Non-specific effects of the putative cholinergic neurotoxin ethylcholine mustard aziridinium ion in the rat brain examined by autoradiography, immunocytochemistry and gel electrophoresis

Autoradiographic localisation of [3H]-ethylcholine mustard aziridinium ion (ECMA) after microinjection into the rat striatum has revealed intracellular sequestration of the toxin by glial and endothelial cells; fewer neuronal cells were labelled. Intrastriatal injection of 200 pmol ECMA caused severe cavitation of the tissue, extensive gliosis and permanent damage to myelinated structures, as revealed by immunocytochemical detection of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). These non-specific effects are in addition to ECMA's irreversible action on the choline carrier associated with cholinergic neurons, and only marginally protected by concomitant administration of the reversible choline transport inhibitor hemicholinium-3. They may instead be attributed to the powerful alkylating action that ECMA has on tissue proteins, as shown by fluorography of synaptosomal proteins treated with [3H]ECMA and separated by SDS-PAGE.     

乙醇对大鼠脑内5-HT能神经体系的影响

目的 观察乙醇处理大鼠脑内色氨酸羟化酶(TPH)、5-羟色胺(5-HT)和5-羟色胺转运体(SERT)的表达改变,判断乙醇对脑内5-HT能神经体系的影响.方法 以20%乙醇代替饮水饲养30只Wistar大鼠6个月;利用免疫组织化学、免疫印迹及流式细胞术等方法,分析乙醇处理大鼠有关脑区5-HT能神经体系相关指标的改变.结果 1.免疫组织化学法可见,乙醇处理组大鼠脑内中缝背核TPH、5-HT免疫反应阳性神经元数量少于对照组(P＜0.01);TPH免疫阳性神经元直径小于对照组(P＜0.01);相关脑区TPH、5-HT和SERT免疫反应灰度值比对照组增高(P<0.05).2.流式细胞术检测可见,乙醇处理组大鼠TPH、5-HT和SERT的表达量低于对照组(P<0.05).3.免疫印迹法检测可见,乙醇处理组大鼠SERT和TPH与β-actin相对吸光度比值均小于相应对照组(P<0.05).结论 乙醇降低脑内TPH、5-HT和SERT的表达,可能改变脑内5-HT能神经体系的功能活动.     

Glucocorticoid hypersecretion following intracerebroventricular injection of ethylcholine mustard aziridinium ion in rats

To investigate whether cholinergic hypofunctions in the brain influence hypothalamic-pituitary-adrenal activity, we examined the effects of cholinergic neurotoxin ethylcholine mustard aziridinium ion on basal and stress-induced levels of corticosterone in rats. Blood sampling from rats following intracerebroventricular injection of saline (5 microl, as a control) or this neurotoxin (5 nmol/5 microl) was performed over a day in one series, and was taken before, during and after an immobilization stress exposure in another series. Plasma levels of corticosterone and adrenocorticotropin were determined by the radioimmunoassay. The basal levels of plasma corticosterone and adrenocorticotropin over a day were significantly higher in the neurotoxin-treated rats, compared with the control rats (corticosterone, P<0.001; adrenocorticotropin, P<0.05). Further, relative adrenal gland weight of the neurotoxin-treated rats was significantly greater than that of the control rats (P<0.05). However, responses in plasma corticosterone level caused by the immobilization stress in the neurotoxin-treated rats were not different from those in the control rats.The present study demonstrated that damage to the cholinergic neurons in the brain increased hypothalamic-pituitary-adrenal activity over a day, probably due to freedom from inhibitory influences of the hippocampal cholinergic system, but that this damage did not influence stress-induced changes in plasma glucocorticoid level.     

Effect of ethylcholine mustard aziridinium (AF64A) and of the monoamine oxidase-B-inhibitor L-deprenyl on the morphology of the rat hippocampus.

The effect of intracerebroventricular (ICV) administration of ethylcholine mustard aziridinium (AF64A) and of the monoamine oxidase (MAO)-B inhibitor L-deprenyl on MAO-A and MAO-B activities and on the morphology of the rat neostriatum and hippocampus were studied. The ICV administration of AF64A was without effect on MAO-A and MAO-B in the neostriatum and caused an increase of MAO-B but not of MAO-A in the hippocampus. No changes in neostriatal micro-anatomy were noticeable in AF64A-injected rats, whereas the neurotoxin caused an impairment in hippocampal micro-anatomy consisting in the loss of nerve cells and of silver-gold impregnated fibres in the CA-1--CA-3 fields. The treatment of AF64A-injected animals with doses of L-deprenyl from 11.17 microM/kg/day significantly reduced MAO-B activity in the hippocampus and improved the morphology of the hippocampus formation. L-deprenyl was without effect on MAO-A activity both in the neostriatum and in the hippocampus, as well as on neostriatal MAO-B activity and morphology. The possibility that MAO-B inhibition may represent a principle for the treatment of age-related physiological and pathological changes characterized by increased MAO-B activity is discussed.     

Effect of aging and acetyl-L-carnitine on energetic and cholinergic metabolism in rat brain regions

The effect of aging and subchronic treatment with acetyl-L-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl-L-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.     

Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro

The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7+/-9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15-17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5-80microM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life-death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose- and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86microM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13h after ethylcholine aziridinium application.The present data indicate that in a model of delayed apoptotic neurodegeneration as induced by ethylcholine aziridinium neuronal cell death in vitro and in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of nitric oxide production and absence of inducible nitric oxide synthase expression. In contrast, evidence for a neuroprotective role of nitric oxide was obtained in vivo as indicated by the exaggeration of the cholinergic lesion after unspecific nitric oxide synthase inhibition by N-nitro-L-arginine methylester.     

Effect of astrocyte-derived factors on ischemic brain edema induced by rat MCA occlusion

To clarify whether astrocyte-derived factors may protect cerebral tissue from ischemia, we examined brain edema, demyelination and astrocyte proliferation in brains with focal ischemia and treated with astrocyte-cultured medium. We occluded the left middle cerebral artery of rats and implanted the Osmotic Minipump, which continuously infused the glial-cultured medium or control medium into the left lateral ventricle. Animals were sacrificed at 3 days or 7 days after occlusion. Brains of both groups were compared by several markers, i.e. extravasation of Evans blue, demyelination by Woelcke's staining and glial proliferation by GFAP staining. We found the astrocyte-cultured medium reduced leakage of Evans blue-plasma protein complex from ischemic lesions and reduced the size of demyelinated lesions. However, the degree of astrocyte proliferation was similar in both groups. From these data, we speculate that humoral factors derived from cultured astrocytes lessened the brain edema by modifying the blood-brain barrier. These factors might also induce proliferation of the microglia, and may protect the neurons from secondary injury by oxygen-free radicals.     

Maternal separation alters serotonergic transporter densities and serotonergic 1A receptors in rat brain

RATIONALE: The basic mechanisms underlying the association between early life maternal separation and adulthood psychiatric disorders are largely unknown. One possible candidate is the central serotonergic system, which is also abnormal in psychiatric illnesses. Neuroadaptational changes in serotonergic transporter and serotonergic 1A receptors may underlie links between early life stress and adulthood psychiatric disorders. OBJECTIVE: The aim of this study was to investigate the consequences of a rat model of maternal separation on serotonergic transporter and serotonergic 1A receptor densities and function in adult rat forebrain. METHODS: Rat pups were separated from dams from postnatal day 2 to postnatal day 14, each day, for zero time, 15 min and 180 min to determine the time-course of effects. A non-handled group was added to control for the effects of handling by an experimenter compared with the animal facility-reared group. Quantitative [(125)I]3beta-(4-iodophenyl)tropan-2beta-carboxylic acid methyl ester and [(125)I]-mPPI autoradiography was used to determine serotonergic transporter and serotonergic 1A densities, respectively. Adult rats were challenged with saline or serotonergic 1A agonist (+) 8-hydroxy-2-(di-n-propylamino)tetralin, 0.4 mg/kg, s.c.) and plasma adrenocorticotropic hormone and corticosterone were determined. RESULTS: serotonergic transporter and serotonergic 1A densities were significantly lower in the non-handled group in the paraventricular, arcuate, dorsomedial and ventromedial nuclei of the hypothalamus. The non-handled group also displayed lower serotonergic transporter and serotonergic 1A densities in the basolateral anterior, basolateral ventral and basomedial amygdaloid nuclei. Serotonergic transporter densities were also decreased in the CA3 area of the hippocampus in the non-handled group. In contrast, the maternal separation 15 min group displayed the highest serotonergic transporter and serotonergic 1A densities in the basomedial nucleus of amygdala, basolateral anterior nucleus of amygdala, basolateral ventral nucleus of amygdala and basomedial nucleus of amygdala amygdaloid nuclei. CONCLUSIONS: Early life maternal separation and the extent of handling can alter adult brain serotonergic transporter and serotonergic 1A levels and function in the forebrain. Alterations in these serotonergic systems by early rearing conditions might increase vulnerability for behavioral disorders in adulthood.     

Natriuresis induced by cholinergic stimulation of the locus coeruleus in the rat

Carbachol injected into the locus coeruleus (LC) induced a dose-dependent natriuresis in the rat. This natriuresis was maintained above control levels during the 120 min of urine sampling. Seizures and arterial blood pressure increase were also observed but they disappeared within 20 min after carbachol injection. Natriuresis was not obtained with either injections of carbachol outside the LC or with hypertonic solutions injected into the LC. Injection of atropine into the LC blocked the natriuresis induced by carbachol. In summary, our data show that carbachol induces natriuresis by an action on muscarinic receptors located in the LC region.     

Effect of dopamine-depleting brain lesions in rat pups: role of striatal serotonergic neurons in behavior

Previous results from our laboratory have demonstrated that 3-day-old rats given dopamine (DA)-depleting brain lesions are spared the severe behavioral dysfunctions seen after comparable brain damage in adults. This behavioral sparing is accompanied by a sprouting of serotonin (5-HT)-containing neurons in the striatum. The present results extend these observations by demonstrating that rats given the brain lesions as 15- or 27-day-olds continue to suckle, wean, and grow into adulthood without exhibiting any obvious behavioral dysfunctions, yet striatal 5-HT levels do not increase. Moreover, combined destruction of DA- and 5-HT-containing neurons in 3-day-old rat pups also produced no obvious behavioral dysfunctions. These and other results indicate that increases in striatal 5-HT are not necessary for the behavioral sparing observed after DA-depleting brain lesions in neonatal rats.     

Effect of cholinergic denervation on hepatic fibrosis induced by carbon tetrachloride in rats

Various factors involved in the development of liver fibrosis, including hepatic stellate cells (HSCs), cholinergic nervous activity and fibrogenetic cytokines. The present study aims to investigate the role of cholinergic regulation in the promoting of liver fibrogenesis relating to bone morphogenetic protein-6 (BMP-6) and/or transforming growth factor-beta1 (TGFbeta1). We treated carbon tetrachloride (CCl(4)) into rats for eight weeks to induce liver fibrosis and arranged these rats for cholinergic denervation, hepatic branch vagotomy or atropine administration. Acetylcholinesterase (AChE) staining showed the distribution of cholinergic nerve around fibrosis scaring septa. The immunohistochemical staining for alpha smooth muscle actin (alphaSMA) indicated the less HSCs in CCl(4) treated rat liver with cholinergic denervation as compared to the sham-operated CCl(4) treated rats. It seems that cholinergic nerve not only innervates around the fibrosis area but also promotes HSCs. We also detected TGFbeta1 and BMP-6 expressions using RT-PCR and immunohistochemistry. The obtained results show that cholinergic denerveration decreases BMP-6 and TGF-beta1 expressions in CCl(4) induced liver fibrosis of rats. In conclusion, cholinergic nerve may influence HSCs in addition to the lowering of BMP-6 and TGF-beta1 gene expressions to modify liver fibrosis.     

In vitro and in vivo modulation of cholinergic muscarinic receptors in rat lymphocytes and brain by cholinergic agents

A binding site for 3H-quinuclidinyl benzylate (QNB) has been identified in rat lymphocytes which has the characteristics of a cholinergic muscarinic receptor (Costa, L. G., Kaylor, G. & Murphy, S. D. (1988). Muscarinic cholinergic binding sites on rat lymphocytes. Immunopharmacology, 16, 139-149.) Here we show that prolonged exposures to cholinergic compounds in vitro and in vivo modulate muscarinic receptor binding in lymphocytes as well as in brain tissue. Exposure of rat splenic lymphocytes in vitro to oxotremorine caused a time- and concentration-dependent decrease in the density of 3H-QNB binding sites. This decrease occurred only when incubation with oxotremorine was carried out at 37 degrees C and not at 0-4 degrees C, suggesting that it was not an artifact due to residual, unwashed, oxotremorine. The effect of oxotremorine was mimicked by two other cholinergic agonists, acetylcholine and carbachol, and was antagonized by atropine, which, when present alone, caused an increase in 3H-QNB binding. In vivo exposures to oxotremorine or atropine (both at 20 mg/kg/day for 14 days via an ALZA minipump) caused a significant decrease (20-30%) and increase (13-30%), respectively, of 3H-QNB binding in various brain areas as well as circulating lymphocytes. Repeated administrations of the organophosphorus insecticide disulfoton (2 mg/kg/day for 14 days, i.p.) caused significant reductions (59-88%) of acetylcholinesterase activity in brain, lymphocytes, plasma and red blood cells, as well as a 23-39% decrease of 3H-QNB binding in brain areas and circulating lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of stimulation of brain serotonergic system on mouse-killing behavior in rats

Mouse-killing behavior was induced in male Wistar rats due to 6 weeks isolation. The selective stimulation of the dorsal raphe nucleus markedly reduced muricide behavior. On the other hand rats with electrodes situated outside of the dorsal raphe (within the substantia grisea centralis) showed no changes in muricide behavior when compared with control (non-stimulated) animals. Pharmacological stimulation of whole serotonergic brain neurons by quipazine, an agonist of serotonin receptors, and compound CGP 6085-A, a selective inhibitor of serotonin uptake, strongly decreased mouse-killing behavior. Our data indicates that serotonergic neurons inhibit predatory aggression in rats.     

Nitric-oxide-induced cGMP synthesis in cholinergic neurons in the rat brain

Nitric oxide (NO)-mediated cGMP synthesis is localized throughout the rat brain in close proximity to the NO-synthase-containing structures. However, characterization of the cGMP synthesizing structures in terms of co-localization with the classical neurotransmitter systems has not yet been reported. Here we present evidence, using double immunostaining for cGMP and the vesicular acetylcholine transporter, that virtually all of the cholinergic fibers in the cerebral cortex and the majority of the cholinergic fibers in the basal ganglia accumulate cGMP in response to a NO donor. In these areas, only few cGMP-containing fibers were observed not to be part of the cholinergic system. Co-localization between cGMP and the vesicular acetylcholine transporter was only observed to a minor degree in the ventral forebrain, the hippocampus, the reticular thalamic nucleus, and the nucleus ambiguus. No association of cGMP synthesis with the cholinergic system was observed to a similar extent in other brain areas. These results, in combination with literature data on the distribution of cholinergic receptors in the rat brain, suggest that NO has an anterograde and/or retrograde signaling function on subsets of cholinergic neurons.     

Behavioral characteristics of rat lines selected for differential hypothermic responses to cholinergic or serotonergic agonists

The present review will describe the formation of two pharmacologically selected lines of rats, their behavioral phenotypes, their responses to select drugs, their possible neurochemical correlates, and their use to detect the therapeutic potential of antidepressant drugs. The Flinders Line rats were established at Flinders University in Australia by selectively breeding for differential responses to an anticholinesterase agent from outbred Sprague-Dawley (SD) rats; the Flinders Sensitive Line (FSL) rats were more sensitive to the hypothermic and behavioral suppressing effects of this agent than the Flinders Resistant Line (FRL) rats. The 8-OH-DPAT line rats were established at the University of North Carolina at Chapel Hill by selectively breeding for differential hypothermic responses to the 5-HT_1A receptor agonist, 8-OH-DPAT; the high DPAT sensitive (HDS) line rats were more sensitive to the hypothermic effects of 8-OH-DPAT than the low DPAT sensitive (LDS) line rats. Studies of these two pairs of lines have indicated that the FSL and HDS rats are both more susceptible to stress-induced behavioral disturbances. Their usefulness in detecting potential antidepressant drugs and the relationship between mood disorders and drug abuse will be discussed.     

Neocortical activation: modulation by multiple pathways acting on central cholinergic and serotonergic systems

 The present experiments examined the central systems which mediate the electrocorticographic activation induced by electrical stimulation of forebrain and midbrain areas known to play a role in cerebral activation. In urethane-anesthetized rats, 100-Hz electrical stimulation of the amygdaloid cmplex or dorsal raphe changed neocortical electrocorticographic activity from large irregular slow activity (LISA; 1–6 Hz, up to 2 mV) to low voltage fast activity (LVFA; less than 0.5 mV, including frequencies above 10 Hz). The LVFA during amygdala stimulation, but not that during dorsal raphe stimulation, was completely abolished and replaced by LISA after administration of the anti-muscarinic agent scopolamine (5 mg/kg, i.p.), confirming previous work suggesting that neocortical LVFA is maintained by two distinct neurochemical inputs to the cortex that can be dissociated using anti-muscarinic drugs. Electrical stimulation of the locus coeruleus area or of the superior colliculus also suppressed LISA and induced LVFA. The LVFA during stimulation of the locus coeruleus area was abolished by the anti-muscarinic drug atropine (50 mg/kg, i.p.), whereas the LVFA during superior colliculus stimulation was atropine-resistant but could be abolished by the serotonergic antagonists methiothepin (5 mg/kg, i.p.) or ketanserin (5 mg/kg, i.p.). Stimulation of 44% of electrode sites in the orbitofrontal cortex produced neocortical LVFA which was reduced by atropine and completely abolished by additional administration of methiothepin. Stimulation of the entorhinal or cingulate cortex was ineffective in producing LVFA and often resulted in the appearance of epileptiform activity. Single-pulse electrical stimulation of those sites that effectively induced atropine-sensitive LVFA (amygdala, locus coeruleus area) produced excitation of over 65% of those extracellularly recorded basal forebrain neurons that fired at higher rates during the presence of neocortical LVFA relative to LISA. About 80% of basal forebrain cells that were more active during LISA relative to LVFA were inhibited by single-pulse stimulation of the amygdala or locus coeruleus area. The present results suggest that widely distributed neuronal systems can produce electrocorticographic activation by acting through mechanisms sensitive to anti-muscarinic or anti-serotonergic drugs, suggeting that these activating influences involve the release of acetylcholine and/or serotonin. Those neural systems that produce atropine-sensitive (i.e., putative cholinergic) LVFA, believed to be dependent on the cholinergic innervation of the cortex arising in the basal forebrain, may produce electrocorticographic activation by exciting basal forebrain cells that appear to contribute to neocortical LVFA, while concurrently inhibiting cells that may contribute to LISA. Received: 28 May 1996 / Accepted: 17 January 1997     

人参皂苷Rb1对缺氧诱导N9细胞应激活化反应的影响

目的 探讨人参皂苷Rb1对缺氧诱导的小胶质细胞活化的的影响. 方法 通过人参皂苷Rb1对缺氧状态下N9细胞的干预,检测细胞形态、增殖活力改变.采用ELISA法、荧光探针DAF-FM DA、Griess Reagent法检测细胞TNF-α、O-2产量以及NO含量改变的影响.借助化学发光法、免疫荧光法分别检测各组细胞线粒体膜电位、细胞色素C含量. 结果 无论是预防性给药还是治疗性给药,人参皂苷Rb1能明显降低缺氧诱导活化的N9细胞NO、O-2以及TNF-α产量,抑制线粒体膜电位的降低,缓解细胞内细胞色素C含量的改变程度. 结论 人参皂苷Rb1均能在一定程度上下调由于缺氧活化导致神经毒性因子的高表达,稳定细胞线粒体的结构和功能,抑制缺氧诱导的N9细胞活化.     

Evaluation of cholinergic markers in Alzheimer's disease and in a model of cholinergic deficit

Cognitive deficits in neuropsychiatric disorders, such as Alzheimer's disease (AD), have been closely related to cholinergic deficits. We have compared different markers of cholinergic function to assess the best biomarker of cognitive deficits associated to cholinergic hypoactivity. In post-mortem frontal cortex from AD patients, acetylcholine (ACh) levels, cholinacetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were all reduced compared to controls. Both ChAT and AChE activity showed a significant correlation with cognitive deficits. In the frontal cortex of rats with a selective cholinergic lesion, all cholinergic parameters measured (ACh levels, ChAT and AChE activities, "in vitro" and "in vivo" basal ACh release) were significantly reduced. AChE activity was associated to ChAT activity, and even more, to "in vivo" and "in vitro" basal ACh release. Quantification of AChE activity is performed by an easy and cheap method and therefore, these results suggest that determination of AChE activity may be used as an effective first step method to evaluate cholinergic deficits.     

Ethanol ingestion in the rat induced by rewarding brain stimulation

An attempt was made to produce ethanol dependence in the rat by rewarding the ingestion of ethanol with reinforcing stimulation of the brain. Each rat was trained to lick a drinkometer tube containing an ethanol solution mixed either in water or sucrose in order to obtain rewarding electrical stimulation which was delivered through a bipolar electrode implanted stereotaxically in the lateral hypothalamus in the region of the medial forebrain bundle (MFB). During a one-hour test session, a volume of ethanol sufficient to produce pharmacological effects was reliably consumed. Following each test session, behavioral signs were noted and a blood-ethanol determination was performed. Reinforcing stimulation failed to elicit the consumption of ethanol mixed either in water or sucrose in a concentration greater than that which was above the normal preference-aversion threshold. Further, an 8-day ethanol-water preference test sequence following the last stimulation session revealed that ethanol intake did not increase significantly above that of the non-stimulated control group. These findings indicate that electrical stimulation of the lateral hypothalamus does not evoke a permanent shift in fluid preference or other signs of ethanol dependence.