Prenatal neuroleptic exposure alters postnatal striatal cholinergic activity in the rat

Previous studies in our laboratory have shown that prenatal exposure to a neuroleptic during a critical period of gestation in the rat results in a marked deficit in the number of striatal dopamine-binding sites and in a diminution of dopamine agonist-induced stereotyped behavior. In the present studies, we examined the effect of prenatal neuroleptic exposure on biochemical parameters of cholinergic activity to determine whether the balance between striatal dopaminergic and cholinergic activity might be altered. The number of muscarinic cholinergic-binding sites and the specific activity of choline acetyltransferase were found to be significantly increased by prenatal treatment with the neuroleptics haloperidol or (+)-butaclamol. From the present studies and previous observations made in our laboratory, it is concluded that the ability of a neuroleptic to affect the number of muscarinic cholinergic receptors in postnatal life may be a result of the phenotypically undifferentiated state of the developing dopamine-binding site. Our findings of increased striatal cholinergic activity accompanied by a marked decrease in dopaminergic activity may have implications for an increased vulnerability to extrapyramidal motor disturbances during postnatal development.     

Perinatal lead exposure alters postnatal cholinergic and aminergic system in rat brain: reversal effect of calcium co-administration

Our earlier studies indicated the role of neurotransmitter systems in lead (Pb) induced behavioral perturbations. In this study, we examined the alterations in synaptosomal acetylcholine (ACh), epinephrine, dopamine, acetylcholinesterase (AChE), and mitochondrial monoamine oxidase (MAO) in the cerebellum and hippocampus of perinatally Pb-exposed rats. Rats (Wistar) were exposed to 0.2% Pb (Pb acetate in drinking water of mother) from gestational day 6 and the pups were exposed lactationally (through mother's milk) to Pb till weaning (postnatal day 21). Studies conducted on different postnatal days (PND 21, 28, 35 and 60) showed significant decreases in synaptosomal AChE and mitochondrial MAO activities, and increases in the levels of ACh, dopamine and epinephrine in the cerebellum and hippocampus of Pb-exposed rats. These alterations were greater at PND 35 and more pronounced in the cholinergic system (ACh and AChE) of hippocampus and the aminergic system (epinephrine, dopamine and MAO) of cerebellum. The total locomotor activity and exploratory behavior were also decreased significantly in Pb-exposed animals corresponding to the alterations observed in cholinergic and aminergic systems. Calcium administration together with Pb, however significantly reversed the Pb-induced alterations in transmitters and enzymes, as well as exploratory and motor behavior suggesting protective effect of calcium in Pb-exposed animals.     

Unilateral odor deprivation: early postnatal changes in olfactory bulb cell density and number

Surgical closure of an external naris of the rat from postnatal day 1 to day 30 results in a 25% decrease in the size of the ipsilateral olfactory bulb. Decreases in size must result from changes in either the number of neurons and/or glia, or their size or both. The present study was designed to quantify cell density (Nv) and number at various early postnatal ages in order to examine both normal patterns of maturation and sequences of change resulting from deprivation. Data from control subjects indicated that numbers of mitral cells remained constant while numbers of external tufted cells increased. Both relay cell populations exhibited increases in nuclear size suggestive of substantial postnatal differentiation. All interneuronal and glial populations increased in number, although differences in maturational patterns were observed between cell species. For example, light and dark subpopulations of granule cells differed in the timing of peak cellular density, and an inside-to-outside gradient of maturation was found for dark granule cells. Growth curves were generally similar in occluded and control pups until approximately day 20, when deprivation resulted in decreased number and nuclear area of external tufted cells and density and number of granule cells and their associated glia. Light granule cells were affected earlier than dark cells, perhaps because of their earlier arrival in the granule cell layer. The affected cell groups represent the last relay and interneuronal populations to be generated, perhaps explaining their particular susceptibility to the effects of experience. Most of the changes emerged late, thus suggesting that they represent the culmination of a series of experience-induced changes within the maturing bulb. The observed effects may result from either altered cellular proliferation or death patterns (or both), alternatives now under investigation.     

Hippocampal cholinergic alterations and related behavioral deficits after early exposure to phenobarbital.

Mice were exposed to phenobarbital (PhB) prenatally and neonatally. Prenatal exposure was accomplished by feeding the mother PhB (3 g/kg milled food) on gestation days 9-18. Neonatal exposure was accomplished by daily injections of 50 mg/kg sodium PhB directly to the pups on days 2-21. Long-term biochemical alterations in the pre- and postsynaptic septohippocampal system, as well as related behavioral deficits, were assessed in the treated animals. Significant increase in B(max) values for binding of [3H]QNB to muscarinic cholinergic receptors was obtained on both ages 22 and 50 in prenatally (40-90%, respectively, p less than 0.001) and neonatally exposed (58-89%, p less than 0.001) mice whereas Kd remained normal. Similarly, a significant increase of inositol phosphate (IP) formation in response to carbachol was found after both prenatal and neonatal exposure to PhB (p less than 0.05). No alterations in choline acetyltransferase (ChAT) activity were observed in the prenatally or neonatally treated animals. The early exposed mice showed deficits in the performance in Morris water maze, a behavior related to the septohippocampal pathway. The results suggest that early exposure to PhB induces alterations in postsynaptic components of the hippocampal cholinergic system and concomitantly to impairment in hippocampus-related behavior.     

Behavioral and biochemical effects of early postnatal cholinergic lesion in the hippocampus.

The effects of early postnatal (PD 8) intracerebroventricular injection of ethylcholine mustard aziridinium ion (AF64A) on development of open-field and cognitive behaviors and cholinergic markers in several brain areas were examined in the rat. The cholinotoxin was bilaterally administered in a dose range of 0.25 to 2.0 nmol. In the open-field tests, the cholinergic lesion caused a dose-dependent increase in activity at 20 days of age, while it resulted in lengthened latency to initiate exploration and decreased rearing activity at adulthood. Hole-board spatial learning was severely inhibited in adult age. The biochemical activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the hippocampus was markedly decreased in a dose-dependent manner, but was unchanged in the neocortex and striatum. Histochemical staining of AChE-positive fibers revealed a severe cholinergic denervation of the granular and pyramidal cell layers of the hippocampus. The results showed that a selective cholinergic deafferentation of the hippocampus at a critical stage of development leads to long-lasting abnormal open-field and spatial learning behaviors.     

Dendritic spine changes in rat hippocampal pyramidal cells after postnatal lead treatment: A golgi study

Newborn rats were exposed to lead (10 mg/ml Pb acetate) from 2 days of age. Control animals received the same concentration of Na acetate. At 20 and 56 days of age the brains were processed for Golgi-Cox impregnation. Hippocampal pyramidal neurons of the CA1 region were analyzed for the spine density of their apical dendrites. A significant decrease was found in the spine density after lead treatment.     

Perturbation of a hippocampal zinc-binding pool after postnatal lead exposure in rats

Morphologic alterations of the hippocampal mossy fiber pathway after postnatal lead exposure have been observed in rats. It is hypothesized that lead might perturb zinc pools found in this pathway. To test this hypothesis, rat pups were exposed to lead indirectly by administering 0.2% lead acetate to dams via the drinking water during lactation for 21 days and control litters were maintained on tap water. To evaluate whether or not the effects of postnatal lead exposure were selective for hippocampal zinc pools, the hippocampus was compared with the cerebellum. There were no significant differences between lead-treated and control rats in total zinc content in either the hippocampus or the cerebellum in rats at 30 and 90 days of age. Furthermore, no differences in the subcellular distribution of zinc were observed between control and lead-treated animals. Because the effects on zinc content may be more subtle, the amounts of cytosolic zinc-binding species, isolated using Ultrogel AcA 34 gel chromatography, were measured in control and lead-treated animals. A striking decrease was observed in the amount of zinc associated with one of the cytosolic zinc-binding species, a putative zinc-glutathione complex, shown in previous studies to be the major hippocampal zinc pool. This effect was observed only in the hippocampus of 30-day-old lead-treated rats. By 90 days of age, the effect was no longer present. These data suggest that lead preferentially affects a zinc pool found in the hippocampus and supports our hypothesis that postnatal lead exposure results in an alteration in hippocampal zinc.     

Effect of prenatal exposure to phencyclidine on the postnatal development of the cholinergic system in the rat

The purpose of this study was to examine the effect of prenatal exposure to phencyclidine (PCP) on the postnatal development of the cholinergic system in the neostriatum and the neocortex. Pregnant females were injected daily with PCP (0.5 mg/kg s.c.) from conception to parturition. On postnatal day 21 or 35, pups were injected with a challenge dose of PCP (0.5 mg/kg s.c.). The levels of acetylcholine and choline were measured, as well as the incorporation of deuterium-labelled choline into newly synthesized acetylcholine in the frontal cortex and neostriatum. The data demonstrate that even at 21 days, rat pups exposed to PCP in utero were more sensitive to a challenge dose of PCP than the saline controls. Thus, a tolerance to PCP, even after a prolonged prenatal exposure did not develop.     

Cholinergic denervation-like changes in rat hippocampus following developmental lead exposure

We investigated the effects of developmental lead exposure from embryonic day 16 (E16) through postnatal day 28 (PN28), on cholinergic and catecholaminergic markers in the septohippocampal pathway in rats through fourth month of age. Lead exposure resulted in a persistent 30–40% reduction of [³H]hemicholinium-3 ([³H]HC-3) binding in the hippocampus through PN120, and 20–30% reduction of septal and hippocampul choline acetyltransferase (ChAT) activity which persisted through PN84 but returned to control levels in both septum and hippocampus at PN112. The muscarinic ligand [³H]quinuclidinyl benzylate ([³H]QNB) binding was reduced in the septum at PN28 but did not differ significantly from controls at PN56–PN112. Neither short- nor long-term effects of Pb exposure on [³H]QNB binding were seen in the hippocampus. Similar to the effects of fimbria-fornix transection, Pb exposure resulted in a long-term 50–90% increase of tyrosine hydroxylase(TH) activity in the hippocampus, although neither treatment affected TH activity in the septum. The lead-induced increase in hippocampul TH was significantly attenuated by superior cervical ganglionectomy. It is concluded that the effects of perinatal lead exposure resemble in several respects those seen following surgical disruption of the septohippocampal pathway in adult animals. The denervation-like effects in the hippocampus may be an important factor in long-term learning and cognitive impairments following developmental exposure to low-levels of lead.     

Time-dependent changes in the binding parameters of muscarinic cholinergic receptors in the rat brain

Using quantitative receptor autoradiography, effects of several incubation times on the binding parameters for [³H]quinuclidinyl benzilate ([³H]QNB) binding were investigated in 5 discrete regions of the rat brain. There were no differences in B_max-values between 3 incubation times. On the other hand, K_d(appl-values markedly depended on the duration of incubation time. Furthermore, Scatchard plots at low [³H]QNB concentrations showed an abnormal binding behavior, i.e. deviation from the straight line representation a single population of the binding site.     

Alterations in septohippocampal cholinergic innervations and related behaviors after early exposure to heroin and phencyclidine.

Mice were exposed to diacetylmorphine (heroin) or phencyclidine (PCP) prenatally or neonatally. At a later age, they were tested for hippocampus-related behavioral deficits and concomitant alterations in the septohippocampal cholinergic innervations. Actually, this is an application of the previously established phenobarbital neuroteratogenicity model to heroin and PCP. Prenatal exposure was accomplished transplacentally by injecting the mother 10 mg/kg heroin or PCP on gestation days 9-18. Neonatal administrations were applied directly by injections of 10 mg/kg of either drug to the pups between neonatal days 2-21. At the age of 50 days, mice exposed to heroin and PCP prenatally exhibited a 107% and 159% increase in their muscarinic cholinergic receptors Bmax, respectively. Neonatal exposure to heroin or PCP caused an 83% and 76% increase in the receptors respectively. On the behavioral level, both prenatal and neonatal exposure to heroin or PCP reduced performance in the hippocampus related eight-arm maze and Morris mazes. Depending on the drug, the test and the period of drug administration, the reduction ranged between 10% and 75%. The results suggest that heroin and PCP induce alterations in the septohippocampal cholinergic innervations and in related behavioral performance. Further studies are necessary in order to connect the biochemical and behavioral events in causal relationships.     

Alterations in hippocampal cholinergic receptors and hippocampal behaviors after early exposure to nicotine

Mice were exposed to nicotine prenatally by injecting the mother with 1.5 mg/kg nicotine SC twice daily on gestation days 9-18 (PreN mice) or neonatally by daily SC injections of 1.5 mg/kg nicotine on postnatal days 2-21 (NeoN mice). At age 50 days, hippocampal muscarinic receptors Bmax of PreN and NeoN mice were 58% and 79% above control, respectively (p less than 0.01); Kd was unaffected by early nicotine exposure. Eight-arm maze performance of nicotine-exposed animals fell behind control level. Both PreN and NeoN made approximately 10% less correct responses in the first eight trials than controls throughout the test period (p less than 0.01). By the last day of testing, PreN needed 23% and NeoN 31% more trials than controls to enter all arms (p less than 0.001). In addition, PreN needed 35 and NeoN 42% more days than controls to reach criterion (p less than 0.05). Similarly, while 61% of controls reached criterion by day 6 only 17% of PreN and 25% of NeoN reached criterion (p less than 0.01). In the Morris maze, PreN needed from 43-119% more time to reach the platform (p less than 0.001). In the spatial probe test, PreN animals made 35% fewer crosses over the area of the missing platform (p less than 0.001). The study suggests that nicotine administered to the fetus or neonate alters septohippocampal chemistry and induces deficits in hippocampus-related behaviors. The possible reversal of the behavioral changes by manipulating the cholinergic innervations should be the subject of future investigations.     

NGF receptor increase in the olfactory bulb of the rat after early odor deprivation

In the olfactory bulb of normal rats, nerve growth factor (NGF) receptor (NGFR) immunoreactivity was largely confined to the glomerular layer. Unilateral closure of the nostril at postnatal day 2 (P2) increased NGFR immunoreactivity in the sealed bulb at both 19 and 60 days after the operation. The increase in NGFR density, measured by autoradiographic immunohistochemistry, was most dramatic 60 days postocclusion. These findings suggest that a compensatory increase in NGFRs may play a role in the maintenance of bulbar function after the early loss of sensory stimulation.     

Hippocampal cholinergic alterations and related behavioral deficits after early exposure to ethanol

The present study was designed to ascertain septohippocampal cholinergic alterations and their related behavioral deficits after early exposure to ethanol. Mouse pups were exposed to ethanol, 3 g/kg by daily subcutaneous injection on postnatal days 2–14. At age 50 days, the ethanol-exposed mice had significant reductions from control levels in eight-arm maze performance. For example, on the fourth testing day, the number of correct entries in the ethanol group was 21% below control levels (P < 0.05) and the number of trials needed to enter all arms was 48% above control (P < 0.001). It took the ethanol-exposed mice twice the time to reach criterion than it did control (P < 0.01). A 33% increase from control level in muscarinic receptor number (β_max) was found in the treated mice of age 22 days and a 64% increase at age 50 days (P < 0.001). However, no differences between control and treated groups could be detected in the presynaptic component of the cholinergic innervation, choline acetyltransferase activity. The results suggest that early ethanol exposure acts on hippocampal function similarly to phenobarbital, probably via alterations in postsynaptic processes in the septohippocampal cholinergic pathways.     

Recovery of the early cellular changes induced by lead in rat peripheral nerves after withdrawal of the toxin

A morphological and quantitative investigation was carried out in the peripheral nerves of adult rats, which were first exposed to 4% lead acetate in the drinking water and then restored to standard laboratory conditions. After six weeks of continuous lead exposure, the only cells involved were Schwann cells (SC) and endothelial cells (EC). As compared to age-matched controls, the most conspicuous changes observed by light and electron microscopy in these cells included the presence of nuclear inclusion bodies (NIB), cytoplasmic hypertrophy, mitochondrial abnormalities, an increased number of myelin-derived SC intracytoplasmic structures, and vesiculation of myelin sheaths. By counting the number of nuclear profiles containing NIB in semithin sections stained with basic fuchsin and methylene blue, we found that SC from predominantly cutaneous (sural) nerves were less vulnerable to lead than SC from mixed (peroneal) and muscular (tibial) nerves. With respect to EC, however, no significant differences were found among these three nerves. After termination of lead exposure, we observed a gradual decrease of most of the above cellular changes, which finally disappeared at day 30 post-intoxication. However, the number of myelin-related SC cytoplasmic bodies still remained above normal levels at the time of the termination of the experiment (60 days post-intoxication). The nature of the changes induced by lead in peripheral nerve cells as well as the rapid and nearly complete recovery suggest that they reflect a compensatory response to overcome the adverse effects of the lead on cell metabolism.     

1000/铅暴露早期诱导成年大鼠视网膜神经元BACE-1表达上调

Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein (Aβ),and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 (BACE-1) in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer’s disease.Adult rats were intraocularly injected with different doses of lead acetate (10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L).The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer’s disease.     

Differential changes in rat cholinergic parameters subsequent to immunotoxic lesion of the basal forebrain nuclei

The degree of lesion produced by 192 IgG-saporin relative to controls was compared using three independent methods. Microdialyzed acetylcholine (ACh), choline acetyltransferase (ChAT) activity, and the rate of ACh synthesis were compared in the frontal cortex and hippocampus. Microdialysis of rats was performed 1 and 15 weeks post-lesion. In week 16, the rats were sacrificed after an injection of deuterated choline (Ch) for determination of the rate of ACh synthesis. ChAT activity was determined at the same timepoints in a separate set of rats. At 1 week, ChAT activity and microdialyzed ACh showed similar degrees of depletion. At 15 weeks, microdialyzed ACh was significantly lower than the synthesis rate in cortex, but not in hippocampus. A small increase in ChAT activity between 1 and 15 weeks was found in the cortex, but not hippocampus. In the hippocampus, however, the rate of ACh synthesis was significantly greater than ChAT activity. This was true for two doses of immunotoxin; the greater compensation occurring with the lesser lesion. Microdialyzed ACh levels were not different from the other measures in hippocampus. Residual cholinergic terminals in the hippocampus, but not frontal cortex, compensate for a selective cholinergic lesion by increasing the rate of synthesis and may thereby alleviate hippocampus-dependent behavioral deficits.     

Development of cholinergic and GABAergic neurons in the rat medial septum: Effect of target removal in early postnatal development

During normal development of the nervous system, the target fields influence the survival and differentiation of projection neurons, but the factors regulating this interaction remain obscure. In the present study, we have raised the question whether the target region is essential for the postnatal development and maintenance of two different types of central projection neurons, cholinergic and GABAergic septohippocampal cells. In early postnatal rats (P5, P10), the hippocampus was eliminated by unilateral intrahippocampal injections of the excitotoxin N-methyl-D-aspartate. After a long survival time (at P70), we have immunostained serial sections of the septal region with antibodies against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, or the calcium-binding protein parvalbumin (PARV) which is known to be contained in GABAergic septohippocampal neurons. In the medial septum ipsilateral to the lesioned side, about 60% of ChAT-immunoreactive neurons and 62% of PARV-immunoreactive neurons were found in adulthood even after complete elimination of the hippocampus. Some immunoreactive cells appeared heavily shrunken, but electron microscopic analysis revealed ultrastructural characteristics typical for medial septal neurons obtained from controls. Our results indicate that target elimination during development affected both types of projection cells, although only the cholinergic cells are known to be responsive to target-derived factors. J. Comp. Neurol. 379:467–481, 1997. © 1997 Wiley-Liss, Inc.     

Maturational changes in neonatal rat motor system with early postnatal administration of nicotine

Experiments were designed to assess the effects of chronic maximum 'smoking dose' nicotine administration on neonatal motor behavior and neuromuscular structural maturation. Thus, Sprague-Dawley rat neonates received daily subcutaneous injections of nicotine (0.05 mg/kg/day) from postnatal days 1 to 14. Motor behavior (ambulations, stop and go movements) was not affected, but a marked increase in grasping duration resulted, indicating increased neuromuscular maturation. Concomitantly, neuromuscular morphological and dynamic contractile changes were observed in the in situ extensor digitorum longus (EDL) muscle. The EDL muscle showed accelerated morphological dimensions of the endplate at postnatal day 7 and a more rapid twitch at 2 weeks of age. The results suggest that this maximum 'smoking dose' of nicotine induces time-dependent maturational changes in the neuromuscular model.