Inactivation of prefrontal cortex abolishes cortical acetylcholine release evoked by sensory or sensory pathway stimulation in the rat

Sensory stimulation and electrical stimulation of sensory pathways evoke an increase in acetylcholine release from the corresponding cortical areas. The pathways by which such sensory information reaches the cholinergic neurons of the basal forebrain that are responsible for this release are unclear, but have been hypothesized to pass through the prefrontal cortex (PFC). This hypothesis was tested in urethane-anesthetized rats using microdialysis to collect acetylcholine from somatosensory, visual, or auditory cortex, before and after the PFC was inactivated by local microdialysis delivery of the GABA-A receptor agonist muscimol (0.2% for 10 min at 2 microl/min). Before PFC inactivation, peripheral sensory stimulation and ventral posterolateral thalamic stimulation evoked 60 and 105% increases, respectively, in acetylcholine release from somatosensory cortex. Stimulation of the lateral geniculate nucleus evoked a 57% increase in acetylcholine release from visual cortex and stimulation of the medial geniculate nucleus evoked a 72% increase from auditory cortex. Muscimol delivery to the PFC completely abolished each of these evoked increases (overall mean change from baseline = -7%). In addition, the spontaneous level of acetylcholine release in somatosensory, visual, and auditory cortices was reduced by 15-59% following PFC inactivation, suggesting that PFC activity has a tonic facilitatory influence on the basal forebrain cholinergic neurons. These experiments demonstrate that the PFC is necessary for sensory pathway evoked cortical ACh release and strongly support the proposed sensory cortex-to-PFC-to-basal forebrain circuit for each of these modalities.     

Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis

Slow firing septal neurons modulate hippocampal and neocortical functions. Electrophysiologically, it is unclear whether slow firing neurons belong to a homogeneous neuronal population. To address this issue, whole-cell patch recordings and neuronal reconstructions were performed on rat brain slices containing the medial septum/diagonal band complex (MS/DB). Slow firing neurons were identified by their low firing rate at threshold (<5 Hz) and lack of time-dependent inward rectification (Ih). Unsupervised cluster analysis was used to investigate whether slow firing neurons could be further classified into different subtypes. The parameters used for the cluster analysis included latency for first spike, slow after-hyperpolarizing potential, maximal frequency and action potential (AP) decay slope. Neurons were grouped into three major subtypes. The majority of neurons (55%) were grouped as cluster I. Cluster II (17% of neurons) exhibited longer latency for generation of the first action potential (246.5+/-20.1 ms). Cluster III (28% of neurons) exhibited higher maximal firing frequency (25.3+/-1.7 Hz) when compared with cluster I (12.3+/-0.9 Hz) and cluster II (11.8+/-1.1 Hz) neurons. Additionally, cluster III neurons exhibited faster action potentials at suprathreshold. Interestingly, cluster II neurons were frequently located in the medial septum whereas neurons in cluster I and III appeared scattered throughout all MS/DB regions. Sholl's analysis revealed a more complex dendritic arborization in cluster III neurons. Cluster I and II neurons exhibited characteristics of "true" slow firing neurons whereas cluster III neurons exhibited higher frequency firing patterns. Several neurons were labeled with a cholinergic marker, Cy3-conjugated 192 IgG (p75NTR), and cholinergic neurons were found to be distributed among the three clusters. Our findings indicate that slow firing medial septal neurons are heterogeneous and that soma location is an important determinant of their electrophysiological properties. Thus, slow firing neurons from different septal regions have distinct functional properties, most likely related to their diverse connectivity.     

Modulators in concert for cognition: modulator interactions in the prefrontal cortex

Research on the regulation and function of ascending noradrenergic, dopaminergic, serotonergic, and cholinergic systems has focused on the organization and function of individual systems. In contrast, evidence describing co-activation and interactions between multiple neuromodulatory systems has remained scarce. However, commonalities in the anatomical organization of these systems and overlapping evidence concerning the post-synaptic effects of neuromodulators strongly suggest that these systems are recruited in concert; they influence each other and simultaneously modulate their target circuits. Therefore, evidence on the regulatory and functional interactions between these systems is considered essential for revealing the role of neuromodulators. This postulate extends to contemporary neurobiological hypotheses of major neuropsychiatric disorders. These hypotheses have focused largely on aberrations in the integrity or regulation of individual ascending modulatory systems, with little regard for the likely possibility that dysregulation in multiple ascending neuromodulatory systems and their interactions contribute essentially to the symptoms of these disorders. This review will paradigmatically focus on neuromodulator interactions in the PFC and be further constrained by an additional focus on their role in cognitive functions. Recent evidence indicates that individual neuromodulators, in addition to their general state-setting or gating functions, encode specific cognitive operations, further substantiating the importance of research concerning the parallel recruitment of neuromodulator systems and interactions between these systems.     

Excessive disgust caused by brain lesions or temporary inactivations: mapping hotspots of the nucleus accumbens and ventral pallidum

Disgust is a prototypical type of negative affect. In animal models of excessive disgust, only a few brain sites are known in which localized dysfunction (lesions or neural inactivations) can induce intense ‘disgust reactions’ (e.g. gapes) to a normally pleasant sensation such as sweetness. Here, we aimed to map forebrain candidates more precisely, to identify where either local neuronal damage (excitotoxin lesions) or local pharmacological inactivation (muscimol/baclofen microinjections) caused rats to show excessive sensory disgust reactions to sucrose. Our study compared subregions of the nucleus accumbens shell, ventral pallidum, lateral hypothalamus, and adjacent extended amygdala. The results indicated that the posterior half of the ventral pallidum was the only forebrain site where intense sensory disgust gapes in response to sucrose were induced by both lesions and temporary inactivations (this site was previously identified as a hedonic hotspot for enhancements of sweetness ‘liking’). By comparison, for the nucleus accumbens, temporary GABA inactivations in the caudal half of the medial shell also generated sensory disgust, but lesions never did at any site. Furthermore, even inactivations failed to induce disgust in the rostral half of the accumbens shell (which also contains a hedonic hotspot). In other structures, neither lesions nor inactivations induced disgust as long as the posterior ventral pallidum remained spared. We conclude that the posterior ventral pallidum is an especially crucial hotspot for producing excessive sensory disgust by local pharmacological/lesion dysfunction. By comparison, the nucleus accumbens appears to segregate sites for pharmacological disgust induction and hedonic enhancement into separate posterior and rostral halves of the medial shell.     

Alterations of Ca-responsive proteins within cholinergic neurons in aging and Alzheimer's disease

The molecular basis of selective neuronal vulnerability in Alzheimer's disease (AD) remains poorly understood. Using basal forebrain cholinergic neurons (BFCNs) as a model and immunohistochemistry, we have demonstrated significant age-related loss of the calcium-binding protein calbindin-D_28K (CB) from BFCN, which was associated with tangle formation and degeneration in AD. Here, we determined alterations in RNA and protein for CB and the Ca²⁺-responsive proteins Ca²⁺/calmodulin-dependent protein kinase I (CaMKI), growth-associated protein-43 (GAP43), and calpain in the BF. We observed progressive downregulation of CB and CaMKI RNA in laser-captured BFCN in the normal-aged-AD continuum. We also detected progressive loss of CB, CaMKIδ, and GAP43 proteins in BF homogenates in aging and AD. Activated μ-calpain, a calcium-sensitive protease that degrades CaMKI and GAP43, was significantly increased in the normal aged BF and was 10 times higher in AD BF. Overactivation of μ-calpain was confirmed using proteolytic fragments of its substrate spectrin. Substantial age- and AD-related alterations in Ca²⁺-sensing proteins most likely contribute to selective vulnerability of BFCN to degeneration in AD.     

Late-Onset,Short-Term Intermittent Fasting Reverses Age-Related Changes in Calcium Buffering and Inhibitory Synaptic Transmission in Mouse Basal Forebrain Neurons

Aging is often associated with cognitive decline and recurrent cellular and molecular impairments. While life-long caloric restriction (CR) may delay age-related cognitive deterioration as well as the onset of neurologic disease, recent studies suggest that late-onset, short-term intermittent fasting (IF), may show comparable beneficial effects as those of life-long CR to improve brain health. We used a new optogenetic aging model to study the effects of late-onset (>18 months), short-term (four to six weeks) IF on age-related changes in GABAergic synaptic transmission, intracellular calcium (Ca²⁺) buffering, and cognitive status. We used male mice from a bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R [VGAT-ChR2(H134R)-EYFP] in a reduced synaptic preparation that allows for specific optogenetic light stimulation on GABAergic synaptic terminals across aging. We performed quantal analysis using the method of failures in this model and show that short-term IF reverses the age-related decrease in quantal content of GABAergic synapses. Likewise, short-term IF also reversed age-related changes in Ca²⁺ buffering and spontaneous GABAergic synaptic transmission in basal forebrain (BF) neurons of aged mice. Our findings suggest that late-onset short-term IF can reverse age-related physiological impairments in mouse BF neurons but that four weeks IF is not sufficient to reverse age-related cognitive decline.SIGNIFICANCE STATEMENT Here, we demonstrate plasticity of the aging brain and reversal of well-defined hallmarks of brain aging using short-term intermittent fasting (IF) initiated later in life. Few therapeutics are currently available to treat age-related neurologic dysfunction although synaptic dysfunction occurs during aging and neurologic disease is a topic of intense research. Using a new reduced synaptic preparation and optogenetic stimulation we are able to study age-related synaptic mechanisms in greater detail. Several neurophysiological parameters including quantal content were altered during aging and were reversed with short-term IF. These methods can be used to identify potential therapies to reverse physiological dysfunction during aging.     

大鼠局灶性短暂脑缺血后前脑室下带的神经发生

目的：观察大鼠短暂性局灶性脑缺血后前脑室下带(SVZ)神经发生的增殖规律。方法：将SD大鼠随机分为正常对照组、假手术组和缺血实验组，缺血实验组再分为缺血后1、4、7、10、14d组。线栓法制作局灶性脑缺血模型；BrdU标记S期细胞并用免疫组织化学方法检测含BrdU的阳性细胞；测量SVZ区域BrdU阳性细胞核的总面积。结果：在缺血侧，缺血后4d BrdU阳性细胞核的总面积明显增加，7d时达到峰值，随后开始下降，在14d时明显下降，但仍高于正常对照组；在缺血对侧，该区域也表现出同样的表达规律，在缺血后10d达到峰值，但增幅较小。结论：短暂性局灶性脑缺血可促进前脑室下带的神经发生，提示成年脑有潜在的自我修复能力。     

Two region‐dependent pathways of eosinophilic neuronal death after transient cerebral ischemia

Various types of eosinophilic neurons (ENs) are found in the post‐ischemic brain. We examined the temporal profile of ENs in the core and peripheral regions of the ischemic cortex, and analyzed the relationship to the expression of various cell death‐related factors. Unilateral forebrain ischemia was induced in Mongolian gerbils by transient common carotid artery occlusions, and the brains from 3 h to 2 weeks post‐ischemia were prepared for morphometric and immunohistochemical analysis of ENs. ENs with minimally abnormal nuclei and swollen cell bodies appeared at 3 h in the ischemic core and at 12 h in the periphery. In both locations multiple cell death‐related factors including calcium, µ‐calpain, cathepsin D, 78 kDa glucose‐regulated protein (GRP78) and ubiquitin were activated. In the ischemic core, pyknosis and irregularly atrophic cytoplasm peaked at 12 h, which was associated with significant increases in staining for calcium and µ‐calpain. ENs with pyknosis and scant cytoplasm peaked at 4 days and were positive for TUNEL and calcium staining. In the ischemic periphery, ENs had slightly atrophic cytoplasm and sequentially developed pyknosis, karyorrhexis and karyolysis over 1 week. These cells were positive for TUNEL and calcium staining. All types of EN were negative for caspase 3. There may be two region‐dependent pathways of EN changes in the post‐ischemic brain: pyknosis with cytoplasmic shrinkage in the core, and nuclear disintegration with slightly atrophic cytoplasm in the periphery. This difference coordinates different activation patterns of cell death‐related factors in ENs.     

大鼠中脑中央灰质与前脑结构的联系——HRP逆、顺行追踪研究

在22只体重140g左右的SD大鼠,用HRP逆行和顺行追踪方法(TMB-ST法成色),研究了中脑中央灰质(CG)与前脑结构的联系。按照泳注中心位置,将实验材料分作背侧区(CGD)、内侧区(CGM)和外侧区(CGL)三组。三组逆行标记细胞分布没有明显差别。在大脑皮质(以扣带前、压后和额叶皮质为著)、终纹床核、视前区、广泛的下丘脑结构(以下丘脑前区、背侧区、灰结节区、外侧区以及下丘脑腹内侧核、背侧乳头体前核为著,下丘脑室旁核亦见标记细胞)以及未定带、缰外侧核等结构察见标记细胞,均以同侧为主。值得注意的CGL组在终板血管器观察到相当丰富的标记细胞,CGM组在邻近弓状核的室管膜内见相当典型的标记细胞。顺行标记纤维主要循同侧背侧纵束、室周纤维系统和内侧前脑束上行,部分向背颅侧或外颅侧方向穿中脑顶盖或网状结构行进。CGD组达束旁核、丘脑室旁核、外侧和内侧缰核、下丘脑脑后区、未定带、乳头体上核、背侧下丘脑区、下丘脑前区、丘脑后核和顶盖前区。CGM和CGL组延伸较远,除至以上部位外,还可抵达未定带下核、Forel区、背侧乳头体前核、下丘脑室旁核、内侧和外侧视前区、斜角带、终纹床核丘脑中线区和内髓板区等部位。