Over a decade of in-vitro data support a critical role for members of the Homer family of postsynaptic scaffolding proteins in regulating the functional architecture of glutamate synapses. Earlier studies of Homer knockout mice indicated a necessary role for Homer gene products in normal mesocorticolimbic glutamate transmission and behaviours associated therewith. The advent of adeno-associated viral vectors carrying cDNA for, or short hairpin RNA against, specific Homer isoforms enabled the site-directed targeting of Homers to neurons in the brain. This approach has allowed our groups to address developmental issues associated with conventional knockout mice, to confirm active roles for distinct Homer isoforms in regulating glutamate transmission in vivo, as well as in mediating a variety of behavioural processes. This review summarizes the existing data derived from our studies using adeno-associated viral vector-mediated neuronal targeting of Homer in rodents, implicating this family of proteins in drug and alcohol addiction, learning/memory and emotional processing.

The central Extended Amygdala (EAc) is an ensemble of highly interconnected limbic structures of the anterior brain, and forms a cellular continuum including the Bed Nucleus of the Stria Terminalis (BNST), the central nucleus of the Amygdala (CeA) and the Nucleus Accumbens shell (AcbSh). This neural network is a key site for interactions between brain reward and stress systems, and has been implicated in several aspects of drug abuse. In order to increase our understanding of EAc function at the molecular level, we undertook a genome-wide screen (Affymetrix) to identify genes whose expression is enriched in the EAc. We focused on the less-well known BNST-CeA areas of the EAc, and identified 121 genes that exhibit more than 2-fold higher expression level in the EAc compared to whole brain. Among these, forty-three genes have never been described to be expressed in the EAc. We mapped these genes throughout the brain, using non-radioactive in situ hybridization, and identified eight genes with a unique and distinct rostro-caudal expression pattern along AcbSh, BNST and CeA. Q-PCR analysis performed in brain and peripheral organ tissues indicated that, with the exception of one (Spata13), all these genes are predominantly expressed in brain. These genes encode signaling proteins (Adora2, GPR88, Arpp21 and Rem2), a transcription factor (Limh6) or proteins of unknown function (Rik130, Spata13 and Wfs1). The identification of genes with enriched expression expands our knowledge of EAc at a molecular level, and provides useful information to towards genetic manipulations within the EAc.

Human studies are necessary to identify and classify the brain systems predisposing individuals to develop alcohol use disorders and those modified by alcohol, while animal models of alcoholism are essential for a mechanistic understanding of how chronic voluntary alcohol consumption becomes compulsive, how brain systems become damaged, and how damage resolves. Our current knowledge of the neuroscience of alcohol dependence has evolved from the interchange of information gathered from both human alcoholics and animal models of alcoholism. Together, studies in humans and animal models have provided support for the involvement of specific brain structures over the course of alcohol addiction, including the prefrontal cortex, basal ganglia, cerebellum, amygdala, hippocampus, and the hypothalamic-pituitary-adrenal axis.

Previous studies using genetic and lesion approaches have shown that the neuropeptide urocortin 1 (Ucn1) is involved in regulating alcohol consumption. Ucn1 is a corticotropin releasing factor (CRF) -like peptide that binds CRF1 and CRF2 receptors. Perioculomotor urocortin-containing neurons (pIIIu), also known as the non-preganglionic Edinger-Westphal nucleus, are the major source of Ucn1 in the brain and are known to innervate the lateral septum. Thus, the present study tested whether Ucn1 could regulate alcohol consumption through the lateral septum. In a series of experiments Ucn1 or CRF was bilaterally injected at various doses into the lateral septum of male C57BL/6J mice. Consumption of 20% volume/volume ethanol or water was tested immediately after the injections using a modification of a 2-h limited access sweetener-free "drinking-in-the-dark" procedure. Ucn1 significantly suppressed ethanol consumption when administered prior to the third ethanol drinking session (the expression phase of ethanol drinking) at doses as low as 6 pmol. Ethanol intake was differentially sensitive to Ucn1, as equivalent doses of this peptide did not suppress water consumption. In contrast, CRF suppressed both ethanol and water intake at 40 and 60 pmol, but not at lower doses. Repeated administration of Ucn1 during the acquisition of alcohol consumption showed that 40 pmol (but not 2 or 0.1 pmol) significantly attenuated ethanol intake. Repeated administration of Ucn1 also resulted in a decrease of ethanol intake in sham-injected animals, a finding suggesting that the suppressive effect of Ucn1 on ethanol intake can be conditioned. Taken together, these studies confirm the importance of lateral septum innervation by Ucn1 in the regulation of alcohol consumption.

Animal models of prenatal ethanol exposure are necessary to more fully understand the effects of ethanol on the developing embryo/fetus. However, most models employ procedures that may produce additional maternal stress beyond that produced by ethanol alone. We employed a daily limited-access ethanol intake model called Drinking in the Dark (DID) to assess the effects of voluntary maternal binge-like ethanol intake on the developing mouse. Evidence suggests that binge exposure may be particularly harmful to the embryo/fetus, perhaps due to the relatively higher blood ethanol concentrations achieved. Pregnant females had mean daily ethanol intakes ranging from 4.2 to 6.4 g/kg ethanol over gestation, producing blood ethanol concentrations ranging from 115 to 182 mg/dL. This level of ethanol intake produced behavioral alterations among adolescent offspring that disappeared by adulthood, including altered sensitivity to ethanol's hypnotic actions. The DID model may provide a useful tool for studying the effects of prenatal ethanol exposure in mice.

BACKGROUND: Structural magnetic resonance imaging (MRI) reveals widespread brain damage manifest as tissue shrinkage and complementary ventriculomegaly in human alcoholism. For an animal model to parallel the human condition, high alcohol exposure should produce similar radiologically detectable neuropathology. Our previous structural MRI study demonstrated only modest brain dysmorphology of the alcohol-preferring (P) rat with average blood alcohol levels(BALs) of 125 mg/dl achieved with voluntary consumption. Here, we tested the hypothesis that wild-type Wistar rats, exposed to vaporized alcohol ensuring higher BALs than typically achieved with voluntary consumption in rodents, would model MRI findings in the brains of humans with chronic alcoholism. METHODS: The longitudinal effects of vaporized alcohol exposure on the brains of 10 wild-type Wistar rats compared with 10 sibling controls were investigated with structural MRI, conducted before (MRI 1) and after (MRI 2) 16 of alcohol exposure and after an additional 8 weeks at a higher concentration of alcohol (MRI 3). RESULTS: Two rats in the alcohol group died prior to MRI 2. The remaining vapor-exposed rats(n = 8) achieved BALs of 293 mg/dl by MRI 2 and 445 mg/dl by MRI 3. Whereas the controls gained 17% of their body weight from MRI 1 to MRI 3, the alcohol-exposed group lost 6%.MRI, quantified with atlas-based parcellation, revealed a profile of significant ventricular expansion,after alcohol vapor exposure, in 9 contiguous slices, extending from the dorsolateral to ventrolateral ventricles. In particular, from MRI 1 to MRI 2, this ventricular volume expanded by an average of 6.5% in the controls and by 27.1% in the alcohol-exposed rats but only an additional 1.5% in controls and 2.4% in alcohol-exposed rats from MRI 2 to MRI 3. The midsagittal volume of the full anterior-to-posterior extent of the corpus callosum grew between the first 2 MRIs in both groups followed by regression in the alcohol group by MRI 3. Although group differences were statistically significant, among animals there was substantial variability of the effects of alcohol exposure on brain morphology; some animals showed profound effects, whereas others were essentially unaffected. CONCLUSIONS: The ventricular dilatation and callosal shrinkage produced in wild-type rats following involuntary alcohol exposure yielded a modestly successful model of neurodysmorphology phenotypes of human alcoholism. As is the case for the human condition, however, in which some individuals express greater alcoholism-related neuropathology than others, some rats maybe more susceptible than others to extreme alcohol exposure.

Inbred strains are genetically stable across time and laboratories, allowing scientists to accumulate a record of phenotypes, including physiological characteristics and behaviors. To date, the C57/C58 family of inbred mouse strains has been identified as having the highest innate ethanol consumption, but some lineages have rarely or never been surveyed. Thus, the purpose of the present experiment was to measure ethanol preference and intake in 22 inbred mouse strains, some of which have never been tested for ethanol consumption. Male and female mice (A/J, BALB/cByJ, BTBR+T(tf/tf), BUB/BnJ, C57BL/6J, C57BLKS/J, C58/J, CZECH/Ei, DBA/2J, FVB/NJ, I/LnJ, LP/J, MA/MyJ, NOD/LtJ, NON/LtJ, NZB/B1NJ, NZW/LacJ, PERA/Ei, RIIIS/J, SEA/GnJ, SM/J, and 129S1/SvlmJ) were individually housed and given unlimited access in a two-bottle choice procedure to one bottle containing tap water and a second containing increasing concentrations of ethanol (3%, 6%, 10%), 0.2% saccharin, and then increasing concentrations of ethanol (3%, 6%, 10%) plus 0.2% saccharin. Mice were given access to each novel solution for a total of 4 days, with a bottle side change every other day. Consistent with previous studies, C57BL/6J (B6) mice consumed an ethanol dose of \textgreater10g/kg/day whereas DBA/2J (D2) mice consumed \textless2g/kg/day. No strain voluntarily consumed greater doses of ethanol than B6 mice. Although the C58 and C57BLKS strains showed high ethanol consumption levels that were comparable to B6 mice, the BUB and BTBR strains exhibited low ethanol intakes similar to D2 mice. The addition of 0.2% saccharin to the ethanol solutions significantly increased ethanol intake by most strains and altered the strain distribution pattern. Strong positive correlations (rs\textgreater or =0.83) were determined between consumption of the unsweetened versus sweetened ethanol solutions. Consumption of saccharin alone was significantly positively correlated with the sweetened ethanol solutions (rs=0.62-0.81), but the correlation with unsweetened ethanol solutions was considerably lower (rs=0.37-0.45). These results add new strains to the strain mean database that will facilitate the identification of genetic relationships between voluntary ethanol consumption, saccharin preference, and other phenotypes.

Few studies have examined the relationship between naturally rewarding behaviors and ethanol drinking behaviors in mice. Although natural and drug reinforcers activate similar brain circuitry, there is behavioral evidence suggesting food and drug rewards differ in perceived value. The primary goal of the present study was to investigate the relationships between naturally reinforcing stimuli and consumption of ethanol in ethanol preferring C57BL/6J mice. Mouse behaviors were observed after the following environmental manipulations: standard or enhanced environment, accessible or inaccessible wheel, and presence or absence of ethanol. Using a high-resolution volumetric drinking monitor and wheel running monitor, we evaluated whether alternating access to wheel running modified ethanol-related behaviors and whether alternating access to ethanol modified wheel running or subsequent ethanol-related behaviors. We found that ethanol consumption remains stable with alternating periods of wheel running. Wheel running increases in the absence of ethanol and decreases upon reintroduction of ethanol. Upon reintroduction of ethanol, an alcohol deprivation effect was seen. Collectively, the results support theories of hedonic substitution and suggest that female C57BL/6J mice express ethanol seeking and craving under these specific conditions.

Chronic ethanol exposure may induce neuroadaptive responses in N-methyl-d-aspartate (NMDA) receptors, which are thought to underlie a variety of alcohol-related brain disorders. Here, we demonstrate that hyperexcitability triggered by withdrawal from chronic ethanol exposure is associated with increases in both synaptic NMDA receptor expression and activation. Withdrawal from chronic ethanol exposure (75 mM ethanol, 5-9 days) elicited robust and prolonged epileptiform activity in CA1 pyramidal neurons from hippocampal explants, which was absolutely dependent upon NMDA receptor activation but independent of chronic inhibition of protein kinase A (PKA). Analysis of Sr(2+)-supported asynchronous NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) was employed to assess changes in NMDA neurotransmission. After chronic exposure, ethanol withdrawal was associated with an increase in mEPSC amplitude 3.38-fold over that after withdrawal from acute ethanol exposure. Analysis of paired evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid EPSCs and spontaneous mEPSCs indicated that withdrawal after chronic exposure was also associated with a selective increase in action potential evoked but not spontaneous transmitter release probability. Immunoblot analysis revealed significant increases in total NR1, NR2A, and NR2B subunit expression after chronic exposure and unaffected by PKA-inhibition manner. Confocal imaging studies indicate that increased NR1 subunit expression was associated with increased density of NR1 expression on dendrites in parallel with a selective increase in the size of NR1 puncta on dendritic spines. Therefore, neuroadaptation to chronic ethanol exposure in NMDA synaptic transmission is responsible for aberrant network excitability after withdrawal and results from changes in both postsynaptic function as well as presynaptic release.

RATIONALE: The Scheduled High Alcohol Consumption (SHAC) binge drinking model is a simple, partial murine model with which to investigate some of the neurobiological underpinnings of alcoholism. OBJECTIVES: The SHAC model was used to characterize monoamine and amino acid adaptations produced in the nucleus accumbens (NAC) by repeated bouts of high alcohol consumption. METHODS: In vivo microdialysis was conducted in the NAC of C57BL/6J (B6) mice during consumption of water, a 5% alcohol (v/v) solution for the first time (SHAC1) or a 5% alcohol solution for the sixth time (SHAC6). A second set of microdialysis experiments assessed the neurotransmitter response to an alcohol challenge injection (1.5 or 2 g/kg, IP). RESULTS: In both drinking experiments, SHAC1 and SHAC6 mice consumed comparable amounts of alcohol during the 40-min period of alcohol availability (approximately 1.5 g/kg) and total fluid intake was similar between water and SHAC1/6 mice. Despite the similarity in alcohol consumption, alcohol-mediated increases in the extracellular concentration of GABA and serotonin were reduced, but glutamate was increased in the NAC of SHAC6 mice, relative to SHAC1 animals. No differences were observed in extracellular dopamine between SHAC1 and SHAC6 mice during alcohol consumption. After alcohol injection, SHAC6 mice also exhibited sensitized glutamate release, but did not differ from water or SHAC1 animals for any of the other neurotransmitters examined. Brain alcohol concentrations did not differ between groups after injection. CONCLUSIONS: Repeated bouts of high alcohol consumption induce an imbalance between inhibitory and excitatory neurotransmission within the NAC that may drive excessive drinking behavior.

RATIONALE: Recently, a simple procedure was described, drinking in the dark (DID), in which C57BL/6J mice self-administer ethanol to the point of intoxication. The test consists of replacing the water with 20% ethanol in the home cage for 2 or 4 h early during the dark phase of the light/dark cycle. OBJECTIVES: To determine whether the model displays predictive validity with naltrexone, and whether opioid or dopaminergic mechanisms mediate excessive drinking in the model. MATERIALS AND METHODS: Naltrexone or GBR 12909 were administered via intraperitoneal injections immediately before offering ethanol solutions, plain tap water, or 10% sugar water to male C57BL/6J mice, and consumption was monitored over a 2- or 4-h period using the DID procedure. RESULTS: Naltrexone (0.5, 1, or 2 mg/kg) dose dependently decreased ethanol drinking but these same doses had no significant effect on the consumption of plain water or 10% sugar water. GBR 12909 (5, 10, and 20 mg/kg) dose dependently reduced the consumption of ethanol and sugar water but had no effect on plain water drinking. CONCLUSIONS: The DID model demonstrates predictive validity. Both opioid and dopamine signaling are involved in ethanol drinking to intoxication. Different physiological pathways mediate high ethanol drinking as compared to water or sugar water drinking in DID. DID may be a useful screening tool to find new alcoholism medications and to discover genetic and neurobiological mechanisms relevant to the human disorder.

Proteomic investigation of normal and diseased brain states has the potential to reveal novel molecular therapeutic and diagnostic targets for a multitude of pathological central nervous system conditions. Due to their unique properties, integral membrane proteins are likely to play a central role in the aetiology of these disorders. These properties, however, have prevented comprehensive analysis of this important class of proteins. Recent advances in sample preparation and proteomic quantification platforms, specifically focused on recovery and enrichment of integral membrane proteins, are discussed.

BACKGROUND: Cirrhosis is the result of chronic liver disease that causes scarring and dysfunction of the liver. The disease is a common concomitant condition resulting from sustained exposure to alcohol. Heavy alcohol use results in brain damage that is generally more severe in cirrhotic compared with noncirrhotic alcoholics. We examined, at the cellular level, gene expression in the frontal cortex of cirrhotic alcoholics. METHODS: Gene expression profiles were compared between cirrhotic and noncirrhotic alcoholics using approximately 47,000 element cDNA microarrays. RESULTS: Widespread differences in transcriptome patterns were observed in cirrhotic compared with noncirrhotic alcoholics and these differences in gene expression accurately distinguished cirrhotic from noncirrhotic alcoholics. Functionally related groups of genes were identified that are involved in cell adhesion, mitochondrial function, synaptic transmission, apoptosis, and cell proliferation. Both astrocytes and neuronal cells were affected at the transcriptional level. The regulated genes are involved in neurite growth, neuronal cell adhesion, synaptic vesicle release, and postsynaptic neurotransmission. CONCLUSIONS: These changes in the transcriptome likely contribute to the more severe brain dysfunction in cirrhotic alcoholics.

The formation of synaptic connections with target cells and maintenance of axons are highly regulated and crucial for neuronal function. The atypical cadherin and G-protein-coupled receptor Flamingo and its orthologs in amphibians and mammals have been shown to regulate cell polarity, dendritic and axonal growth, and neural tube closure. However, the role of Flamingo in synapse formation and function and in axonal health remains poorly understood. Here we show that fmi mutations cause a significant increase in the number of ectopic synapses on muscles and result in the formation of novel en passant synapses along axons, and unique presynaptic varicosities, including active zones, within axons. The fmi mutations also cause defective synaptic responses in a small subset of muscles, an age-dependent loss of muscle innervation and a drastic degeneration of axons in 3rd instar larvae without an apparent loss of neurons. Neuronal expression of Flamingo rescues all of these synaptic and axonal defects and larval lethality. Based on these observations, we propose that Flamingo is required in neurons for synaptic target selection, synaptogenesis, the survival of axons and synapses, and adult viability. These findings shed new light on a possible role for Flamingo in progressive neurodegenerative diseases.

We describe a comprehensive translational approach for identifying candidate genes for alcoholism. The approach relies on the cross-matching of animal model brain gene expression data with human genetic linkage data, as well as human tissue data and biological roles data, an approach termed convergent functional genomics. An analysis of three animal model paradigms, based on inbred alcohol-preferring (iP) and alcohol-non-preferring (iNP) rats, and their response to treatments with alcohol, was used. A comprehensive analysis of microarray gene expression data from five key brain regions (frontal cortex, amygdala, caudate-putamen, nucleus accumbens and hippocampus) was carried out. The Bayesian-like integration of multiple independent lines of evidence, each by itself lacking sufficient discriminatory power, led to the identification of high probability candidate genes, pathways and mechanisms for alcoholism. These data reveal that alcohol has pleiotropic effects on multiple systems, which may explain the diverse neuropsychiatric and medical pathology in alcoholism. Some of the pathways identified suggest avenues for pharmacotherapy of alcoholism with existing agents, such as angiotensin-converting enzyme (ACE) inhibitors. Experiments we carried out in alcohol-preferring rats with an ACE inhibitor show a marked modulation of alcohol intake. Other pathways are new potential targets for drug development. The emergent overall picture is that physical and physiological robustness may permit alcohol-preferring individuals to withstand the aversive effects of alcohol. In conjunction with a higher reactivity to its rewarding effects, they may able to ingest enough of this nonspecific drug for a strong hedonic and addictive effect to occur.

Currently, measuring ethanol behaviors in flies depends on expensive image analysis software or time intensive experimental observation. We have designed an automated system for the collection and analysis of locomotor behavior data, using the IEEE 1394 acquisition program dvgrab, the image toolkit ImageMagick and the programming language Perl. In the proposed method, flies are placed in a clear container and a computer-controlled camera takes pictures at regular intervals. Digital subtraction removes the background and non-moving flies, leaving white pixels where movement has occurred. These pixels are tallied, giving a value that corresponds to the number of animals that have moved between images. Perl scripts automate these processes, allowing compatibility with high-throughput genetic screens. Four experiments demonstrate the utility of this method, the first showing heat-induced locomotor changes, the second showing tolerance to ethanol in a climbing assay, the third showing tolerance to ethanol by scoring the recovery of individual flies, and the fourth showing a mouse's preference for a novel object. Our lab will use this method to conduct a genetic screen for ethanol-induced hyperactivity and sedation, however, it could also be used to analyze locomotor behavior of any organism.

Conditional gene knockout represents an extremely powerful approach to study the function of single genes in the nervous system. The Cre-LoxP system is the most advanced technology for spatial and temporal control of genetic inactivation, and there is rapid progress using this methodology in neuroscience research. In this approach, mice with LoxP sites flanking the gene of interest (floxed mice) are bred with transgenic mice expressing Cre recombinase under the control of a selected promoter (Cre mice). This promoter is critical in that it determines the time and site of Cre expression. Cre enzyme, in turn, recombines the floxed gene and produces gene knockout. Here we review Cre mouse lines that have been developed to target either the entire brain, selected brain areas, or specific neuronal populations. We then summarize phenotypic consequences of conditional gene targeting in the brain for more than 40 genes, as reported to date. For many broadly expressed genes, brain-restricted knockout has overcome lethality of conventional knockout (KO) and has highlighted a specific role of the encoded protein in some aspect of brain function. In the case of neural genes, data from null mutants in specific brain sites or neurons has refined our understanding of the role of individual molecules that regulate complex behaviors or synaptic plasticity within neural circuits. Among the many developing functional genomic approaches, conditional gene targeting in the mouse has become an excellent tool to elucidate the function of the approximately 5000 known or unknown genes that operate in the nervous system.

Monoamine transporters play a key role in neuronal signaling by mediating reuptake of neurotransmitters from the synapse. The function of the dopamine transporter (DAT), an important member of this family of transporters, is regulated by multiple signaling mechanisms, which result in altered cell surface trafficking of DAT. Protein-protein interactions are likely critical for this mode of transporter regulation. In this study, we identified proteins associated with DAT by immunoprecipitation (IP) followed by mass spectrometry. We identified 20 proteins with diverse cellular functions that can be classified as trafficking proteins, cytoskeletal proteins, ion channels and extracellular matrix-associated proteins. DAT was found to associate with the voltage-gated potassium channel Kv2.1 and synapsin Ib, a protein involved in regulating neurotransmitter release. An in silico analysis provided evidence for common transcriptional regulation of the DAT proteome genes. In summary, this study identified a network of proteins that are primary candidates for functional regulation of the DAT, an important player in mechanisms of mental disorders and drug addiction.

Models of dependence-induced increases in ethanol self-administration will be critical in increasing our understanding of the processes of addiction and relapse, underlying mechanisms, and potential therapeutics. One system that has received considerable attention recently is the CRF1 system that may mediate the link between anxiety states and relapse drinking. C57BL/6J mice were trained to lever press for ethanol, were made dependent and then were allowed to self-administer ethanol following a period of abstinence. The effect of the CRF1 antagonist, antalarmin, was examined on this abstinence-induced self-administration in a separate group of mice. Finally, dependence-induced changes in ethanol self-administration were examined in CRF1 knockout and wild type mice. The results indicated that ethanol self-administration was increased following the induction of dependence, but only after a period of abstinence. This increase in ethanol self-administration was blocked by antalarmin. Furthermore, CRF1 knockout mice did not display this increased ethanol self-administration following dependence and abstinence. These studies, using both a pharmacological and genetic approach, support a critical role for the CRF1 system in ethanol self-administration following dependence. In addition, a model is presented that may be useful for studies examining underlying mechanisms of the ethanol addiction process as well as for testing potential therapeutics.

Wernicke's encephalopathy (WE) is characterized by lesions in thalamus, hypothalamus (including mammillary nuclei), and inferior colliculi, results in serious disabilities, has an etiology of thiamine deficiency, is treatable with thiamine, and occurs most commonly with alcoholism. Despite decades of study, whether alcohol exposure exacerbates the neuropathology or retards its resolution remains controversial. To examine patterns of brain damage and recovery resulting from thiamine deprivation with and without alcohol exposure, we conducted in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) at 3 T in alcohol-preferring (P) rats, which had voluntarily consumed large amounts of alcohol before thiamine manipulation. A total of 18 adult male P rats (nine alcohol-exposed) received a thiamine-deficient diet for 2 weeks: 10 (five alcohol-exposed) received intraperitoneal (i.p.) pyrithiamine (PT) and eight (four alcohol-exposed) received i.p. thiamine supplementation. Neurological signs developed by day 14. Rats were scanned before thiamine depletion and 18 and 35 days after thiamine repletion. Two-dimensional J-resolved MRS single-voxel spectra with water reference were collected in a voxel subtending the thalamus; metabolite quantification was corrected for voxel tissue content. MRI identified significant enlargement of dorsal ventricles and increase in signal intensities in thalamus, inferior colliculi, and mammillary nuclei of PT compared with thiamine-treated (TT) groups from MRI 1-2, followed by significant normalization from MRI 2-3 in thalamus and colliculi, but not mammillary nuclei and lateral ventricles. Voxel-by-voxel analysis revealed additional hyperintense signal clusters in the dorsal and ventral hippocampus and enlargement of the fourth ventricle. MRS showed a significant decline and then partial recovery in thalamic N-acetylaspartate, a marker of neuronal integrity, in PT compared with TT rats, with no change detected in creatine, choline, or glutamate. PT rats with prior alcohol exposure exhibited attenuated recovery in the thalamus and arrested growth of the corpus callosum; further, two of the five alcohol-exposed PT rats died prematurely. Parenchymal and ventricular changes with thiamine manipulation concur with human radiological signs of WE. The enduring macrostructural and neurochemical abnormalities involving critical nodes of Papez circuit carry liabilities for development of amnesia and incomplete recovery from other cognitive and motor functions subserved by the affected neural systems.