neurons damage.

OR-034 The parvalbumin positive neuron involved the regulation of motor cortex excitability in the exercise-induced fatigue

Objective Objective:Cortical parvalbumin-expressing inhibitory neurons(PV) control the activity of excitatory neurons and regulate their spike output. The present experiment is to determine the role of PV neuron in the reglution of excitability of primary motor cortex (M1) during the exercise-induced fatigue and possible molecular mechanism. Methods Methods: Male Wistar rats randomly divided into control group(C),exhaustive exercise group(E) and repeated exhaustive exercise group(RE). The gradually increasing load treadmill exercise-induced fatigue model was employed in the Group E and RE.The in vivo multi-channel recording methods was used for recording the neuronal electrophysiological activities of primary motor cortex.To observe the neuron firing rate changes during the rest state,immediately after exhausted exercise and after repeated exhaustive exercise.We also detected the expression of PV positive neurons in the primary motor cortex by the immunofluorescence method. The western blot method was used to determine the expression of calmodulin-dependent protein kinase II (CaMKII)、phosphorylated calmodulin-dependent protein kinase II( pCaMKII) and extracellular signal regulated kinase (ERK) in the primary motor cortex. Results Results：The electrophysioligical results indicated that the neuron firing rate after repeated exhausted excise the neuron firing rate significantly decreased compared with the rest state (P<0.05),but have no significantly changes as compared with exhausted excise;The expression of PV positive neurons in the group of E and RE significantly increased compared with the group C(P<0.01);The western blot results indicated that the protein expression of ERK in group REsignificantly decreased compared with group C, the pCaMKII expression of group RE decreased,but have no statistical difference. Conclusions Conclusion: After exercise-indued fatigue ,the increase of PV positive neuron maybe one reason for the excitability changes in primary motor cortex.the alteraions in the electrical signal may be participate in the regluation of exercise-induced fatigue. pCaMKII and ERK signal pathway may invloved in the molecular mechanism of exercise-induced fatigue.