Effects of alcohol exposure during early life on neuron numbers in the rat hippocampus. I. Hilus neurons and granule cells

We previously showed that 16-day-old rats exposed to a relatively high dose of ethanol at 10-15 postnatal days of age have fewer neurons in the hilus region of the hippocampus compared with controls. Dentate gyrus granule cell numbers, however, showed no statistically significant changes attributable to the ethanol treatment. It is possible that some of the changes in brain morphology, brought about as a result of the exposure to ethanol during early life, may not be manifested until later in life. This question has been further addressed in an extension to our previous study. Wistar rats were exposed to a relatively high daily dose of ethanol on postnatal days 10-15 by placement in a chamber containing ethanol vapour, for 3 h/day. The blood ethanol concentration was found to be similar to430 mg/dl at the end of the period of exposure. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anaesthetised and killed either at 16 or 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle and the physical disector methods were used to estimate, respectively, the regional volumes and neuron cell numerical densities in the hilus and granule cell regions of the dentate gyrus. The total numbers of neurons in the hilus region and granule cell layer were computed from these estimates. It was found that 16-day-old animals had 398,000-441,000 granule cells, irrespective of group. The numbers of granule cells increased such that by 30 days of age, rats had 487,000-525,500 granule cells. However, there were no significant differences between ethanol-treated rats and their age-matched controls in granule cell numbers. In contrast, ethanol-treated rats had slightly but significantly fewer neurons in the hilus region than did control animals at 16 days of age, but not at 30 days of age. Therefore, it appears that a short period of ethanol exposure during early life can have effects on neuron numbers of some hippocampal neurons, but not others. The effects on hilar neuron numbers, observed as a result of such short periods of ethanol treatment, appeared to be transitory. (C) 2003 Wiley-Liss, Inc.

The sit-up: complex kinematics and muscle activity in voluntary axial movement

This paper describes the kinematics and muscle activity associated with the standard sit-up, as a first step in the investigation of complex motor coordination. Eight normal human subjects lay on a force table and performed at least 15 sit-ups, with the arms across the chest and the legs straight and unconstrained. Several subjects also performed sit-ups with an additional weight added to the head. Support surface forces were recorded to calculate the location of the center of pressure and center of gravity; conventional motion analysis was used to measure segmental positions; and surface EMG was recorded from eight muscles. While the sit-up consists of two serial components, 'trunk curling' and 'footward pelvic rotation', it can be further subdivided into five phases, based on the kinematics. Phases I and II comprise trunk curling. Phase I consists of neck and upper trunk flexion, and phase II consists of lumbar trunk lifting. Phase II corresponds to the point of peak muscle contraction and maximum postural instability, the 'critical point' of the sit-up. Phases III-V comprise footward pelvic rotation. Phase III begins with pelvic rotation towards the feet. phase W with leg lowering, and phase V with contact between the legs and the support surface. The overall pattern of muscle activity was complex with times of EMG onset, peak activity, offset, and duration differing for different muscles. This complex pattern changed qualitatively from one phase to the next, suggesting that the roles of different muscles and, as a consequence, the overall form of coordination, change during the sit-up. (C) 2003 Elsevier Science Ltd. All rights reserved.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

pH dependence of the progression in NMR T-2 relaxation times in post-mortem muscle

Continuous NMR T-2 relaxation measurements were carried out on seven rabbit longissimus muscle samples in the period from 25 min to 28 h post-mortem at 200 MHz for H-1. To display differences in post-mortern pH progress and extent of changes in water characteristics during conversion of muscle to meat, three of the seven animals were pre-slaughter injected with adrenaline (0.5 mg/kg live weight 4 h before sacrifice) to differentiate muscle glycogen stores at the time of slaughter. Distributed analysis of T-2 data displayed clear differences in the characteristics of the various transverse relaxation components dependent on progress in pH, as did the water-holding capacity of samples 24 h postmortem. This reveals a pronounced effect of the progressive change in pH on the subsequent development in physical/chemical states of water during the conversion of muscle to meat. Finally, the relaxation characteristics are discussed in relation to supposed post-mortem processes of protein denaturation.

Vitamin D receptor expression in the embryonic rat brain

We are interested in determining whether low maternal vitamin D-3 affects brain development in utero. Whilst the vitamin D receptor (VDR) has been identified in embryonic rat brains, the timing and magnitude of its expression across the brain remains unclear. In this study we have quantitated VDR expression during development as well correlated the timing of its appearance with two vital developmental events, apoptosis and mitosis. Brains from embryonic rats (embryonic days 15-23) were examined. We show that the well-described increase in apoptotic cells and decrease in mitotic cells during development correlates with the appearance of the VDR in brain tissue. Given that vitamin D-3 regulates mitosis and apoptosis in non-neuronal tissue we speculate that the timing of VDR expression in embryonic brain may directly or indirectly mediate features of neuronal apoptosis and mitosis.

The force-velocity relationship of the human soleus muscle during submaximal voluntary lengthening actions

In experiments on isolated animal muscle, the force produced during active lengthening contractions can be up to twice the isometric force, whereas in human experiments lengthening force shows only modest, if any, increase in force. The presence of synergist and antagonist muscle activation associated with human experiments in situ may partly account for the difference between animal and human studies. Therefore, this study aimed to quantify the force-velocity relationship of the human soleus muscle and assess the likelihood that co-activation of antagonist muscles was responsible for the inhibition of torque during submaximal voluntary plantar flexor efforts. Seven subjects performed submaximal voluntary lengthening, shortening(at angular, velocities of +5, -5, +15, -15 and +30, and -30degrees s(-1)) and isometric plantar flexor efforts against an ankle torque motor. Angle-specific (90degrees) measures of plantar flexor torque plus surface and intramuscular electromyography from soleus, medial gastrocnemius and tibialis anterior were made. The level of activation (30% of maximal voluntary isometric effort) was maintained by providing direct visual feedback of the soleus electromyogram to the subject. In an attempt to isolate the contribution of soleus to the resultant plantar flexion torque, activation of the synergist and antagonist muscles were minimised by: (1) flexing the knee of the test limb, thereby minimising the activation of gastrocnemius, and (2) applying an anaesthetic block to the common peroneal nerve to eliminate activation of the primary antagonist muscle, tibialis anterior and the synergist muscles, peroneus longus and peroneus brevis. Plantar flexion torque decreased significantly (P<0.05) after blocking the common peroneal nerve which was likely due to abolishing activation of the peroneal muscles which are synergists for plantar flexion. When normalised to the corresponding isometric value, the force-velocity relationship between pre- and post-block conditions was not different. In both conditions, plantar flexion torques during shortening actions were significantly less than the isometric torque and decreased at faster velocities. During lengthening actions, however, plantar flexion torques were not significantly different from isometric regardless of angular velocity. It was concluded that the apparent inhibition of lengthening torques during voluntary activation is not due to co-activation of antagonist muscles. Results are presented as mean (SEM).

Localization of a brain sulfotransferase, SULT4A1, in the human and rat brain: An immunohistochemical study

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.

Effects of age and alcohol exposure during early life on pyramidal cell numbers in the CA1-CA3 region of the rat hippocampus

We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life can result in an alteration in spatial learning ability. The hippocampal formation is known to be involved in the control of this ability. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life had either immediate or delayed effects on the numbers of pyramidal cells in the CA1-CA3 subregion of the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol at postnatal day 10-15 by placing them for 3 h/day in a chamber containing ethanol vapor. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anesthetized and killed at 16 and 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle was used to determine the volumes of the CA1 and CA2+CA3 regions. The physical disector method was used to estimate the numerical density of neurons in each of the subdivisions. The total number of pyramidal cells was calculated by multiplying the appropriate estimates of the numerical density by the volume. There were significant age-related reductions in the total numbers of pyramidal cells at 16-30 days of age irrespective of the groups examined. Ethanol treated rats were found to have slightly but significantly fewer pyramidal cell neurons than either the MRC or SC groups. These observations indicate that pyramidal cells in the hippocampus may be vulnerable to a relatively high dose of ethanol exposure during this short period of early postnatal life. (C) 2003 Wiley-Liss, Inc.

Recruitment of single human low-threshold motor units with increasing loads at different muscle lengths

We investigated the recruitment behaviour of low threshold motor units in flexor digitorum superficialis by altering two biomechanical constraints: the load against which the muscle worked and the initial muscle length. The load was increased using isotonic (low load), loaded dynamic (intermediate load) and isometric (high load) contractions in two studies. The initial muscle position reflected resting muscle length in series A, and a longer length with digit III fully extended in series B. Intramuscular EMG was recorded from 48 single motor units in 10 experiments on five healthy subjects, 21 units in series A and,27 in series B, while subjects performed ramp up, hold and ramp down contractions. Increasing the load on the muscle decreased the force, displacement and firing rate of single motor units at recruitment at shorter muscle lengths (P < 0.001, dependent t-test). At longer muscle lengths this recruitment pattern was observed between loaded dynamic and isotonic contractions, but not between isometric and loaded dynamic contractions. Thus, the recruitment properties of single motor units in human flexor digitorum superficialis are sensitive to changes in both imposed external loads and the initial length of the muscle. (C) 2003 Elsevier Ltd. All rights reserved.

Firing properties and connectivity of neurons in the rat lateral central nucleus of the amygdala

Using whole cell recordings from acute slices of the rat amygdala, we have examined the physiological properties of and synaptic connectivity to neurons in the lateral sector of the central amygdala (CeA). Based on their response to depolarizing current injections, CeA neurons could be divided into three types. Adapting neurons fired action potentials at the start of the current injections at high frequency and then showed complete spike-frequency adaptation with only six to seven action potentials evoked with suprathreshold current injections. Late-firing neurons fired action potentials with a prolonged delay at threshold but then discharged continuously with larger current injections. Repetitive firers discharged at the start of the current injection at threshold and then discharged continuously with larger current injections. All three cells showed prolonged afterhyperpolarizations (AHPs) that followed trains of action potentials. The AHP was longer lasting with a larger slow component in adapting neurons. The AHP in all cell types contained a fast component that was inhibited by the SK channel blocker UCL1848. The slow component, not blocked by UCL1848, was blocked by isoprenaline and was significantly larger in adapting neurons. Blockade of SK channels increased the discharge frequency in late firers and regular-spiking neurons but had no effect on adapting neurons. Blockade of the slow AHP with isoprenaline had no effect on any cell type. All cells received a mixed glutamatergic and GABAergic input from a medial pathway. Electrical stimulation of the lateral (LA) and basolateral (BLA)nuclei evoked a large monosynaptic glutamatergic response followed by a disynaptic inhibitory postsynaptic potential. Activation of neurons in the LA and BLA by puffer application of glutamate evoked a small monosynaptic response in 13 of 55 CeA neurons. Local application of glutamate to the CeL evoked a GABAergic response in all cells. These results show that at least three types of neurons are present in the CeA that can be distinguished on their firing properties. The firing frequency of two of these cell types is determined by activation of SK channels. Cells receive a small input from the LA and BLA but may receive inputs that course through these nuclei en route to the CeA.

Low prenatal vitamin D alters morphology, cell density and gene expression in adult rat brain: Correlations with schizophrenia

Statement of the study: Based on data from ecological and analytic epidemiological studies, we have proposed that low prenatal vitamin D is a candidate risk-modifying factor for schizophrenia. Previously, we demonstrated that low prenatal vitamin D adversely affected brain development in neonatal rats (Eyles et al, 2003). Here we examine the impact of both prenatal and early life hypovitaminosis D on various outcomes in the adult rat brain. Methods: Female Sprague-Dawley rats were made vitamin D deficient via the use of a special diet (Dyets CA) and lighting conditions that excluded UVB radiation. Animals were kept under these conditions for 6 weeks then mated with males kept under normal conditions. Vitamin deplete dams were kept under these conditions during pregnancy. Offspring from two test groups were examined. Offspring were either reared with dams repleted with vitamin D at birth or remained under deplete conditions till weaning. Both test groups were weaned under normal vitamin D conditions and remained so till testing at adulthood. We compared the brains of adult offspring kept under both test conditions with animals from control environments. Summary of results: We found a significant persistent dose-related increase in lateral ventricle volume and alterations in anterior cingulate and prefrontal cortical cell densities (consistent with the known prodifferentiation properties of this steroid). In both test groups we observed a reduced expression of NGF as well as a down-regulation of transcripts coding for GABAA alpha 4 receptor and two neuronal structural elements; MAP2 and Neurofilament L. Conclusion: These findings provide further evidence that vitamin D is involved in brain development. An increase in prefrontal cortical cell density, a reduction neuronal structural elements and persistent ventriculomegaly are all common anatomical findings in the brains of patients with schizophrenia. The specific reduction in transcripts for neuronal structural proteins but not GFAP is also in accordance with the proposal that frontal cortical architecture in schizophrenia reflects a reduction in connectivity rather than a reduction in glial processes(Goldman-Rakic and Selemon, 1997). These findings confirm the biological plausibility of early life hypovitaminosis D as a risk factor for schizophrenia.

Rat osseous plate alkaline phosphatase as Langmuir monolayer - An infrared study at the air-water interface

A glycosylphosphatidylinositol (GPI)-anchored enzyme (rat osseous plate alkaline phosphatase-OAP) was studied as monolayer (pure and mixed with lipids) at the air-water interface. Surface pressure and surface potential-area isotherms showed that the enzyme forms a stable monolayer and exhibits a liquid-expanded state even at surface pressure as high as 30 mN m(-1). Isotherms for mixed dimyristoylphosphatidic acid (DMPA)-OAP monolayer showed the absence of a liquid-expanded/liquid-condensed phase transition as observed for pure DMPA monolayer. In both cases, pure or mixed monolayer, the enzyme preserves its native conformation under compression at the air-water interface as observed from in situ p-polarized light Fourier transform-infrared reflection-absorption spectroscopic (FT-IRRAS) measurements. Changes in orientation and conformation of the enzyme due to the presence or absence of DMPA, as well as due to the surface compression, are discussed. (C) 2008 Published by Elsevier Inc.