DARPP-32 and NCS-1 Expression is not Altered in Brains of Rats Treated with Typical or Atypical Antipsychotics

Dopamine-mediated neurotransmission imbalances are associated with several psychiatry illnesses, such as schizophrenia. Recently it was demonstrated that two proteins involved in dopamine signaling are altered in prefrontal cortex (PFC) of schizophrenic patients. DARPP-32 is a key downstream effector of intracellular signaling pathway and is downregulated in PFC of schizophrenic subjects. NCS-1 is a neuronal calcium sensor that can inhibit dopamine receptor D₂ internalization and is upregulated in PFC of schizophrenic subjects. It is well known that dopamine D₂ receptor is the main target of antipsychotic. Therefore, our purpose was to study if chronic treatment with typical or atypical antipsychotics induced alterations in DARPP-32 and NCS-1 expression in five brain regions: prefrontal cortex, hippocampus, striatum, cortex and cerebellum. We did not find any changes in DARPP-32 and NCS-1 protein expression in any brain region investigated.     

Motor Skill Learning Is Associated with Phase-Dependent Modifications in the Striatal cAMP/PKA/DARPP-32 Signaling Pathway in Rodents

Abundant evidence points to a key role of dopamine in motor skill learning, although the underlying cellular and molecular mechanisms are still poorly understood. Here, we used a skilled-reaching paradigm to first examine changes in the expression of the plasticity-related gene Arc to map activity in cortico-striatal circuitry during different phases of motor skill learning in young animals. In the early phase, Arc mRNA was significantly induced in the medial prefrontal cortex (mPFC), cingulate cortex, primary motor cortex, and striatum. In the late phase, expression of Arc did not change in most regions, except in the mPFC and dorsal striatum. In the second series of experiments, we studied the learning-induced changes in the phosphorylation state of dopamine and cAMP-regulated phosphoprotein, 32k Da (DARPP-32). Western blot analysis of the phosphorylation state of DARPP-32 and its downstream target cAMP response element-binding protein (CREB) in the striatum revealed that the early, but not late, phase of motor skill learning was associated with increased levels of phospho-Thr34-DARPP-32 and phospho-Ser133-CREB. Finally, we used the DARPP-32 knock-in mice with a point mutation in the Thr34 regulatory site (i.e., protein kinase A site) to test the significance of this pathway in motor skill learning. In accordance with our hypothesis, inhibition of DARPP-32 activity at the Thr34 regulatory site strongly attenuated the motor learning rate and skilled reaching performance of mice. These findings suggest that the cAMP/PKA/DARPP-32 signaling pathway is critically involved in the acquisition of novel motor skills, and also demonstrate a dynamic shift in the contribution of cortico-striatal circuitry during different phases of motor skill learning.     

The Cdk5 inhibitor Roscovitine increases LTP induction in corticostriatal synapses

In corticostriatal synapses, LTD (long-term depression) and LTP (long-term potentiation) are modulated by the activation of DA (dopamine) receptors, with LTD being the most common type of long-term plasticity induced using the standard stimulation protocols. In particular, activation of the D1 signaling pathway increases cAMP/PKA (protein kinase A) phosphorylation activity and promotes an increase in the amplitude of glutamatergic corticostriatal synapses. However, if the Cdk5 (cyclin-dependent kinase 5) phosphorylates the DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) at Thr⁷⁵, DARPP-32 becomes a strong inhibitor of PKA activity. Roscovitine is a potent Cdk5 inhibitor; it has been previously shown that acute application of Roscovitine increases striatal transmission via Cdk5/DARPP-32. Since DARPP-32 controls long-term plasticity in the striatum, we wondered whether switching off CdK5 activity with Roscovitine contributes to the induction of LTP in corticostriatal synapses. For this purpose, excitatory population spikes and whole cell EPSC (excitatory postsynaptic currents) were recorded in striatal slices from C57/BL6 mice. Experiments were carried out in the presence of Roscovitine (20 μM) in the recording bath. Roscovitine increased the amplitude of excitatory population spikes and the percentage of population spikes that exhibited LTP after HFS (high-frequency stimulation; 100Hz). Results obtained showed that the mechanisms responsible for LTP induction after Cdk5 inhibition involved the PKA pathway, DA and NMDA (N-methyl-D-aspartate) receptor activation, L-type calcium channels activation and the presynaptic modulation of neurotransmitter release.     

In Vivo Stimulation of D1 Receptors Increases the Phosphorylation of Proteins in the Striatum

In vivo changes in levels of DARPP-32 [dopamine (DA)- and cyclic AMP-regulated phosphoprotein, Mr = 32,000] protein phosphorylation in response to DA agonists in the rat striatum were measured using a novel assay that combines the benefits of rapid quenching of enzyme activity by focused microwave irradiation with a back-phosphorylation assay. The basal level of phospho-DARPP-32 was 5.6% of total DARPP-32. Injections of L-3,4-dihydroxyphenylalanine (100 mg/kg) increased this level to 44.4%. This effect was not as great if focused microwave irradiation was not used. The D1-specific agonist SKF 38393 (10 mg/kg) increased the level of phospho-DARPP-32 to 36.4%. A further modification of the back-phosphorylation assay was used to detect other phosphoproteins that appear to be regulated by DA. These results establish an assay for in vivo studies of postsynaptic responses involving second messengers in the DA system and provide direct in vivo evidence for the hypothesis that stimulation of D1 receptors increases the phosphorylation of DARPP-32, as well as several other proteins.     

DARPP-32 genomic fragments drive Cre expression in postnatal striatum

To direct Cre-mediated recombination to differentiated medium-size spiny neurons (MSNs) of the striatum, we generated transgenic mice that express Cre recombinase under the regulation of DARPP-32 genomic fragments. In this reported line, recombination of an R26R reporter allele occurred postnatally in the majority of medium-size spiny neurons of the dorsal and ventral striatum (caudate nucleus and nucleus accumbens/olfactory tubercle), as well as in the piriform cortex and choroid plexus. Although regulatory fragments were selected to target MSNs, low levels of Cre-recombinase expression, as detected by beta-galactosidase activity from the R26R reporter gene, were also apparent in widely dispersed areas or cells of the forebrain and hindbrain. These included the primary and secondary motor cortex, and association cortex, as well as in the olfactory bulb and cerebellar Purkinje cells. Notably, expression in these regions was well below that of endogenous DARPP-32. Analysis of colocalization of beta-galactosidase, as detected either by histochemistry or immunocytochemistry, and DARPP-32 revealed double-labeling in almost all DARPP-32-expressing MSNs in the postnatal striatum, but not in extrastriatal regions. The DARPP-32Cre transgenic mouse line thus provides a useful tool to specifically express and/or inactivate genes in mature MSNs of the striatum.     

Subcellular location of PKA controls striatal plasticity: stochastic simulations in spiny dendrites

Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA), which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity.     

Studies on the relationship of tyrosine hydroxylase,dopamine and cyclic amp-regulated phosphoprotein-32 immunoreactive neuronal structures and d1 receptor antagonist binding sites in various brain regions of the male rat—mismatches indicate a role of d1 receptors in volume transmission

The relationship between tyrosine hydroxylase (TH), dopamine (DA) and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) immunoreactive (IR) neuronal structures and D1 receptor antagonist binding sites has been analysed in various brain regions in the male rat, using immunocytochemistry and receptor autoradiography with the iodinated analogue of SCH 23390 ([¹²⁵I]SCH 23982) as radioligand. Two-colour immunocytochemistry was used to establish in detail the relationship between DARPP-32 and the TH IR neuronal structures in mes-, di- and telencephalon. The analysis reveals complex matches and mismatches between central DARPP-32 immunoreactive neurones, DA neurones and D1 DA receptors.The results inter alia indicate a probable release of DA from the dendritic plexus of the zona reticulata of the substantia nigra to reach D1 DA receptors via extracellular pathways. DA released from the few DA terminals present in the entopeduncular nucleus and from adjacent dopamine axons may also reach D1 DA receptors in this nucleus by extracellular diffusion. A similar situation may also exist in the globus pallidus. Thus, DA may in some regions be released as a paracrine signal to reach distant D1 DA receptors. This type of chemical transmission has been called volume transmission and D1 receptors may thus participate in volume transmission.The mismatch obtained in, for example, the amygdaloid cortex and hypothalamus between D1 receptor antagonist binding sites and DARPP-32 IR nerve cell profiles, is compatible with the possibility that some D1 receptors linked to adenylate cyclase may not involve DARPP-32 as a substrate protein for the cyclic AMP-dependent protein kinase. In addition the possibility should be considered that D1 receptors may not always be linked to adenylate cyclase.Finally, the mismatch in the median eminence between [¹²⁵I]SCH 23982 binding sites and DARPP-32 IR profiles may indicate the existence of D1 receptors which are masked under basal conditions in the male rat.     

Glial fibrillary acidic protein in rat brain structures under conditions of training for a passive defensive habit

Under conditions of training for a passive defensive habit in rats, changes were observed in the content of an intermediate filament marker of the astroglial cellular cytoskeleton, glial fibrillary acidic protein (GFAP), in different brain structures (frontal cortex, medial thalamus, striatum, hippocampus, and pons). It is hypothesized that GFAP is involved in the control of synaptic plasticity underlying memory formation.     

Cholinergic hyperinnervation in the cerebral cortex of microencephalic rats does not result in muscarinic receptor down-regulation or in alteration of receptor-stimulated phosphoinositide metabolism

Administration of methylazoxymethanol (MAM; 25 mg/kg) to pregnant rats at gestational day 15 (GD 15) induces a marked reduction of telencephalic areas of the offspring brain. Previous neurochemical studies demonstrated a marked cholinergic hyperinnervation in the cerebral cortex of microencephalic rats. In this study we have evaluated whether this cholinergic hyperinnervation could result in altered functionality of muscarinic receptors. Acetylcholinesterase activity (AChE) was increased by 69% in the cerebral cortex of MAM treated rats confirming a relative hyperinnervation, whereas in the hippocampus and striatum no significant changes were observed. Despite the marked hyperinnervation, in the cerebral cortex of microencephalic rats neither muscarinic receptor-stimulated phosphoinositide metabolism nor muscarinic, receptor density were altered. No differences in receptor density were also observed in the hippocampus and striatum. Chronic diisopropylfluorophosphate (DFP) administration induced a marked decrease of AChE activity and down-regulation of muscarinic receptors whereas atropine administration resulted in receptor up-regulation in cerebral cortex, striatum and hippocampus of both control and MAM rats. The results confirm a relative cholinergic hyperinnervation in the cerebral cortex of microencephalic rats and demonstrate that the regulation of muscarinic receptor-stimulated phosphoinositide metabolism and muscarinic receptor plasticity is not modified in a condition of increased cholinergic presynaptic terminals.     

Il1-β Involvement in Cognitive Impairment after Sepsis

Sepsis is defined as the host's reaction to infection and characterised by a systemic inflammatory response with important clinical implications. Central nervous system dysfunction secondary to sepsis is associated with local generation of pro- and anti-inflammatory cytokines, impaired cerebral microcirculation, an imbalance of neurotransmitters, apoptosis and cognitive impairment. It's known that the IL-1β is one of the first cytokines to be altered. Thus, the objective of this study was to evaluate the role of IL-1β in cognitive parameters in brain tissue through the use of an IL-1β (IL-1ra) receptor antagonist up to 10 days and to assess blood–brain barrier permeability, cytokine levels, oxidative parameters and energetic metabolism up to 24 h, after sepsis induction. To this aim, we used sham-operated Wistar rats or submitted to the cecal ligation and perforation (CLP) procedure. Immediately after, the animals received one dose of 10 μg of IL-1ra. After 24 h, the rats were killed and were evaluated for biochemical parameters in the pre-frontal cortex, hippocampus and striatum. After 10 days, the animals were submitted to the habituation to the open field and step-down inhibitory avoidance task. We observed that the use of IL-1ra reverted the increase of blood–brain barrier permeability in the pre-frontal cortex, hippocampus and striatum; the increase of IL-1β, IL1-6 and TNF-α levels in the pre-frontal cortex and striatum; the decrease of complex I activity in the pre-frontal, hippocampus and striatum; the increase of oxidative parameters in pre-frontal cortex, hippocampus and striatum; and cognitive impairment. In conclusion, the results observed in this study reinforce the role of acute brain inflammatory response, in particular, the IL1β response, in the cognitive impairment associated with sepsis.     

Chronic Treatment of Rats with SCH-23390 or Raclopride Does Not Affect the Concentrations of DARPP-32 or Its mRNA in Dopamine-Innervated Brain Regions

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a neuronal phosphoprotein that is enriched in neurons which possess dopamine D1 receptors, particularly striatonigral neurons. In rat brain slices, the phosphorylation state of DARPP-32 is regulated by dopamine, acting through the dopamine D1 receptor and the adenylyl cyclase system. This study reports that chronic blockade (21 days) of either dopamine D1 receptors by SCH-23390 or dopamine D2 receptors by raclopride does not affect the concentrations of DARPP-32 in specific rat brain regions (striatum, thalamus, hippocampus, frontal cerebral cortical pole). Northern blot analysis indicates that the steady-state level of DARPP-32 mRNA in striatum is also unchanged by these treatments.     

Effects of chronic mild stress on the oxidative parameters in the rat brain

Major depression is characterized for symptoms at the psychological, behavioral and physiological levels. The chronic mild stress model has been used as an animal model of depression. The consumption of sweet food, locomotor activity, body weight, lipid and protein oxidation levels and superoxide dismutase and catalase activities in the rat hippocampus, prefrontal cortex and cortex were assessed in rats exposed to chronic mild stress. Our findings demonstrated a decrease on sweet food intake, no effect on locomotor activity, lack of body weight gain, increase in protein (prefrontal, hippocampus, striatum and cortex) and lipidic peroxidation (cerebellum and striatum), and an increase in catalase (cerebellum, hippocampus, striatum, cortex) and a decrease in superoxide dismutase activity (prefrontal, hippocampus, striatum and cortex) in stressed rats. In conclusion, our results support the idea that stress produces oxidants and an imbalance between superoxide dismutase and catalase activities that contributes to stress-related diseases, such as depression.     

Differential Response of Central Dopaminergic System in Acute and Chronic Unpredictable Stress Models in Rats

We aimed to evaluate the response of dopaminergic system in acute stress (AS) and chronic unpredictable stress (CUS) by measuring dopamine (DA) levels, its receptor densities in the frontal cortex, striatum, hippocampus, amygdala and orbito-frontal cortex regions of rat brain, and investigated the corresponding behavioral locomotor changes. Involvement of D₁ receptor was also examined during AS and CUS using A 68930, a D₁ selective agonist. Rats were exposed to AS (single immobilization for 150 min) and CUS (two different stressors for 7 days). AS significantly decreased the DA levels in the striatum and hippocampus, and A 68930 pretreatment significantly reverted these changes. However, in the frontal cortex significantly increased DA levels were remain unchanged following A 68930. CUS led to a decrease of DA levels in the frontal cortex, striatum and hippocampus, which were normalized by A 68930. Saturation radioligand binding assays revealed a significant decrease in the number of D₁-like receptors in the frontal cortex during CUS, which were further decreased by A 68930 pretreatment. However, in the striatum and hippocampus, A 68930 pretreatment reduced the CUS induced increase in the number of D₁-like receptors. No significant changes were observed in the amygdala and orbito-frontal cortex during AS and CUS, while D₂-like receptors were unchanged in all the brain regions studied. Locomotor activity was significantly decreased in both the stress models, A 68930 pretreatment significantly increased stereotypic counts and horizontal activity. Thus, present investigation provide insights into the differential regional response of dopaminergic system during AS and CUS. Further, neurochemical and behavioral effects of D₁ agonist pretreatment suggest specific modulatory role of D₁ receptor under such stressful episodes.     

Psychological Stress Expands Low Molecular Weight Iron Pool in Cerebral Cortex,Hippocampus, and Striatum of Rats

We have previously demonstrated that psychological stress (PS) can cause iron to accumulate in the cerebral cortex, hippocampus, and striatum of rats. However, why iron accumulates and in what oxidation state iron it accumulates in the brain of PS-exposed rats has not been well elucidated. In the present study, we investigated the influence of PS on the low molecular weight iron pool (LMWIP) in the rat brain. The results showed that: (1) PS significantly expanded LMWIP in the cerebral cortex, hippocampus, and striatum in rats; (2) PS caused derangement of pyramidal cells and reduced the layers of pyramidal CA1 and CA2 neurons; (3) PS exposure greatly lowered the expression of ferritin (Fn) and hephaestin (HP) in the rat cortex and hippocampus; and (4) PS decreased superoxide dismutase, glutathione peroxidase, and glutathione level and increased malondialdehyde level in the cerebral cortex, hippocampus, and striatum in rats. These results indicated that PS could expand LMWIP significantly, which may be attributed to PS-induced decrease in Fn, HP expression, and the subsequent reduction in iron storage and utilization, and expansion of LMWIP could in turn lead to aggravation of oxidative damage.     

Transient calcium and dopamine increase PKA activity and DARPP-32 phosphorylation

Reinforcement learning theorizes that strengthening of synaptic connections in medium spiny neurons of the striatum occurs when glutamatergic input (from cortex) and dopaminergic input (from substantia nigra) are received simultaneously. Subsequent to learning, medium spiny neurons with strengthened synapses are more likely to fire in response to cortical input alone. This synaptic plasticity is produced by phosphorylation of AMPA receptors, caused by phosphorylation of various signalling molecules. A key signalling molecule is the phosphoprotein DARPP-32, highly expressed in striatal medium spiny neurons. DARPP-32 is regulated by several neurotransmitters through a complex network of intracellular signalling pathways involving cAMP (increased through dopamine stimulation) and calcium (increased through glutamate stimulation). Since DARPP-32 controls several kinases and phosphatases involved in striatal synaptic plasticity, understanding the interactions between cAMP and calcium, in particular the effect of transient stimuli on DARPP-32 phosphorylation, has major implications for understanding reinforcement learning. We developed a computer model of the biochemical reaction pathways involved in the phosphorylation of DARPP-32 on Thr34 and Thr75. Ordinary differential equations describing the biochemical reactions were implemented in a single compartment model using the software XPPAUT. Reaction rate constants were obtained from the biochemical literature. The first set of simulations using sustained elevations of dopamine and calcium produced phosphorylation levels of DARPP-32 similar to that measured experimentally, thereby validating the model. The second set of simulations, using the validated model, showed that transient dopamine elevations increased the phosphorylation of Thr34 as expected, but transient calcium elevations also increased the phosphorylation of Thr34, contrary to what is believed. When transient calcium and dopamine stimuli were paired, PKA activation and Thr34 phosphorylation increased compared with dopamine alone. This result, which is robust to variation in model parameters, supports reinforcement learning theories in which activity-dependent long-term synaptic plasticity requires paired glutamate and dopamine inputs.     

左旋千金藤啶碱D1激动作用增加6-OHDA损毁大鼠的DARPP-32磷酸化效应

为了进一步阐明SPD对大鼠纹状体突触后D1受体的激动作用特性,本文应用反磷酸化在体内测定及放射配体结合方法,分别观察SPD对6-OHDA损毁大鼠纹状体DARPP-32体内磷酸化作用及突触后D1受体密度的影响.结果表明:皮下给予SPD(20,40 mg/kg,21 d),损毁侧纹状体DARPP-32体外[32P]的掺入量较健侧下降50%(P<0.01).换言之,损毁侧纹状体内DARPP-32的磷酸化程度增加了.然而,SPD使损毁导致D1受体上调的作用减弱(Bmax 从385.0±26.1 fmol/mg 降至319.7±20.1 fmol/mg水平).因此,SPD激动D1受体,使6-OHDA损毁大鼠纹状体内DARPP-32磷酸化作用加强,而受体密度减少.这是SPD调节脑内D1受体信号转导功能的重要机制.     

Plasticity in the Rat Prefrontal Cortex: Linking Gene Expression and an Operant Learning with a Computational Theory

The plasticity in the medial Prefrontal Cortex (mPFC) of rodents or lateral prefrontal cortex in non human primates (lPFC), plays a key role neural circuits involved in learning and memory. Several genes, like brain-derived neurotrophic factor (BDNF), cAMP response element binding (CREB), Synapsin I, Calcium/calmodulin-dependent protein kinase II (CamKII), activity-regulated cytoskeleton-associated protein (Arc), c-jun and c-fos have been related to plasticity processes. We analysed differential expression of related plasticity genes and immediate early genes in the mPFC of rats during learning an operant conditioning task. Incompletely and completely trained animals were studied because of the distinct events predicted by our computational model at different learning stages. During learning an operant conditioning task, we measured changes in the mRNA levels by Real-Time RT-PCR during learning; expression of these markers associated to plasticity was incremented while learning and such increments began to decline when the task was learned. The plasticity changes in the lPFC during learning predicted by the model matched up with those of the representative gene BDNF. Herein, we showed for the first time that plasticity in the mPFC in rats during learning of an operant conditioning is higher while learning than when the task is learned, using an integrative approach of a computational model and gene expression.     

Distinctions in metabolism of serotonin and dopamine in rat brain during latent inhibition

Latent inhibition (LI) is a behavioral phenomenon, in which repeated presenting of a non-reinforced stimulus retards conditioning to this stimulus when it is coupled with a reinforcer. In order to find specific serotonin (5-HT- and dopamine (DA) changes mediating the LI, the 5-HT and DA metabolism was investigated in certain brain regions. Oxidative deamination of 5-HT and DA by monoamine oxidase (MAO) was determined in the prefrontal cortex, striatim, amygdala, and hippocampus at preexposure and testing stages of the LI using the passive avoidance procedure in rats. Preexposed animals demonstrated high MAO activity for 5-HT deamination in the amygdala and striatum and lower MAO activity for DA deamination in the amygdala and hippocampus. After testing the LI, a high level of 5-HT deamination by MAO was revealed in the amygdala, white the lower level of 5-HT deamination by MAO was shown in the prefrontal cortex. At the same time, no changes in DA metabolism were found in all the brain regions studied. Thus, the role of dopaminergic system in the LI effect may be limited by the preexposure stage. The obtained evidence suggests that the enhanced 5-HT activity in the amygdala and striatum induced by the preexposed stimulus is a principal biochemical mechanism underlying the LI.     

Effects of ethanol on brain monoamine content of spontaneously hypertensive rats (SHR)

Spontaneously hypertensive rats (SHR) were administered either 2.4 g/kg ethanol or an isocaloric glucose daily for 4 weeks and the levels of norepinephrine (NE), epinephrine (EP), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in different brain regions were determined. Results indicated a 3-fold increase in NE level in brain stem and hypothalamus and more than 2-fold increase in DA in corpus striatum in alcohol-treated rats as compared to controls. There was a significant increase in the level of DA in the corpus striatum but the levels in cerebral cortex, brain stem and hippocampus were decreased instead. Decreases in 5-HT levels were found in hypothalamus, brain stem, cortex and cerebellum of alcohol-treated brain as compared to untreated controls. These results indicate alterations of the biogenic amine contents in different regions of the SHR brain after chronic ethanol ingestion. Since stimulated release of biogenic amines in the SHR brain has been implicated in the regulation of blood pressure, changes due to ethanol ingestion may be a risk factor in hypertensive patients.