Prenatal and neonatal exposure to low-dose of bisphenol-A enhance the morphine-induced hyperlocomotion and rewarding effect

Bisphenol-A has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. In the previous study, we reported that prenatal and neonatal exposure to high-dose of bisphenol-A affects the development of central dopaminergic system in the mouse limbic area. The present study was then undertaken to investigate whether prenatal and neonatal exposure to lower dose of bisphenol-A could change the morphine-induced several pharmacological actions such as rewarding effect and hyperlocomotion in mice. Prenatal and neonatal exposure to low-dose of bisphenol-A enhanced the morphine-induced hyperlocomotion and rewarding effect. Additionally, the treatment with bisphenol-A produced an up-regulation of dopamine receptor function to activate G-protein in the mouse limbic forebrain, which is thought to play a critical role for hyperlocomotion and rewarding effects by drugs of abuse. These findings suggest that prenatal and neonatal exposure to low-dose of bisphenol-A can potentiate the central dopamine receptor-dependent neurotransmission, resulting in the supersensitivity of the morphine-induced hyperlocomotion and rewarding effects in the mouse.     

Memory formation as related to genotypic or experimental variations of hippocampal cholinergic activity in mice.

Two successive experiments were designed to compare learning and retention and the post-training effect of hippocampal electrical stimulation on retention of a lever-press conditioning among several groups of mice. In parallel experiments, we studied the enzymes involved in acetylcholine metabolism in the dorsal hippocampus. Results showed that either a genotypic or an experimentally induced decrease of choline acetyltransferase activity weakened both long-term retention and the facilitatory effect of post-trial stimulation but improved short-term retention. The role of hippocampal cholinergic synapses in response execution and memory consolidation is discussed.     

The distribution of cholinergic perikarya with respect to enkephalin-rich patches in the caudate nucleus of the adult cat

The distribution of cholinergic interneurons with respect to enkephalin-rich patches in the caudate nucleus of the cat was examined using both computer-assisted 3-D reconstruction and immunocytochemical techniques. Examination of the 3-D distribution of perikarya staining for choline acetyltransferase (ChAT) revealed that these cells were not evenly distributed within the caudate nucleus but exhibited areas of increased and decreased density. Comparison of the 3-D distribution of cholinergic perikarya to that of the enkephalin-rich patches indicated that areas of increased ChAT+ cell density often corresponded to the positions of enkephalin-rich patches within the dorsal-lateral caudate nucleus. At more ventral regions, there was no clear correspondence between areas of increased ChAT+ cell density and enkephalin-rich patches. In agreement with these observations, a quantitative analysis of sections double-labeled for ChAT and enkephalin revealed that the density of cholinergic neurons within enkephalin-rich patches was twice that in the surrounding tissue in the dorsal region of the caudate nucleus. In contrast at more ventral levels, the difference in the density of ChAT+ cells in enkephalin-rich patches did not significantly differ from that in the surrounding striatal tissue. Both the results of the 3-D and the double-labeling analysis suggest that cholinergic neurons are not evenly distributed within the caudate nucleus of the cat but form loose clusters which are associated dorsally with the enkephalin-rich patches. These results also provide further evidence of heterogeneity within the striosomal compartment in the cat.     

Expression and localization of pChAT as a novel method to study cholinergic innervation of rat adrenal gland

Cholinergic innervation of the rat adrenal gland has been analyzed previously using cholinergic markers including acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). In the present study, we demonstrate putative cholinergic neurons in the rat adrenal gland using an antibody to pChAT, which is the product of a splice variant of ChAT mRNA that is preferentially localized in peripheral cholinergic nerves. Most of the ganglionic neurons as well as small single sporadic neurons in the adrenal gland were stained intensely for pChAT. The density of pChAT-immunoreactive (IR) fibers was distinct in the adrenal cortex and medulla. AChE-, cChAT- and VAChT-immunoreactivities were also observed in some cells and fibers of the adrenal medulla, while the cortex had few positive nerve fibers. These results indicate that ganglionic neurons of the adrenal medulla and nerve fibers heterogeneously express cholinergic markers, especially pChAT. Furthermore, the innervation of the adrenal gland, cortex and medulla, by some cholinergic fibers provides additional morphological evidence for a significant role of cholinergic mechanisms in adrenal gland functions.     

Collateral axons of cholinergic pontine neurones projecting to midline, mediodorsal and parafascicular thalamic nuclei in the rat

The organization of collateral axons projecting from neurones in the pontine laterodorsal tegmental nucleus (LDTg) has been examined using combinations of retrograde neuronal tracers with immunocytochemical markers for the acetylcholine-synthesizing enzyme choline acetyltranferase (CHAT), focussing on projections to the midline, mediodorsal and parafascicular thalamic nuclei and the ventral tegmental area. 25–59% of LDTg neurones projecting to the mediodorsal nucleus provided collaterals to the midline nuclei. Virtually all (87–96%) of these double retrogradely labelled neurones appeared cholinergic. 9–18% of LDTg neurones projecting to the parafascicular nuclei also provided a collateral to the midline nuclei and 50–78% of these double retrogradely labelled neurones stained for CHAT. 26–29% of the single LDTg neurones which projected collaterals to both the mediodorsal and midline nuclei, were found to project a third collateral to the ventral tegmental area. These anatomical findings, taken together with functional evidence, suggest that cholinergic terminals arising from LDTg are involved in coordinating thalamic mechanisms of brain state control; and in regulating dopaminergic pathways, both directly and via the thalamus.     

Learning impairment induced by lesion of the CA1 field of the primate hippocampus: attempts to ameliorate the impairment by transplantation of fetal CA1 tissue

 Monkeys with bilateral excitotoxic lesion of the CA1 field of the hippocampus were severely impaired at learning visuospatial conditional tasks. This was not a general spatial impairment, because the animals were not impaired on serial spatial reversal, which requires response flexibility in the spatial domain; they were not impaired at learning to choose the position furthest away from a single stimulus, which requires analysis of spatial layout of the test area, and they were not impaired at discriminating between two patterns that differed only in orientation. CA1-lesioned monkeys were impaired at learning a visuospatial conditional task when trials of the two component types ”if AA go left” and ”if BB go right” were presented according to either a pseudorandom or alternating schedule; but they were not impaired if one component type of trial was presented until three consecutive correct responses were made, followed by the other type of trial, to three consecutive correct responses. In all cases testing continued until a criterion of 27 of 30 consecutive correct responses across both types of trial was achieved. Although this suggests that CA1-lesioned animals are particularly prone to interference effects, they had no difficulty in learning ten concurrent visual discriminations presented against either a uniform background or with each discrimination presented against its own distinctive background, a condition that might reduce interference in unoperated monkeys. Interference following hippocampal damage might occur at a deeper level than stimulus identification such that animals with hippocampal damage may be able to learn about many aspects of different stimuli in parallel but may be unable to learn about multiple related aspects of the same subject matter. Monkeys with grafts of fetal CA1 tissue in the lesioned CA1 field showed significant improvement relative to CA1-lesioned animals on those tasks on which CA1-lesioned animals were impaired, although they remained impaired relative to control animals. This suggests that the grafts had produced some improvement in performance. Grafted monkeys did not differ from unoperated control monkeys or from CA1-lesioned monkeys on those tasks that were not sensitive to CA1 damage. This demonstrates that the grafts did not have an additional deleterious effect on cognitive performance. Received: 15 August 1996 / Accepted: 9 December 1996     

Age-related spatial cognitive impairment is correlated with a decrease in ChAT in the cerebral cortex,hippocampus and forebrain of SAMP8 mice

At present, the mechanisms underlying cognitive disorders remain unclear. The senescence-accelerated mice (SAM) prone/8 (P8) has been proposed as a useful model for the study of aging, and SAM resistant/1 (R1) is its control as a normal aging strain. The purpose of this study was to investigate choline acetyltransferase (ChAT) expression in SAM brain. The age-related decline of learning and memory ability in P8 mice (4, 8 and 12 months old, n = 10 for each group) was proved in Morris water maze test (MWM). After the behavioral test, protein and mRNA levels of ChAT were determined in the cerebral cortex, hippocampus and forebrain by means of immunostaining, Western blotting, and real time quantitative PCR (QPCR). Comparing with 4-month-old P8 and R1, 8- and 12-month-old P8 showed age-related cognitive impairment in MWM test. The latencies of the 4-month-old P8 in a hidden platform trial were significantly shorter, and the retention time was significantly longer than that of the older P8 groups. In addition, significantly low level of ChAT protein was observed in older P8 groups. Comparing with the 4-month-old P8, ChAT mRNA in the 12-month-old P8 declined significantly in all three regions of P8 brain. Pearson correlation test showed that the latencies in the MWM were positively correlated with the level of ChAT in P8. Such phenomenon could not be detected in normal aging R1 mice. These findings suggest that the decrease of ChAT in P8 mice was responsible for the age-related learning and memory impairments in some sense.     

Ghrelin induced memory facilitation implicates nitric oxide synthase activation and decrease in the threshold to promote LTP in hippocampal dentate gyrus

Although the hypothalamus has been long considered the main ghrelin (Ghr) target organ mediating orexigenic effects, recently it has been shown that in-vivo Ghr hippocampus administration improves learning and memory in the inhibitory avoidance paradigm. However, the possible mechanisms underlying this memory facilitation effect have not been clarified. Given that the biochemical memory cascade into the hippocampus involves nitric oxide (NO) synthesis via NO synthase (NOS) activation, we investigated 1) if Ghr administration modulated NOS activity in the hippocampus; and 2) if hippocampal NOS inhibition influenced Ghr-induced memory facilitation, using a behavioral paradigm, biochemical determinations and an electrophysiological model. Our results showed that intra-hippocampal Ghr administration increased the NOS activity in a dose dependent manner, and reduced the threshold for LTP generation in dentate gyrus of rat hippocampus. Moreover, pre-administration of NG-nitro-l-arginine (l-NOArg) in the hippocampus partially prevented the Ghr-induced memory improvement, abolished the increase in NOS activity, and prevented the decreased threshold to generate LTP induced by Ghr. These findings suggest that activation of the NOS/NO pathway in hippocampus participates in the effects of Ghr on memory consolidation and is related with plastic properties of the hippocampal three-synaptic loop.     

The cholinergic innervation of the rat substantia nigra: a light and electron microscopic immunohistochemical study

Summary Putative cholinergic axons and synaptic endings were demonstrated in the substantia nigra (SN) of the rat by light and electron microscopy on the basis of the localization of choline acetyltransferase (ChAT) immunoreactivity. The distribution of ChAT immunoreactivity in the SN as demonstrated by light microscopy revealed a modest network of ChAT-immunoreactive beaded axons in the SNc, in comparison to a relatively sparse distribution in the SNr. These axonal profiles were most dense in the middle of the rostral-caudal extent of the SNc and appeared to be concentrated in the middle third of the medial-lateral extent. By electron microscopy, unmyelinated, small diameter (0.25 m) ChAT-immuno-reactive axons were observed interspersed among numerous other non-immunoreactive axons in the SNc. ChAT-immunoreactive synaptic endings were observed in juxtaposition to small caliber (0.5 m) non-immunoreactive dendrites, and contained numerous spheroidal synaptic vesicles and occasional mitochondria. Synaptic contact zones were characterized by an accumulation of synaptic vesicles along the presynaptic membrane, and a prominent postsynaptic densification producing an asymmetrical pre-/postsynaptic membrane profile typical of excitatory synapses. These findings provide direct evidence for a cholinergic innervation of the SN, and suggest that this input may have an excitatory effect on neuronal elements in the SNc.     

Localization of two cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter in the central nervous system of the rat: in situ hybridization histochemistry and immunohistochemistry

Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are proteins that are required for cholinergic neurotransmission. Present knowledge concerning the organization of cholinergic structures has been derived primarily from immunohistochemistry for ChAT. In the present study, we investigated the distribution of mRNAs and the corresponding proteins for ChAT and VAChT by in situ hybridization histochemistry and immunohistochemistry. The patterns of distribution of perikarya containing ChAT mRNA, ChAT protein, VAChT mRNA and VAChT protein were similar in most regions, and co-localization in the same neuron of mRNAs for ChAT and VAChT, that of ChAT mRNA and ChAT protein, and that of VAChT mRNA and VAChT protein were demonstrated. However, in the cerebral cortex and hypothalamus, ChAT-immunoreactive perikarya were present, but they did not contain mRNAs for ChAT and VAChT, and VAChT protein. On the other hand, in the cerebellum, Purkinje cell bodies contained VAChT mRNA and VAChT protein, but they did not contain either ChAT mRNA or ChAT protein. Axon bundles were clearly revealed by immunohistochemistry for ChAT, but they were not detected by that for VAChT. Both ChAT and VAChT antibodies revealed preterminal axons and terminal-like structures. In the forebrain, they were present in the olfactory bulb, nucleus of the lateral olfactory tract, olfactory tubercle, lateral septal nucleus, amygdala, hippocampus, neocortex, caudate-putamen, thalamus and median eminence of the hypothalamus. In the brainstem, they were localized in the superior colliculus, interpeduncular nucleus and some cranial nerve motor nuclei, and further in the ventral horn of the spinal cord. These results indicate strongly that ChAT and VAChT are expressed in most of the cholinergic neurons, and that immunohistochemistry for VAChT is as useful to detect cholinergic terminal fields as that for ChAT.     

摘除松果体对大鼠学习记忆及基底前脑胆碱能系统的影响

目的　探讨松果体功能减退对大鼠学习记忆及基底前脑胆碱能系统的影响。方法　选用 3月龄SD大鼠 2 4只 ,随机分为对照组、去松果体组和褪黑素 (MT)组。手术摘除松果体。饲养 1个月后用Morris水迷宫测试学习记忆功能 ,同时用组织化学和免疫组化方法测定海马、前额叶皮质AchE纤维和内侧隔核、斜角带核的ChAT神经元的数量。结果　与对照组比较 ,去松果体组逃避潜伏期明显增加 ,海马、前额叶皮质AchE纤维数量明显减少 ,但内侧隔核、斜角带核的ChAT神经元数量变化不明显。结论　大鼠去松果体可引起大鼠学习记忆能力减弱 ,这可能与基底前脑胆碱能神经元的功能下降有关     

Recognition of novel objects and their location in rats with selective cholinergic lesion of the medial septum

The importance of cholinergic neurons projecting from the medial septum (MS) of the basal forebrain to the hippocampus in memory function has been controversial. The aim of this study was to determine whether loss of cholinergic neurons in the MS disrupts object and/or object location recognition in male Sprague-Dawley rats. Animals received intraseptal injections of either vehicle, or the selective cholinergic immunotoxin 192 IgG-saporin (SAP). 14 days later, rats were tested for novel object recognition (NOR). Twenty-four hours later, these same rats were tested for object location recognition (OLR) (recognition of a familiar object moved to a novel location). Intraseptal injections of SAP produced an 86% decrease in choline acetyltransferase (ChAT) activity in the hippocampus, and a 31% decrease in ChAT activity in the frontal cortex. SAP lesion had no significant effect on NOR, but produced a significant impairment in OLR in these same rats. The results support a role for septo-hippocampal cholinergic projections in memory for the location of objects, but not for novel object recognition.     

Cholinergic innervation of the main and the accessory olfactory bulbs of the rat as revealed by a monoclonal antibody against choline acetyltransferase

Summary The main and accessory olfactory bulbs (MOB and AOB) of the rat were immunohistochemically stained with a monoclonal antibody against choline acetyltransferase (ChAT) in order to know the difference in the distribution patterns of cholinergic fibers between these two structures. A few ChAT-immunoreactive cell bodies were found in the superficial and middle parts of the external plexiform layer (EPL) of the MOB, in the granule cell layer (GCL) of the MOB, and in the GCL of the AOB. The frequency in appearance of these cells was 0.9 cells/section in the MOB and 0.3 cells/section in the AOB. While the glomerular layer (GL) and the superficial part of the EPL were most densely innervated in the MOB, the internal plexiform layer received the richest innervation in the AOB. There were no immunoreactive structures in the olfactory nerve layer of the MOB and in the vomeronasal nerve layer and glomerular layer of the AOB. In addition to a relatively homogenous distribution of cholinergic fibers in the MOB and AOB, there were several foci of very dense network of immunoreactive fibers at the posterior level of the OB. These foci formed a part of the modified glomerular complex that was recently identified using 2-deoxyglucose method and was presumed to be related to suckling behaviour in the neonatal rat.     

Distribution of catecholaminergic afferent fibres in the rat globus pallidus and their relations with cholinergic neurons

The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase–antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.     

Granule cells are the main source of excitatory input to a subpopulation of GABAergic hippocampal neurons as revealed by electron microscopic double staining for zinc histochemistry and parvalbumin immunocytochemistry

Immunocytochemistry was combined with a recent modification of Timm's method to evaluate semiquantitatively the mossy fiber innervation of dendrites and somata of parvalbumin-containing neurons of the hilus of the dentate gyrus and the CA3 area of Ammon's horn. Using this electron microscopic double staining technique, it was found that (1) the overwhelming majority (95%) of terminals forming asymmetric synapses with parvalbumin-positive dendrites in the dentate hilus, and the strata pyramidale and lucidum of the CA3 area of Ammon's horn, originated from granule cells; (2) two-thirds of the asymmetric axosomatic terminals of parvalbumin-positive neurons contained zinc; and (3) no zinc-containing axon terminals formed synapses with somata or main dendritic shafts of the granule cells.     

The possible impairment of respiratory-related neural loops may be associated with the silent pneumonia induced by SARS-CoV-2

As compared to many other viral pulmonary infections, there existed several peculiar manifestations in the COVID-19 patients, including the “silence” of pneumonia in both mild and severe cases and a long intensive care unit stay for those requiring invasive mechanical ventilation. Similar silent pneumonia has been documented in the infectioninduced by H5N1 influenza virus HK483 and was found to result from the direct attack of the virus on the bronchopulmonary C-fibers at the early stage and the final infection in the brainstem at the late stage. The long stay of critical patients in the intensive care unit is possibly due to the depression of central respiratory drive, which resulted in the failure to wean from the mechanic ventilation. Carotid and aortic bodies and bronchopulmonary C-fibers are two key peripheral components responsible for the chemosensitive responses in the respiratory system, while triggering respiratory reflexes depends predominantly on the putative chemosensitive neurons located in the pontomedullary nuclei. In view of the findings for the H5N1 influenza virus, the silence of pneumonia induced by SARS-CoV-2 may be due to the possible impairment of peripheral chemosensitive reflexes as well as the damage to the respiratory-related central neurons.     

P3a and P3b components associated to the neurocognitive evaluation of invalidly cued targets

The present report focuses on evaluating the neurocognitive consequences of the correct or incorrect spatial prediction induced by a spatial cue. Positions in the vertical meridian were cued in order to evaluate the cognitive consequences in the processing of the validly (VC) or invalidly cued (IC) targets. The behavioural responses and the 64 EEG channel were recorded. The late endogenous event-related potential (ERP) induced by target stimuli in VC and IC targets were compared in voltage amplitude, voltage and current source density topographies. The P3a and a late positive complex, possibly P3b were increased in a statistically significant manner in the IC targets with regard to the VC targets. The previous result suggests that subjects prepare to accomplish the task upon specification of the cue, and when the IC target appeared it is treated as a low probability stimulus in a similar manner to deviant stimuli in odd-ball paradigms.     

Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation

The accumulation of protein deposits in neurons, in vitro proteasome assays and over-expression studies suggest that impairment of the ubiquitin-proteasome system (UPS) may be a common mechanism of pathogenesis in polyglutamine diseases such as Huntington disease and spinocerebellar ataxias (SCAs). Using a knock-in mouse model that recapitulates the clinical features of human SCA7, including selective neuronal dysfunction, we assessed the UPS at cellular resolution using transgenic mice that express a green fluorescent protein (GFP)-based reporter substrate (Ub(G76V)-GFP) of the UPS. The levels of the reporter remained low during the initial phase of disease, suggesting that neuronal dysfunction occurs in the presence of a functional UPS. Late in disease, we observed a significant increase in reporter levels specific to the most vulnerable neurons. Surprisingly, the basis for the increase in Ub(G76V)-GFP protein can be explained by a corresponding increase in Ub(G76V)-GFP mRNA in the vulnerable neurons. An in vitro assay also showed normal proteasome proteolytic activity in the vulnerable neurons. Thus, no evidence for general UPS impairment or reduction of proteasome activity was seen. The differential increase of Ub(G76V)-GFP among individual neurons directly correlated with the down-regulation of a marker of selective pathology and neuronal dysfunction in SCA7. Furthermore, we observed a striking inverse correlation between the neuropathology revealed by this reporter and ataxin-7 nuclear inclusions in the vulnerable neurons. Altogether, these data show a protective role against neuronal dysfunction for polyglutamine nuclear inclusions and exclude significant impairment of the UPS as a necessary step for polyglutamine neuropathology.     

Depression-like state in maternal rats induced by repeated separation of pups is accompanied by a decrease of cell proliferation and an increase of apoptosis in the hippocampus

Stressful experiences, such as an unsatisfactory mother–infant relationship after delivery, can induce depressive disorders, and it is well-known that stressors impair memory function. The hippocampus plays a crucial role in memory processes. In the present study, we determined whether a depressed-like state induced by repeated separation of pups affects the memory capability of the maternal rats. We also determined the effects of repeated separation from pups on cell proliferation, apoptosis, and serotonin expression in the brains of maternal rats. In the present results, the immobility time in the forced swim test was increased and the climbing time was decreased in the mothers separated from their pups. The latency in the step-down avoidance task was increased in the mothers separated from their pups. Also, the expressions of serotonin (5-hydroxytryptamine) and tryptophan hydroxylase in the dorsal raphe were decreased in the mothers separated from their pups. The number of Ki-67-positive cells was decreased, while the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells in the hippocampal dentate gyrus was increased in the mothers separated from their pups. Based on the present results, it is suggested that separation of pups might induce a depressed-like state in the maternal rats with reduced cell proliferation and increased apoptosis in the hippocampus, resulting in memory impairment of maternal rats.