Donald F. Siwek

Involvement of free radicals in excitotoxicity in vivo

Abstract: Recent evidence has linked excitotoxicity with the generation of free radicals. We examined whether free radical spin traps can attenuate excitotoxic lesions in vivo. Pretreatment with N‐tert‐butyl‐α‐(2‐sulfophenyl)‐nitrone (S‐PBN) significantly attenuated striatal excitotoxic lesions in rats produced by N‐methyl‐d‐aspartate (NMDA), kainic acid, and α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid (AMPA). In a similar manner, striatal lesions produced by 1‐methyl‐4‐phenylpyridinium (MPP+), malonate, and 3‐acetylpyridine were significantly attenuated by either S‐PBN or α‐phenyl‐N‐tert‐butylnitrone (PBN) treatment. Administration of S‐PBN in combination with the NMDA antagonist MK‐801 produced additive effects against malonate and 3‐acetylpyridine toxicity. Malonate injections resulted in increased production of hydroxyl free radicals (•OH) as assessed by the conversion of salicylate to 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA). This increase was significantly attenuated by S‐PBN, consistent with a free radical scavenging effect. S‐PBN had no effects on malonate‐induced ATP depletions and had no significant effect on spontaneous striatal electrophysiologic activity. These results provide the first direct in vivo evidence for the involvement of free radicals in excitotoxicity and suggest that antioxidants may be useful in treating neurologic illnesses in which excitotoxic mechanisms have been implicated.

Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy.

Objective: To characterize the motor neuron dysfunction in two models by performing physiologic and morphometric studies. Background: Mutations in the gene encoding cytosolic superoxide dismutase 1 (SOD1) account for 25% of familial ALS (FALS). Transgenes with these mutations produce a pattern of lower motor neuron degeneration similar to that seen in patients with FALS. In contrast, mice lacking SOD1 develop subtle motor symptoms by approximately 6 months of age. Methods: Physiologic measurements, including motor conduction and motor unit estimation, were analyzed in normal mice, mice bearing the human transgene for FALS (mFALS mice), and knockout mice deficient in SOD1 (SOD1-KO). In addition, morphometric analysis was performed on the spinal cords of SOD1-KO and normal mice. Results: In mFALS mice, the motor unit number in the distal hind limb declined before behavioral abnormalities appeared, and motor unit size increased. Compound motor action potential amplitude and distal motor latency remained normal until later in the disease. In SOD1-KO mice, motor unit numbers were reduced early but declined slowly with age. In contrast with the mFALS mice, SOD1-KO mice demonstrated only a modest increase in motor unit size. Morphometric analysis of the spinal cords from normal and SOD1-KO mice showed no significant differences in the number and size of motor neurons. Conclusions: The physiologic abnormalities in mFALS mice resemble those in human ALS. SOD1-deficient mice exhibit a qualitatively different pattern of motor unit remodeling that suggests that axonal sprouting and reinnervation of denervated muscle fibers are functionally impaired in the absence of SOD1.

Single cell activity patterns of pedunculopontine tegmentum neurons across the sleep-wake cycle in the freely moving rats

Microinjections of the excitatory amino acid, L‐glutamate into the cholinergic cell compartment of the pedunculopontine tegmentum (PPT) of the rat induces both wakefulness and/or rapid eye movement (REM) sleep depending on the glutamate dosage. However, no studies have systematically recorded the electrical activity of these cells in the freely moving rat across the sleep‐wake cycle. In this study, we have recorded the spontaneous activity patterns of single PPT cells (n = 70) in the freely moving rat across the sleep‐wake cycle. PPT neurons were classified into three groups based on patterns in their spontaneous activity. The first group of cells (12.86%) was more active during REM sleep than they were during wakefulness or slow‐wave sleep (SWS). The second group of cells (60.0%) was more active during REM and wakefulness than during SWS. The firing rate of the third group of cells (27.14%) did not change as a function of behavioral state. This study also demonstrated that the level of activity within the cholinergic cell compartment of the PPT during SWS drops to 7.4% of that observed during wakefulness and that during REM sleep it changes to 65.5% of wakefulness levels. These findings indicate that in the freely moving rat, the discharging of PPT neurons correlates with wakefulness and REM sleep. Additionally, these neurons may be an integral part of the brainstem wakefulness and REM sleep‐generating mechanisms in the rat.

Endogenous and exogenous nitric oxide in the pedunculopontine tegmentum induces sleep

Mesopontine cholinergic cells in the pedunculopontine tegmental (PPT) nuclei modulate the control of the wake‐sleep cycle by releasing acetylcholine to their target structures. These cells also synthesize nitric oxide (NO) which diffuses into the extracellular space and acts as a neuronal messenger. The present study is based on the hypothesis that NO synthesis and its presence in the extracellular space in the PPT play a functional role in regulating the behavioral states of waking and sleep. This hypothesis was tested by microinjecting a control vehicle, NO donor, S‐Nitroso‐N‐acetyl‐penicillamine (SNAP) and a competitive inhibitor of NO synthase enzyme (NOS), NG‐Nitro‐L‐arginine methylester hydrochloride (L‐NAME) into the PPT while quantifying the effects on wakefulness and sleep. Six cats were implanted with bilateral guide tubes for PPT microinjection and with standard electrodes to measure waking, slow‐wave sleep (SWS), and rapid eye movement (REM) sleep. Five‐hour free‐moving polygraphic recordings were made following each microinjection (0.25 μl) of control saline, SNAP or L‐NAME.

Complex transcallosal interactions in visual cortex.

Reversible inactivation by cooling of the transcallosal projecting neurons in areas 17 and 18 of one hemisphere bring about complex changes in the spontaneous and evoked activity of neurons in the callosal receiving zone of the opposite hemisphere. These changes include increases and decreases in evoked and spontaneous activities. Overall, 90% of neurons in layers II and III, 50% in layer IV, and 100% in layers V and VI were affected by the block of transcallosal input. The complexity of the changes was greatest in layers II and III, which are the major callosal recipient layers. The results indicate that many excitatory and inhibitory circuits are under the direct control of transcallosal fibers in the normally functioning brain.

Choline availability during embryonic development alters the localization of calretinin in developing and aging mouse hippocampus

Abstract Choline availability in the diet during pregnancy alters fetal brain biochemistry with resulting behavioral changes that persist throughout the lifetime of the offspring. In the present study, the effects of dietary choline on the onset of GABAergic neuronal differentiation in developing fetal brain, as demarcated by the expression of calcium binding protein calretinin, are described. In these studies, timed-pregnant mice were fed choline supplemented, control or choline deficient AIN-76 diet from day 12-17 of pregnancy and the brains of their fetuses were studied on day 17 of gestation. In the primordial dentate gyrus, we found that pups from choline deficient-dams had more calretinin protein (330% increase), and pups from choline supplemented-dams had less calretinin protein (70% decrease), than did pups from control-dams. Importantly, decreased calretinin protein was still detectable in hippocampus in aged, 24-month-old mice, born of choline supplemented-dams and maintained since birth on a control diet. Thus, alterations in the level of calretinin protein in fetal brain hippocampus could underlie the known, life long effects of maternal dietary choline availability on brain development and behavior.

IDENTIFICATION OF CHOLINERGIC AND NON-CHOLINERGIC NEURONS IN THE PONS EXPRESSING PHOSPHORYLATED CYCLIC ADENOSINE MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN AS A FUNCTION OF RAPID EYE MOVEMENT SLEEP

Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of rapid eye movement (REM) sleep. In the present study performed on rats, phosphorylated cyclic AMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high rapid eye movement sleep (HR, approximately 27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With HR, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low rapid eye movement sleep (LR, approximately 2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr=0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr=0.88), PnO (Rsqr=0.87), and SubCD (Rsqr=0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr=0.70) and DRN (Rsqr=0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are inactive. More importantly, the regression analysis indicated that pCREB activation in approximately 98% of PPT cholinergic neurons, was caused by REM sleep. Moreover the results indicate that during REM sleep, PPT intracellular PKA activation and a transcriptional cascade involving pCREB occur exclusively in the cholinergic neurons.

Effects of passive-avoidance training on sleep-wake state-specific activity in the basolateral and central nuclei of the amygdala.

This study examined the effects of intense emotional learning on the sleep-wake state-specific electroencephalographic (EEG) activities of the basolateral (BLA) and central (CeA) nuclei of the amygdala. Rats were trained on a passive-avoidance learning (PAL) protocol that was followed by 6 hr of undisturbed polygraphic recording and a PAL test. After PAL training, the total amount of REM sleep decreased: high-frequency EEG power decreased in the CeA during REM sleep and increased in the BLA during all sleep-wake stages. These results suggest that there is no homeostatic demand for REM sleep after intense emotional learning. However, the PAL-specific changes in the local EEG suggest that some form of memory processing may occur within the amygdala during REM sleep.

Prenatal choline deficiency decreases the cross-sectional area of cholinergic neurons in the medial septal nucleus.

Levels of dietary choline in utero influence postnatal cognitive performance. To better understand this phenomenon, forebrain cholinergic neurons were studied in the 8-9 month old offspring of dams fed a control or choline-deficient diet from EDs 11-17. Serial sections were immunostained with antibodies against p75, a cholinergic marker. Neuronal morphology was analyzed in the basal forebrain, a heterogeneous area composed of several structures including the medial septal nucleus (MSN), nucleus of the diagonal band (DB), and the nucleus basalis of Meynert (NB). Neuronal cross-sectional areas were selectively reduced in the MSN of choline-deficient animals, compared to controls, but cell counts were not altered. Our findings suggest that cholinergic medial septal neurons may be selectively vulnerable to in utero choline deficiency.

The temporal degradation of bone collagen: A histochemical approach

As forensic anthropologists are currently unable to estimate reliably and quantitatively the postmortem interval (PMI) of skeletonized remains, the current study was conducted to determine if degradation of bone collagen over time could be quantified using sirius red/fast green staining, and whether the degradation would occur at a predictive rate such that it may be used to estimate the PMI of skeletonized individuals. Resin embedded 200-300μm cross-sections of pig (Sus scrofa) long bones with known provenience and PMIs ranging from fresh to 12 months were stained using a histochemical reaction which differentially stains collagenous (Co) and non-collagenous (NCo) proteins. Spectrophotometry was used to determine the concentration of Co and NCo proteins in each bone section, after which the ratio of these proteins was calculated. The results of this study revealed a significant decline in the ratios of Co/NCo protein concentrations over the time period studied (p<0.001). Furthermore, a significant negative correlation between the ratios of Co/NCo protein concentrations and time (r=-0.563, p<0.0001) was observed. Despite a significant correlation, the moderate r-value obtained suggests that, at present, this method is useful primarily for detecting and quantifying the degradation of Co and NCo proteins in bones. Future studies that include shorter time intervals and environmental factors, such as soil pH, temperature, and hydrology may prove to be critical for using this method for PMI estimation.