Norepinephrine drives persistent activity in prefrontal cortex via synergistic α1 and α2 adrenoceptors

Optimal norepinephrine levels in the prefrontal cortex (PFC) increase delay-related firing and enhance working memory, whereas stress-related or pathologically high levels of norepinephrine are believed to inhibit working memory via α1 adrenoceptors. However, it has been shown that activation of Gq-coupled and phospholipase C-linked receptors can induce persistent firing, a cellular correlate of working memory, in cortical pyramidal neurons. Therefore, despite its importance in stress and cognition, the exact role of norepinephrine in modulating PFC activity remains elusive. Using electrophysiology and optogenetics, we report here that norepinephrine induces persistent firing in pyramidal neurons of the PFC independent of recurrent fast synaptic excitation. This persistent excitatory effect involves presynaptic α1 adrenoceptors facilitating glutamate release and subsequent activation of postsynaptic mGluR5 receptors, and is enhanced by postsynaptic α2 adrenoceptors inhibiting HCN channel activity. Activation of α2 adrenoceptors or inhibition of HCN channels also enhances cholinergic persistent responses in pyramidal neurons, providing a mechanism of crosstalk between noradrenergic and cholinergic inputs. The present study describes a novel cellular basis for the noradrenergic control of cortical information processing and supports a synergistic combination of intrinsic and network mechanisms for the expression of mnemonic properties in pyramidal neurons.

Neuroprotection through excitability and mTOR required in ALS motoneurons to delay disease and extend survival.

Delaying clinical disease onset would greatly reduce neurodegenerative disease burden, but the mechanisms influencing early preclinical progression are poorly understood. Here, we show that in mouse models of familial motoneuron (MN) disease, SOD1 mutants specifically render vulnerable MNs dependent on endogenous neuroprotection signaling involving excitability and mammalian target of rapamycin (mTOR). The most vulnerable low-excitability FF MNs already exhibited evidence of pathology and endogenous neuroprotection recruitment early postnatally. Enhancing MN excitability promoted MN neuroprotection and reversed misfolded SOD1 (misfSOD1) accumulation and MN pathology, whereas reducing MN excitability augmented misfSOD1 accumulation and accelerated disease. Inhibiting metabotropic cholinergic signaling onto MNs reduced ER stress, but enhanced misfSOD1 accumulation and prevented mTOR activation in alpha-MNs. Modulating excitability and/or alpha-MN mTOR activity had comparable effects on the progression rates of motor dysfunction, denervation, and death. Therefore, excitability and mTOR are key endogenous neuroprotection mechanisms in motoneurons to counteract clinically important disease progression in ALS.

Genetics of recurrent airway obstruction (RAO)

Recurrent airway obstruction (RAO) is a multifactorial and polygenic disease. Affected horses are typically 7 years of age or older and show exercise intolerance, increased breathing effort, coughing, airway neutrophilia, mucus accumulation and hyperreactivity as well as cholinergic bronchospasm. The environmental factors responsible are predominantly allergens and irritants in haydust, but the immunological mechanisms underlying RAO are still unclear. Several studies have demonstrated a familiar predisposition for RAO and it is now proven that the disease has a genetic basis. In offspring, the risk of developing RAO is 3-fold increased when one parent is affected and increases to almost 5-fold when both parents have RAO. Segregation analysis in two high-prevalence families demonstrated a high heritability and a complex inheritance with several major genes. A whole genomescan showed chromosome-wide significant linkage of seven chromosomal regions with RAO. Of the microsatellites, which were located near atopy candidate genes, those in a region of chromosome 13 harboring the IL4R gene were strongly associated with the RAO phenotype in the offspring of one RAO-affected stallion. Furthermore, IgE-levels are influenced by hereditary factors in the horse, and we have evidence that RAO-affected offspring of the same stallion have increased levels of specific IgE against moldspore allergens. The identification of genetic markers and ultimately of the responsible genes will not only allow for an improved prophylaxis, i.e. early identification of susceptible individuals and avoidance of high-risk matings, but also improve our ability to find new therapeutic targets and to optimize existing treatments.

Aberrant association of misfolded SOD1 with Na+/K+ATPase-α3 impairs its activity and contributes to motor neuron vulnerability in ALS.

Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron excitability and spinal circuits are pathological hallmarks of ALS, but the underlying molecular mechanisms remain unresolved. Here, we sought to understand whether the expression of mutant SOD1 protein could contribute to altering processes governing motor neuron excitability. We used the conformation specific antibody B8H10 which recognizes a misfolded state of SOD1 (misfSOD1) to longitudinally identify its interactome during early disease stage in SOD1G93A mice. This strategy identified a direct isozyme-specific association of misfSOD1 with Na+/K+ATPase-α3 leading to the premature impairment of its ATPase activity. Pharmacological inhibition of Na+/K+ATPase-α3 altered glutamate receptor 2 expression, modified cholinergic inputs and accelerated disease pathology. After mapping the site of direct association of misfSOD1 with Na+/K+ATPase-α3 onto a 10 amino acid stretch that is unique to Na+/K+ATPase-α3 but not found in the closely related Na+/K+ATPase-α1 isozyme, we generated a misfSOD1 binding deficient, but fully functional Na+/K+ATPase-α3 pump. Adeno associated virus (AAV)-mediated expression of this chimeric Na+/K+ATPase-α3 restored Na+/K+ATPase-α3 activity in the spinal cord, delayed pathological alterations and prolonged survival of SOD1G93A mice. Additionally, altered Na+/K+ATPase-α3 expression was observed in the spinal cord of individuals with sporadic and familial ALS. A fraction of sporadic ALS cases also presented B8H10 positive misfSOD1 immunoreactivity, suggesting that similar mechanism might contribute to the pathology.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

STUDIES OF DISULFOTON TOLERANCE IN RATS

Disulfoton (O,O, diethyl S-2-(ethylthio)ethyl phosphorodithioate) and other organophosphorus ester compounds are insecticides which inhibit acetylcholinesterase. Chemicals of this class cause signs of toxicity in mammals which are referable to acculmulation of acetylcholine at neuroeffector sites. A tolerance to this toxic action can be induced in experimental animals by giving multiple, sublethal doses of the compounds. There is strong evidence that disulfoton tolerance occurs because of a reduction in the sensitivity of tissues in the affected animals to acetylcholine.^ Experiments were designed to test the possibility that a decrease in the number of muscarinic cholinergic receptors could be downmodulating the sensitivity of tissues to acetylcholine. It was found that, concomitant with the onset of disulfoton tolerance, there was a decrease relative to control values in the specific binding of {('3)H} quinuclidinyl benzilate ({('3)H}QNB, a compound which selectively labels muscarinic cholinergic receptors) to homogenates of rat brain and ileal muscle. The decrease in {('3)H}QNB binding was due to a reduction in the density of muscarinic receptors. There was, however, no alteration in the binding of {('3)H} QNB, or the muscarinic agonists {('3)H} oxotremorine-M and oxotremorine to atria from disulfoton-tolerant rats. The possibility that cardiac tissue was not subsensitive to cholinergic agonists was ruled out in experiments testing the effect of the muscarinic agonist carbachol on heart rate in vivo, and the negative chronotropic effect of oxotremorine on atria from disulfoton-tolerant rats: a clear reduction in the sensitivity to cholinergic agonists was seen in each case. It was, therefore concluded that the specificity and temporal correlation of {('3)H}QNB binding decreases suggested that the loss of muscarinic receptors might play a role in modulating the sensitivity of several tissues to acetylcholine, but that other mechanisms also contribute to the tolerance phenomenon.^ Other experiments revealed that disulfoton tolerance, as measured by resistance to the lethal effects of carbachol, could be induced by feeding rats low levels of the organophosphorus ester in the diet. The concentration of disulfoton used inhibited acetylcholinesterase, but not to the extent that overt signs of toxicity were observed. These results suggested that tolerance to organophosphorus ester insecticides could be induced in rodents with a dosing scheme which more closely modeled the sort of low level exposures which would be expected in humans environmentally or occupationally in contact with these compounds. ^

Regulation of tyrosine hydroxylase gene expression by mRNA stabilization

Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 μM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3′UTR using RNA electro-mobility shift assays show the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains two proteins. Northwesterns yielded similar results. Transfection studies reveal an increase in expression of the full-length TH transcript due to DMPP-treatment similar to that of endogenous TH mRNA. This finding suggests the increased expression is due primarily to mRNA stabilization. Transfection of luciferase reporter constructs containing regions of the TH 3′UTR reveal only the full-length 3′UTR influenced the expression level of reporter transcripts. ^

(Table 2) Mercury and selenium concentration in the brain stem of wild polar bears (Ursus maritimus), east Greenland

Polar bears (Ursus maritimus) are exposed to high concentrations of mercury because they are apex predators in the Arctic ecosystem. Although mercury is a potent neurotoxic heavy metal, it is not known whether current exposures are of neurotoxicological concern to polar bears. We tested the hypotheses that polar bears accumulate levels of mercury in their brains that exceed the estimated lowest observable adverse effect level (20 µg/g dry wt) for mammalian wildlife and that such exposures are associated with subtle neurological damage, as determined by measuring neurochemical biomarkers previously shown to be disrupted by mercury in other high-trophic wildlife. Brain stem (medulla oblongata) tissues from 82 polar bears subsistence hunted in East Greenland were studied. Despite surprisingly low levels of mercury in the brain stem region (total mercury = 0.36 ± 0.12 µg/g dry wt), a significant negative correlation was measured between N-methyl-D-aspartate (NMDA) receptor levels and both total mercury (r = -0.34, p < 0.01) and methylmercury (r = -0.89, p < 0.05). No relationships were observed among mercury, selenium, and several other neurochemical biomarkers (dopamine-2, gamma-aminobutyric acid type A, muscarinic cholinergic, and nicotinic cholinergic receptors; cholinesterase and monoamine oxidase enzymes). These data show that East Greenland polar bears do not accumulate high levels of mercury in their brain stems. However, decreased levels of NMDA receptors could be one of the most sensitive indicators of mercury's subclinical and early effects.

Transfer of human serum IgG to nonobese diabetic Igμnull mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren’s syndrome

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren’s syndrome. NOD.Igμnull mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igμnull mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab′)2 fragments from parental NOD mice or human primary Sjögren’s syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren’s syndrome.

Neuronal nicotinic threonine-for-leucine 247 α7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the α7 mutant receptor for 5HT is not modified by the presence of dihydro-β-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates α7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

Muscarinic stimulation of synaptic activity by protein kinase C is inhibited by adenosine in cultured hippocampal neurons

We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1–43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca2+ signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca2+ concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca2+ concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4β-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.

Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome

To study the pathogenesis of central nervous system abnormalities in Down syndrome (DS), we have analyzed a new genetic model of DS, the partial trisomy 16 (Ts65Dn) mouse. Ts65Dn mice have an extra copy of the distal aspect of mouse chromosome 16, a segment homologous to human chromosome 21 that contains much of the genetic material responsible for the DS phenotype. Ts65Dn mice show developmental delay during the postnatal period as well as abnormal behaviors in both young and adult animals that may be analogous to mental retardation. Though the Ts65Dn brain is normal on gross examination, there is age-related degeneration of septohippocampal cholinergic neurons and astrocytic hypertrophy, markers of the Alzheimer disease pathology that is present in elderly DS individuals. These findings suggest that Ts65Dn mice may be used to study certain developmental and degenerative abnormalities in the DS brain.

Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid β (Aβ) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes Aβ deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of Aβ in the pathogenesis of the disease.

Effects of muscarinic blockade in perirhinal cortex during visual recognition

Stimulus recognition in monkeys is severely impaired by destruction or dysfunction of the perirhinal cortex and also by systemic administration of the cholinergic-muscarinic receptor blocker, scopolamine. These two effects are shown here to be linked: Stimulus recognition was found to be significantly impaired after bilateral microinjection of scopolamine directly into the perirhinal cortex, but not after equivalent injections into the laterally adjacent visual area TE or into the dentate gyrus of the overlying hippocampal formation. The results suggest that the formation of stimulus memories depends critically on cholinergic-muscarinic activation of the perirhinal area, providing a new clue to how stimulus representations are stored.

Neurosteroids: Deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus

Pregnenolone sulfate (PREG S) is synthesized in the nervous system and is a major neurosteroid in the rat brain. Its concentrations were measured in the hippocampus and other brain areas of single adult and aged (22–24 month-old) male Sprague–Dawley rats. Significantly lower levels were found in aged rats, although the values were widely scattered and reached, in about half the animals, the same range as those of young ones. The spatial memory performances of aged rats were investigated in two different spatial memory tasks, the Morris water maze and Y-maze. Performances in both tests were significantly correlated and, accompanied by appropriate controls, likely evaluated genuine memory function. Importantly, individual hippocampal PREG S and distance to reach the platform in the water maze were linked by a significant correlation, i.e., those rats with lower memory deficit had the highest PREG S levels, whereas no relationship was found with the PREG S content in other brain areas (amygdala, prefrontal cortex, parietal cortex, striatum). Moreover, the memory deficit of cognitively impaired aged rats was transiently corrected after either intraperitoneal or bilateral intrahippocampal injection of PREG S. PREG S is both a γ-aminobutyric acid antagonist and a positive allosteric modulator at the N-methyl-d-aspartate receptor, and may reinforce neurotransmitter system(s) that decline with age. Indeed, intracerebroventricular injection of PREG S was shown to stimulate acetylcholine release in the adult rat hippocampus. In conclusion, it is proposed that the hippocampal content of PREG S plays a physiological role in preserving and/or enhancing cognitive abilities in old animals, possibly via an interaction with central cholinergic systems. Thus, neurosteroids should be further studied in the context of prevention and/or treatment of age-related memory disorders.