Sodium-dependent, high-affinity choline uptake in hippocampus and frontal cortex of the rat affected by acute restraint stress

Acute (45-min) restraint stress decreased sodium-dependent, high-affinity choline uptake in the hippocampus and frontal cortex of rats. The effect of restraint on the hippocampus was blocked, whereas that on the frontal cortex was not significantly affected, by pretreatment of the rats with the narcotic antagonist naltrexone (1 mg/kg, i.p.) immediately prior to restraint.     

Corticotropin-releasing factor immunoreactive neurons persist throughout the brain in Alzheimer's disease

Corticotropin-releasing factor (CRF) immunoreactivity was examined in the hippocampal formation, cerebellum and hypothalamus of normal aged and Alzheimer's disease (AD) brains. Immunoreactive non-pyramidal neurons were located in the polymorphic layer of the dentate gyrus and CA fields. A plexus of CRF terminals was seen in the supragranular layer of the dentate gyrus. In AD, occasional senile plaques contained CRF-immunoreactive fiber terminals but the pattern of staining was otherwise unchanged aside from a suggestion of increased staining intensity. Similarly, the pattern of immunoreactive cerebellar climbing fibers and paraventricular hypothalamic neurons was preserved in AD brains aside from increased perikaryal staining intensity in the hypothalamus.     

Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer's disease: An autoradiographic study using3H-hemicholinium-3

Summary ³H-hemicholinium-3 (³H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer's disease (AD) patients and of 11 matched controls. A significant decrease in the density of³H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I–III of the frontal cortex. By contrast, in the caudate-putamen the number of³H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (–40%), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD.     

Characterization of [H]hemicholinium-3 binding associated with neuronal choline uptake sites in rat brain membranes

Hemicholinium-3 (HCh-3) is a potent and specific inhibitor of the high-affinity choline transport process (HAChT) localized on cholinergic neurons. In this study, the specific binding of [³H]HCh-3 (120 Ci/mmol) was characterized in crude synaptic membranes prepared from rat brain. The binding of [³H]HCh-3 to forebrain membranes was saturable, reversible and specific with an apparent K_d under optimal conditions of 35 nM and a B_max of 56 fmol/mg protein. The potency of various HAChT inhibitors correlated with their apparent affinities for the specific [³H]HCh-3 binding site. The specific binding of [³H]HCh-3 exhibited an uneven regional distribution in the adult rat brain that corresponded to the activity of the HAChT in these regions. Transsection of the fornix, which causes a degeneration of the septal hippocampal cholinergic pathway, resulted in comparable reductions of the specific [³H]HCh-3 binding and the specific activity of choline acetyltransferase, a presynaptic marker for cholinergic terminals in the hippocampal formation; the lesion did not affect the specific activity of glutamic acid decar☐ylase, a presynaptic marker for GABAergic neurons within the hippocampus. Maximal binding occurred in the presence of 200 mM NaCl: potassium, lithium, rubidium and calcium substituted poorly for sodium; and bromide, fluoride, iodide, sulfate and phosphate were less effective anions than chloride. Increasing concentrations of NaCl increased the affinity of the site for [³H]HCh-3 with no significant effect on the maximal number of sites; the enhancement of affinity was due to a selective slowing of the rate of dissociation of the ligand from its binding site. These findings indicate that [³H]HCh-3 binds to the carrier site mediating the HAChT on cholinergic neurons; thus, this radioligand may be a useful probe for investigating this presynaptic component (HAChT) of cholinergic neurons.     

Decreased [H]hemicholinium binding to high-affinity choline uptake sites in aged rat brain

The binding of [³H]hemicholinium ([³H]HCh-3) to sodium-dependent high-affinity choline uptake sites provides a useful neuroanatomical and functional marker of the cholinergic system. We examined the autoradiographic distribution of [³H]HCh-3 binding sites in the forebrain of young (4–6 months) and old (32 months) rats. There was a widespread reduction of [³H]HCh-3 binding site density in the aged rat brain. This loss presented regional differences with maximal reduction in the medial and posterior striatum (55%) and in the dentate gyrus (47%), in limbic areas such as basolateral amygdala, tubercle olfactorium and piriform cortex the autoradiographic signal was about 25–30% lower. In aged hippocampus and cerebral cortex the density of [³H]HCh-3 binding sites was about 40% lower, the difference between young and senescent animals being less evident in the medial septum and basal nucleus. No significant alterations were observed in interpeduncular nucleus from old rats. These data are in agreement with the functional results obtained by measuring other cholinergic parameters in the aged rat and confirm the vulnerability of cholinergic system during aging     

Changes in high affinity choline uptake in rat cortex following lesions of the magnocellular forebrain nuclei

High affinity choline uptake (HACU) and choline acetyltransferase (CAT) were measured in the cerebral cortex of rats 4 and 20 days after placing electrolytic lesions in the magnocellular forebrain nuclei (MFN) or in the pallidum.Four days after MFN lesion a 40–50% decrease in ipsilateral cortical HACU was found and a slightly smaller decrease was found 4 days after the pallidum lesion. Twenty days after the lesion, HACU activity returned to control values in the ipsilateral parietal cortex, its decrease was smaller than 4 days postlesion in the ipsilateral frontal cortex and a significant increase was found in the contralateral cortex.CAT activity showed a 40% decrease in the frontal, parietal and occipital ipsilateral cortex 4 days after MFN lesion. The same decrease was found 20 days postlesion. However, at this time a significant increase in CAT activity was detected in the contralateral cortex.The ipsilateral recovery of HACU activity 20 days after the lesions and the contralateral increase in HACU and CAT activity demonstrate the remarkable and widespread functional adjustment associated with discrete brain lesions. The existence of a large cholinergic pathway projecting to the neocortex from the basal forebrain region is also confirmed.     

MKC-231, a choline uptake enhancer: (3) mode of action of MKC-231 in the enhancement of high-affinity choline uptake

MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.     

High-affinity uptake system for cysteine in crude synaptosomal fractions of rat cerebral cortex

The in vitro uptake of [³⁵S] cysteine was studied in crude synaptosomal preparation of the cerebral cortex of rat. The accumulation of cysteine was found to be temperature- and time-dependent. It was linear at least for four minutes at 37 C with characteristics of saturable kinetics. Uptake of cysteine was Na⁺- and K⁺-dependent. Increasing the Na⁺ ion concentration increased the accumulation of cysteine in synaptosomal preparations; unlike the Na⁺ ion, an increase in the K⁺ ion, an increase in the K⁺ ion inhibited cysteine uptake. Cysteine was accumulated against concentration gradients by a saturable mechanism. Double reciprocal plot of the cysteine uptake suggests two types of affinity systems, with K_m values for the high-affinity uptake of about 12.2 μM and for the low-affinity uptake of about 4 mM. The high-affinity uptake was also significantly inhibited by ouabain, a potent inhibitor of the Na⁺-K⁺-dependent ATPase, and other metabolic inhibitors. The results of the effects of cysteine analogues on uptake also suggested that it is a substrate-specific high-affinity uptake system for cysteine.     

Comparative ontogenic profile of cholinergic markers,including nicotinic and muscarinic receptors,in the rat brain

The ontogenic profiles of several cholinergic markers were assessed in the rat brain by using quantitative in vitro receptor autoradiography. Brain sections from animals at different stages of development were processed with [³H]AH5183 (vesamicol; vesicular acetylcholine transport sites), [³H]N-methylcarbamylcholine (α₄β₂ nicotinic receptor sites), [³H]hemicholinium-3 (high-affinity choline uptake sites), [³H]3-quinuclidinyl benzilate (total population of muscarinic receptor sites), [³H]4-DAMP (muscarinic M₁/M₃ receptor sites), [³H]pirenzepine (muscarinic M₁ receptor sites), and [³H]AF-DX 116 and [³H]AF-DX 384 (muscarinic M₂ receptor sites) as radiolabeled probes. The results revealed that, by the end of the prenatal period (embryonic day 20), the densities of nicotinic receptor and vesicular acetylcholine transport sites already represented a considerable proportion of those observed in adulthood (postnatal day 60) in different laminae of the frontal, parietal, and occipital cortices, in the layers of Ammon's horn fields and the dentate gyrus of the hippocampal formation, as well as in the amygdaloid body, the olfactory tubercle, and the striatum. In contrast, at that stage, the densities of total muscarinic, M₁/M₃, M₁, and possibly M₂ receptor and high-affinity choline uptake sites represent only a small proportion of levels seen in the adult. Differences were also observed in the postnatal ontogenic profiles of nicotinic, muscarinic, vesamicol, and high-affinity choline uptake sites. For example, between postnatal weeks 3 and 5, the levels of M₁/M₃ and M₁ sites were at least as high as in the adult, whereas M₂ and high-affinity choline uptake site densities appeared to be delayed and to reach adult values only after postnatal week 5. With regard to cholinergic innervation in the developing rat brain, the present findings suggest a temporal establishment of several components of the cholinergic systems. The first components are the vesicular acetylcholine transporter and nicotinic sites; these are followed by M₁/M₃ and M₁ sites and, finally, by M₂ and high-affinity choline uptake sites. © 1996 Wiley-Liss, Inc.     

Age dependent changes in choline uptake of the chick iris

The kinetics of the uptake of choline, the rate-limiting substrate in the in vivo synthesis of acetylcholine, were studied during the period 1–7 years in the iris of the chick. These changes were correlated to the endogenous levels of acetylcholine and choline in the same organ. V_max values per iris decrease significantly at 5 years and continue to decrease at 7 years, to 64% and 37% of the one year value, respectively. If the variation in V_max is calculated per protein the decline is 34% between 1 and 5 years.K_m does not change significantly during the period 1–7 years. Total acetylcholine and choline levels follow a similar trend, decreasing progressively from 1 to 7 years.Sensitivity to hemicholinium (5.5 × 10⁻⁵M) decrease significantly between 5 days of incubation (d.i.) and 5 years. Inhibition by ouabain (10⁻⁴ M) shows an opposite trend, increasing significantly from 20% at 5 d.i. to 52% at 3 months. At 5 years sensitivity to ouabain is the same as at 3 months. Na⁺ dependence decreases significantly between 5 d.i. and 5 years but no significant changes are seen between 5 d.i. and 3 months. Uptake at 27 °C decreases from 59% of control at 5 d.i. to 45% at 3 months, and is not changed at 5 years.Our observations suggest that the effect of aging on peripheral cholinergic neurons is not generalized, but is specifically directed toward the neuronal perophery (terminals) as opposed to cell bodies.     

Interaction between nerve growth factor and GM1 monosialoganglioside in preventing cortical choline acetyltransferase and high affinity choline uptake decrease after lesion of the nucleus basalis

Monosialoganglioside GM₁ and nerve growth factor (NGF) were administered alone or concomitantly to adult male rats with a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM). High-affinity choline uptake (HACU) rate and choline acetyltransferase (ChAT) activity were measured, 4 and 21 days after surgery, respectively, in the frontal and parietal cortices of both hemispheres. A 33–34% decrease in HACU rate and a 43-39% decrease in ChAT activity was found in the ipsilateral cortices 4 and 21 days, respectively, after the lesion. If the lesioned rats received NGF (10 μg i.c.v.) twice a week or daily administrations of GM₁ (30 mg/kg, i.p.), beginning immediately after surgery the decrease in HACU rate and ChAT activity was smaller. If NGF and GM, were given concomitantly no decrease in HACU rate and ChAT activity was detected in the lesioned hemisphere and a slight increase occurred in the contralateral hemisphere. However, after the concurrent administration of NGF (10 μg i.c.v.) and the inactive dose of GM₁ 10 mg/kg i.p. no decrease in HACU and ChAT activity was also found in the lesioned rats. The latter finding indicates a potentiation by GM₁ of NGF effects on the cholinergic neurons of the IBM. The two drugs may either antagonize the neurotoxic effects of ibotenic acid or stimulate a compensatory activity in the remaining neurons.     

Corticotropin-releasing factor administered into the locus coeruleus, but not the parabrachial nucleus, stimulates norepinephrine release in the prefrontal cortex

Previous studies have indicated that intracerebroventricular application of corticotropin-releasing factor (CRF) activates noradrenergic neurons in the brain stem locus coeruleus (LC) and norepinephrine (NE) metabolism in several brain regions. To assess whether CRF has direct effects on LC noradrenergic neurons, CRF was infused into the LC and concentrations of NE and its metabolites were measured in microdialysates collected from the medial prefrontal cortex (PFM). Infusion of 100 ng of CRF into the LC significantly increased dialysate concentrations of NE and of its catabolite MHPG in the ipsilateral PFM, whereas no significant changes were observed following infusion of artificial CSF. No response was observed when the infusions of CRF occurred outside of the LC, including those in the parabrachial nucleus. Although CRF administered into the LC slightly increased dialysate concentrations of NE in the contralateral PFM, this effect was not statistically significant. The effect of CRF injected into the LC on dialysate NE was prevented by combination with a 10-fold excess of the CRF antagonist, alpha-helical CRF_9–41 indicating some specificity in the response. These results are consistent with anatomical and electrophysiological evidence suggesting that CRF may directly activate noradrenergic neurons in or close to the LC.     

PKC isoforms were reduced by lead in the developing rat brain

A plethora of protein kinase C (PKC) isoforms play important roles in regulating synaptic plasticity and neurotransmitter release. Even though, most PKC isoforms are involved in Pb-induced neuronal toxicity, its mechanism is still unclear. The current study addresses the effect of Pb on PKC isoforms in different regions of the developing rat brain. Sprague-Dawley (SD) pregnant rats were exposed to 0.1% Pb as lead acetate dissolved in distilled deionized water (DDW) from gestation day 6 through 21 postnatal day (PND). Control rats were allowed to drink DDW. Pups were sacrificed on PND 1, 5, 10 and 45. Rat brain was immediately excised and separated into the brain stem (BS), the cerebellum (CB), the hippocampus (HC) and the frontal cortex (FC). The Pb level in different regions of the brain was determined using an analytical graphite tube atomizer (Varian). Typical PKC (alpha, beta, gamma), novel PKC (epsilon) and atypical PKC (mu) in the above brain regions were enriched by immunoprecipitation and later were assayed by Western blotting. The total, calcium-dependent and -independent PKC activities were determined by the radioactivity of total gamma-32P transferred to histone. The results indicated that on PND 1, Pb reduced the PKC-gamma protein in HC and FC, whereas on PND 5 the proteins of PKC isoforms (alpha, beta, gamma, epsilon, mu) in HC and FC were significantly reduced. These reductions in PKC proteins were higher in membrane fractions than in cytosolic fractions. On PND 10, Pb reduced all PKC isoforms. However, on PND 45, Pb had no significant effect on all PKC isoforms except epsilon. Pb inhibited the total PKC activity by 70% on PND 1 and 5, the bulk of these PKC activities were calcium-dependent. The results suggest that during early stages of the rat brain development, Pb exposure decreased PKC activities and also reduced PKC isoforms including PKC-gamma and epsilon which are reported to have roles in the memory formation and long-term potentiation (LTP).     

Regional adaptation of muscarinic receptors and choline uptake in brain following repeated administration of diisopropylfluorophosphate and atropine

The specific [³H]QNB binding and high-affinity uptake of [¹⁴C]choline in 8 brain regions (cerebral cortex, hippocampus, hypothalamus, thalamus, striatum, midbrain, cerebellum and brainstem) after repeated administration of DFP and atropine to guinea-pigs were simultaneously measured. Following repeated DFP administration, AChE was markedly depressed in each brain region. In these animals, there was a significant decrease in specific [³H]QNB binding in the cerebral cortex, hippocampus and striatum, whereas the [³H]QNB binding in the rest of brain regions was unchanged. Scatchard analysis revealed a 36% decrease in the B_max value for the striatal [³H]QNB binding without a change in theK_d value, suggesting a change in the receptor density. In contrast, repeated atropine administration produced a significant enhancement in the [³H]QNB binding only in the hippocampus and striatum. The B_max value in the striatum increased by 21% without a change in theK_d value.In addition to the receptor alteration, high affinity uptake of [¹⁴C]choline in the hippocampus and striatum was significantly decreased by DFP treatment, while that in the striatum increased by atropine treatment. Thus the present study has demonstrated that a prolonged activation and blockade of central muscarinic receptors resulted in specific adaptation in both the receptor density and ACh availability at the synapses in the cerebral cortex, hippocampus and striatum.     

Dexamethasone affects radioactive choline uptake in rat diaphragm nerve endings and not in muscle fibres

The initial rate of radioactive choline (Ch) uptake in the endplate-rich area (EPA) of both stimulated and unstimulated hemidiaphragms is significantly increased by 0.2 microM dexamethasone (Dex) in the presence of 10 microM Ch. In autoradiographs, the mean grain densities above the muscle fibres are not altered by Dex. The mean grain densities above the nerve endings are significantly increased in the presence of Dex in stimulated tissue, and slightly but not significantly increased in unstimulated tissue. There is a positive correlation between the initial rate of Ch uptake in the EPA and the amount of isotope in the nerve terminals, in the absence and presence of Dex. Without correcting for the large amount of diffusion which occurs, the ratio of the grain densities above the nerve terminals to that above the muscle fibres in the presence of Dex is 2.12 in stimulated tissue, and 1.40 in unstimulated tissue. The ratio in the stimulated tissue is significantly greater than the control ratio in the absence of Dex (1.66). Therefore, Dex affects radioactive Ch uptake in nerve endings and not in muscle fibres in the rat diaphragm. The stimulation-induced increase in the uptake of isotope into the nerve endings is abolished in a Na+-depleted medium, and in the absence of Ca2+. Dex has no effect on this abolition. We conclude that relatively low concentrations of Dex affect Ch transport in rat diaphragm nerve endings by a mechanism as yet to be defined.     

Aging and stress-induced changes in choline and glutamate uptake in hippocampus and septum of two rat strains differing in longevity and reactivity to stressors

Stress induced changes in neurochemical indices of neurotransmission are more pronounced in the septohippocampal cholinergic system of Wistar Kyoto rats, which are behaviorally more reactive to Stressors and have a shorter life span, than in Brown Norway rats. Moreover, pronounced degeneration of septohippocampal cholinergic neurons occurs earlier in life in Wistar Kyoto rats. In the present study the high affinity synaptosomal uptakes of choline and glutamate were used as indices for cholinergic and glutamatergic systems respectively. Following 2 hr of mild restraint stress increases in both uptake systems were observed in all regions examined (hippocampus, septum and frontal cortex). The stress-induced increases were generally similar in young (3 months) and aged (20 months) rats of both strains. The noted exception was that choline uptake levels, which were reduced in the hippocampus of unhandled aged WKY rats, remained unchanged after stress. The results confirm the involvement of the septohippocampal cholinergic system in the response to acute stress and extend the findings to include the hippocamposeptal glutamatergic system activation as well. It is suggested that in spite of neuronal degeneration during aging, these responses to stress can be maintained by compensatory efforts of neurons that remain intact.     

Corticotropin-releasing factor mRNA increases in the inferior olivary complex during harmaline-induced tremor

This study reports that corticotropin-releasing factor (CRF) expression within the inferior olivary complex (IOC) of the cat is increased 8 h after administration of the tremor-inducing β-carboline harmaline. Following harmaline treatment, hybridization of an oligodeoxynucleotide complementary to CRF mRNA increased significantly in the dorsal accessory olive, subnuclei A and C of the medial accessory olive and the dorsal cap of Kooy, a subnucleus thought previously to be unresponsive physiologically to harmaline. At this early time point, greater increases in CRF mRNA hybridization were present in the caudal than the rostral IOC. These results support published reports that harmaline-mediated effects are more profound within the caudal than the rostral IOC, but also suggest that harmaline mediates cellular responses in inferior olivary neurons which are not related to activation of rhythmic firing.     

Evidence for a frontocortical-septal glutamatergic pathway and compensatory changes in septal glutamate uptake after cortical and fornix lesions in the rat

To determine the source of glutamatergic input to the septum and to the nucleus accumbens septi, glutamate uptake was assessed after transections of the frontal cortex and/or fornix. Uptake in the septum and accumbens was reduced by 25 and 30% respectively, 6 days after bilateral frontal cortex transections. Both indices returned to control levels 30 days postoperatively. In contrast, while unilateral fornix transection did not affect uptake in the accumbens at either day 6 or 30, uptake in the septum was significantly reduced (25–35%) at both times. When a unilateral transection of the fornix was performed in rats with a pre-existing bilateral ablation of the frontal cortex, a further reduction in uptake was observed in the septum (50–60% at both 6 and 30 days after the fornix transection). The data implicate glutamate as a neurotransmitter in frontocortico-septal projections and suggest that the contribution of the hippocampo-septal system to total glutamate uptake in the septum is increased following ablation of the frontocortico-septal system.