Jeffery W. Brock

Stress-related behavior and central norepinephrine concentrations in the REM sleep-deprived rat

Rapid eye movement sleep deprivation (REMd) is a potent stressor in the rat. Behavioral abnormalities are among the earliest overt symptoms of REMd, the mechanisms for which remain largely unknown. The phenomena of hyperphagia and weight loss that are associated with REMd may contribute to its later morbidity; however, little is known about the onset of these phenomena or the neurotransmitter mechanisms that are involved. The aim of this study was to determine whether the earliest effects of REMd on consumatory behavior in the rat and its performance in the swimming cylinder of Porsolt are related to changes in norepinephrine (NE) concentrations in the cerebral cortex and selected areas of the hypothalamus. Sprague-Dawley rats were divided into three groups (n = 6): the REMd group resided in a water tank on 6.5-cm diameter pedestals for 96 h; the tank control (TC) group resided in the water tank on 15-cm pedestals for 96 h; the cage controls (CC) remained in their home cages for the duration of the study. In the first series of experiments, body weights and caloric intake were recorded daily, along with the performance of all animals in the swimming cylinder of Porsolt. In the second series of experiments, body weights and caloric intake were recorded, but the Porsolt test was not employed and the brains were dissected after 96 h for NE analysis by HPLC. It was observed that the REMd group had lower immobility times (p < 0.05) in the Porsolt test after only 24 h, compared to groups TC and CC.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in dendritic spine density in the rat brain associated with protein malnutrition

Rats that consume a high-protein diet are hyperactive, hyperresponsive to noxious stimuli, and demonstrate elevated basal arousal levels. Although the mechanism involved in dietary protein-induced changes in behavior is unclear, it may involve many changes including abnormal dendrite morphology in the brain. Three groups of rats were pair-fed with isocaloric diets containing 8%, 20% and 50% casein for 4 weeks. Their brains were processed for examination of dendritic spine densities in the frontal, parietal, and entorhinal cortices, the striatum, and the septum. Animals on the 50% casein diet showed increased spine densities in all areas investigated (P less than 0.05), compared to the animals on normal (20%) casein. In contrast, animals maintained on the 8% casein diet showed increased spine densities (P less than 0.05) only in the striatum and entorhinal cortex, and exhibited normal densities in the frontal and parietal cortices, and the medial septum.

Antibodies against synthetic peptides predicted from the nucleotide sequence of D2 receptor recognize native dopamine receptor protein in rat striatum.

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG‐KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24–34 and 176–185 from the NH2 terminus, respectively, were conjugated to keyhole limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross‐reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90‐kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I‐AP‐azidospiperone (125I‐NAPS). Photoaffinity labeling of the 90‐kDa protein by 125I‐NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90‐kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H] YM‐09151‐2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration‐dependent manner, a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H] YM‐09151‐2 binding to striatal membranes were examined in the presence of antibody and showed a 25–30% decrease in Bmax (203.5 ± 11.0 and 164.6 ±3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD. These results suggest that polyclonal antisera raised against peptide I exhibited specific antibodies for the dopamine D2 receptor protein. The primary epitope for this antibody is at or near the ligand binding site which can be recognized in both denatured and native receptor protein in striatal membranes.

Effects of rapid eye movement sleep deprivation on the properties of striatal dopaminergic system

Using the water tank procedure, we have examined the effects of rapid eye movement (REM) sleep deprivation and associated stress on the properties of striatal dopaminergic system. While stress decreased the number of D1 and D2 dopamine receptors, a combination of REM sleep deprivation attenuated the decrease. The ratio of D1 to D2 densities, however, increased on both the stress and REM sleep deprivation groups. In contrast, the number of dopamine uptake sites remained unchanged. The enhanced behavioral responses to dopaminergic stimulants after REM sleep deprivation are discussed.

REM sleep deprivation alters dopamine D2 receptor binding in the rat frontal cortex.

REM sleep deprivation (RSD) of rats results in facilitation of dopaminergic behavior and an increase in striatal D2 receptor density. To determine whether RSD results in changes in D2 receptor in other brain regions, receptor affinity (Kd) and density (Bmax) were measured in the anteromediofrontal (AM), cingulate (CN), and sulcal cortex (SL) in four groups of rats: 1), RSD96 group (RSD for 96 h; small pedestal/water tank method), 2) RSD24 group (large pedestals for 72 h then small pedestals for 24 h), 3) tank control group (TC; large pedestals for 96 h), and 4) cage control group. In separate groups, ambulation was recorded for 30 min following treatments. Group RSD96 showed an increase in activity compared to TC, and TC was increased compared to CC (p < 0.05 for all). In group RSD24, the AM showed an increase in Bmax and Kd (p < 0.05), but there were no effects by RSD96. In the CN, Bmax and Kd were decreased by RSD96 (p < 0.05) but not RSD24. In the SL, Bmax was increased by RSD96, but not RSD24, whereas Kd was increased in both RSD groups (p < 0.05).

Differential modulation of dopaminergic systems in the rat brain by dietary protein

Rats that consume a diet 50% rich in protein exhibit hyperactivity and hyperresponsiveness to nociceptive stimuli, in which facilitation of dopaminergic activity has been implicated. We studied the regional changes in the concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brains of rats that were maintained on high-protein (HP, 50% casein), normal-protein (NP, 20% casein), and low-protein (LP, 8% casein) diets for 36 weeks. Brain nuclei that represented different DAergic systems were punchdissected and analyzed using HPLC. In the substantia nigra, the striatum, and the dentate gyrus, DA concentrations decreased and increased, respectively, with a decrease and increase in dietary protein (p<0.05 compared to the NP diet). Similar trends in the effect of the HP diet were observed in the ventral tegmental area, amygdala, frontal cortex, subiculum, centromedial nucleus (CM) of the thalamus, and inferior colliculi (IC), although the differences in DA concentrations were not statistically significant. These brain areas also showed a pattern of decreased DA concentration in association with the LP diet, and the differences were statistically significant (p<0.05) in the CM and IC. DA concentrations in most regions of the midbrain and brainstem were not different between the diet groups, nor were consistent trends observed in those regions. Also, there were no consistent relationships between DOPAC/DA and HVA/DA ratios and dietary protein level. These data suggest that only discrete dopaminergic neuronal circuits in the rat forebrain were sensitive to changes in dietary protein level.

Dietary protein levels alter rat behavior

Abstract The performance of rats in a battery of behavioral test systems following long-term, dietary protein manipulation was evaluated. Groups of rats were pair-fed with isocaloric diet containing low (8%), normal (20%) and high (50%) casein for 20 weeks. The high-casein group was more responsive compared to the normal- and low-casein groups in sensorimotor function, negative geotaxis, and spontaneous locomotor activity. In addition, the high-casein fed rats exhibited reduced aversion as measured by the elevated plus-maze test of anxiety, and hyperalgesia as shown by the tail-flick reaction time. Results from the animals which were fed the low-casein diet were not different from those fed the normal-casein diet in all of the behavioral tests employed except the tail-flick, in which the low-casein group exhibited hypoalgesia. These data suggest that consumption of a long-term, high-protein diet leads to hyperactivity, hyper-responsiveness, and anxiolysis in rats. Only the results of the tail-flick test were compatible with protein: carbohydrate ratio effect on behavior.

A paradoxical elevation of brain cyclo(His-Pro) levels in hyperphagic obese Zucker rats

Several studies suggest a role for endogenous cyclo(His-Pro) or CHP in appetite regulation. In the present study, we have examined the regional brain distribution of CHP in hyperphagic obese Zucker rats and their lean littermates. The data show a significant elevation in the levels of CHP in many brain regions, including hypothalamus of the obese rat. Within the hypothalamus, the lateral hypothalamic (LH) nucleus of obese rats had significantly higher levels of CHP when compared to that of the lean littermates. Administration of dehydroepiandrosterone, a steroid hormone known to decrease food intake and body weight gain, to obese rats led to decrease in the levels of CHP in the LH. These data further suggest a role for the endogenous CHP in attenuating food intake.

Motor, but not sensory, cortical potentials are amplified by high-protein diet

Animals fed a high-protein diet (50% casein) are hyperactive and more responsive to nociceptive stimuli than those fed either a normal- or low-protein diet. The mechanisms mediating dietary protein-induced behavior are unknown and may include both central and peripheral neural effects. Adult, Sprague-Dawley rats were fed 50% casein (treatment group) and 24% casein (control group) ad lib for 36-40 weeks. The animals were anesthetized with alpha-chloralose and urethane (50 mg/kg and 1.5 mg/kg, IP). EEG recordings were averaged while the anesthetized animal was conditioned using an alerting stimulus-imperative stimulus (AS-IS) paradigm. AS consisted of a 1.5 kHz, 90 dB tone cue. This was followed 2 seconds later by IS, an electrical tail stimulation (11 V, 1.4 s duration). Two negative deflections (N1 and N2) were generated by the frontal cortex during the AS-IS interstimulus interval. N1, an alerting response, was not different between the two groups. N2 amplitude and peak latency were significantly increased in the high-protein group (205% and 117% of control, respectively; p less than 0.05). N2 represents the activation of cells in the motor cortex. Brainstem auditory-evoked responses and somatosensory-evoked potentials also were recorded, but no differences were observed between the two diet groups. These data suggest that consumption of a high-protein diet results in an increase in central arousal mechanisms (measured by cortical negativity response), specifically involving increased excitability of the motor cortex, that is not associated with a disorder of information processing in the cerebral cortex (measured by brainstem auditory-evoked responses and somatosensory-evoked potentials).

Dietary Protein and Central Monoamine Concentrations in the Rat.

In a previous report we demonstrated that rats that consumed a high-protein diet (HP; 50% casein) for 36 weeks were hyperactive and hyperresponsive to nociceptive stimuli, compared to rats that consumed normal (NP; 20% casein) or low-protein (LP; 8% casein) diets. In addition, we have also previously, reported that dopamine concentrations in the nigrostriatal system of the rats were decreased and increased, respectively, with a decrease and increase in dietary protein. In the present study, rats were maintained on the HP, NP and LP diets and regional changes in the concentrations of norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) were assessed. Concentrations of 5-HT in the medial raphe, dorsal raphe, and several of their target tissues, revealed no consistent effect of manipulating dietary protein over the range of 5-HT levels measured. NE concentrations in most of the brain regions innervated by neurons of the locus coeruleus and lateral tegmentum showed no significant differences among the diet groups. However, NE concentrations in the parietal cortex were significantly increased in rats that consumed the HP diet. The present study indicates that the brain NE pathways, particularly that innervating the parietal cortex, is susceptible to dietary protein manipulation.