Martin I. Smith

The novel brain neuropeptide, orexin-A, modulates the sleep–wake cycle of rats

Orexin‐A is a novel neuropeptide initially isolated from hypothalamic extracts but now known to be present in fibres distributed throughout the rat CNS including many regions associated with sleep–wake regulation. The recognition of a particularly dense innervation of orexinergic nerves in the locus coeruleus, together with the observed increase in firing rate of locus coeruleus neurons following application of orexin‐A in vitro, further highlighted a potential involvement of the peptide in modulating the arousal state. The present study was undertaken to determine the effects of intracerebroventricularly (ICV) administered orexin‐A on the sleep–wake cycle of conscious rats using electroencephalographic and electromyographic recordings. When administered at the onset of the normal sleep period, orexin‐A (1, 10 or 30 μg/rat ICV) produced a dose‐dependent increase in the time rats spent awake during the second and third hours after dosing. The enhancement of arousal was accompanied by a marked reduction in paradoxical sleep and deep slow wave sleep at the highest dose. The latency to the first occurrence of paradoxical sleep was also prolonged. This overall profile of increased arousal and decreased paradoxical sleep is consistent with a high rate of firing of locus coeruleus neurons as would be expected to occur following ICV administration of orexin‐A. It is concluded that orexin‐A may play an important physiological role in regulating the sleep–wake state, a hypothesis considerably strengthened by the recently reported narcoleptic phenotype of prepro‐orexin (the precursor for orexin‐A) knockout mice.

Characterization of SB-269970-A, a selective 5-HT7 receptor antagonist

The novel 5‐HT7 receptor antagonist, SB‐269970‐A, potently displaced [3H]‐5‐CT from human 5‐HT7(a) (pKi 8.9±0.1) and 5‐HT7 receptors in guinea‐pig cortex (pKi 8.3±0.2). 5‐CT stimulated adenylyl cyclase activity in 5‐HT7(a)/HEK293 membranes (pEC50 7.5±0.1) and SB‐269970‐A (0.03–1 μM) inhibited the 5‐CT concentration‐response with no significant alteration in the maximal response. The pA2 (8.5±0.2) for SB‐269970‐A agreed well with the pKi determined from [3H]‐5‐CT binding studies. 5‐CT‐stimulated adenylyl cyclase activity in guinea‐pig hippocampal membranes (pEC50 of 8.4±0.2) was inhibited by SB‐269970‐A (0.3 μM) with a pKB (8.3±0.1) in good agreement with its antagonist potency at the human cloned 5‐HT7(a) receptor and its binding affinity at guinea‐pig cortical membranes. 5‐HT7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB‐269970‐A was CNS penetrant (steady‐state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min−1 kg−1). Following a single dose (3 mg kg−1) SB‐269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea‐pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5‐CT (0.3 mg kg−1 i.p.) induced hypothermia in guinea‐pigs was blocked by SB‐269970‐A (ED50 2.96 mg kg−1 i.p.) and the non‐selective 5‐HT7 receptor antagonist metergoline (0.3–3 mg kg−1 s.c.), suggesting a role for 5‐HT7 receptor stimulation in 5‐CT induced hypothermia in guinea‐pigs. SB‐269970‐A (30 mg kg−1) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3  h of EEG recording in conscious rats.

A STUDY OF ROTATIONALLY INVARIANT AND SYMMETRIC INDICES OF DIFFUSION ANISOTROPY

This study investigated the properties of a class of rotationally invariant and symmetric (relative to the principal diffusivities) indices of the anisotropy of water self-diffusion, namely fractional anisotropy (FA), relative anisotropy (RA), and volume ratio (VR), with particular emphasis to their measurement in brain tissues. A simplified theoretical analysis predicted significant differences in the sensitivities of the anisotropy indices (AI) over the distribution of the principal diffusivities. Computer simulations were used to investigate the effects on AI image quality of three magnetic resonance (MR) diffusion tensor imaging (DTI) acquisition schemes, one being novel: the schemes were simulated on cerebral model fibres varying in shape and spatial orientation. The theoretical predictions and the results of the simulations were corroborated by experimentally determined spatial maps of the AI in a normal feline brain in vivo. We found that FA mapped diffusion anisotropy with the greatest detail and SNR whereas VR provided the strongest contrast between low- and high-anisotropy areas at the expense of increased noise contamination and decreased resolution in anisotropic regions. RA proved intermediate in quality. By sampling the space of the effective diffusion ellipsoid more densely and uniformly and requiring the same total imaging time as the published schemes, the novel DTI scheme achieved greater rotational invariance than the published schemes, with improved noise characteristics, resulting in improved image quality of the AI examined. Our findings suggest that significant improvements in diffusion anisotropy mapping are possible and provide criteria for the selection of the most appropriate AI for a particular application.

Evidence implicating a role for orexin-1 receptor modulation of paradoxical sleep in the rat

The neuropeptide orexin-A modulates the sleep-wake cycle such that central administration to rats increases arousal, reduces slow-wave-sleep (SWS) and paradoxical sleep (PS) and delays PS onset. The contribution of orexin-1 and -2 receptor (OXR) activation to this orexin-A response is still unknown. Using the OX(1)R antagonist SB-334867-A we investigated the role of this receptor in orexin-A-induced PS alteration. Male rats prepared for frontal-occipital electroencephalograph, nuchal muscle electromyograph recording and lateral ventricle cannulae received vehicle or orexin-A (10 microg icv) at lights on in combination with vehicle or SB-334867-A (10 or 30 mg/kg ip) 30 min pre-icv injection. The amount of arousal, SWS 1, SWS 2 and PS was determined during the 1st h post icv administration along with the latency to onset of the first> or =10 s epoch of PS. Orexin-A administration reduced the amount and increased the latency to onset of PS. SB-334867-A reversed this effect of orexin-A. The present study demonstrates that the OX(1)R also has a role in orexinergic sleep modulation.

Cortical spreading depression in the gyrencephalic feline brain studied by magnetic resonance imaging

1 Time‐lapse diffusion‐weighted magnetic resonance imaging (DWI) was used to detect and characterize complex waves of cortical spreading depression (CSD) evoked with KCl placed upon the suprasylvian gyrus of anaesthetized cats. 2 The time‐lapse representations successfully demonstrated primary CSD waves that propagated with elliptical wavefronts selectively over the ipsilateral cerebral hemispheres with a velocity of 3.8 ± 0.70 mm min−1 (mean ± s.e.m. of 5 experiments). 3 In contrast, the succeeding secondary waves often remained within the originating gyrus, were slower (velocity 2.0 ± 0.18 mm min−1), more fragmented and varied in number. 4 Computed traces of the apparent diffusion coefficients (ADCs) showed negative deflections followed by monotonic decays (amplitudes: primary wave, ‐19.9 ± 2.8 %; subsequent waves, ‐13.6 ± 1.9 %; duration at half‐maximal decay, 150‐200 s) when determined from regions of interest (ROIs) through which both primary and succeeding CSD waves propagated. 5 The passage of both the primary and the succeeding waves often correlated with transient DC potential deflections recorded from the suprasylvian gyrus. 6 The detailed waveforms of the ADC and the T2*‐weighted (blood oxygenation level‐dependent: BOLD) traces showed a clear reciprocal correlation. These imaging features that reflect disturbances in cellular water balance agree closely with BOLD measurements that followed the propagation velocities of the first and subsequent CSD events. They also provide a close physiological correlate for clinical observations of cortical blood flow disturbances associated with human migraine.

Diffusion-Weighted MRI Used to Detect in Vivo Modulation of Cortical Spreading Depression: Comparison of Sumatriptan and Tonabersat

Spreading cortical depolarization and depression of electroencephalographic activity (SD) may underlie the aura and spreading neurovascular events of migraine. Cortical depolarization may also precipitate the progressive development of cerebral pathology following ischemia. However, data on SD in the human brain are sparse, most likely reflecting the technical difficulties involved in performing such clinical studies. We have previously shown that the transient cerebral water disturbances during SD can be quantitatively investigated in the gyrencephalic brain using repetitive diffusion-weighted magnetic resonance imaging (DWI). To investigate whether DWI could detect modulation of the spatiotemporal properties of SD in vivo, the effects of the antimigraine drug sumatriptan (0.3 mg/kg iv) and the novel anticonvulsant tonabersat (10 mg/kg ip) were evaluated in the cat brain. Supporting previous findings, sumatriptan did not affect the numbers of events (range, 4-8), the duration of SD activity (39.8 +/- 4.4 min, mean +/- SEM), and event velocity (2.2 +/- 0.4 mm min(-1)); tonabersat significantly reduced SD event initiation (range, 0-3) and duration (13.2 +/- 5.0 min) and increased primary event velocity (5.4 +/- 0.7 mm min(-1)). However, both drugs significantly decreased, by >50%, the spatial extent of the first KCl-evoked SD event, and sumatriptan significantly increased event propagation across the suprasylvian sulcus (5.5 +/- 0.6 vs 2.4 +/- 0.4 events in controls). These results demonstrate (1) the feasibility of using DWI to evaluate therapeutic effects on SD, and (2) that sumatriptan may directly modulate the spatial distribution of SD activity in the gyrencephalic brain.

Effect of SB-243213, a selective 5-HT2C receptor antagonist, on the rat sleep profile: A comparison to paroxetine

5-HT(2) receptor antagonists promote slow wave sleep (SWS) in humans and rats, conversely 5-HT(2) agonists inhibit SWS in rats. These alterations are thought to be predominantly mediated via the 5-HT(2C) receptor subtype. It is evident that 5-HT(2) receptor function also plays an important role in depression. Here, we examine the acute effect of the selective 5-HT(2C) receptor antagonist 5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-triflouromethylindoline hydrochloride (SB-243213-A) on rat sleep in comparison to the selective serotonin reuptake inhibitor (SSRI) paroxetine. Both SB-243213-A (10 mg/kg po) and paroxetine (3 mg/kg po) significantly increased deep SWS (SWS2) quantity (27% and 24%, respectively) and reduced paradoxical sleep (PS) quantity (35%) during the sleep period. Following SB-243213-A, SWS2 occurrence frequency was reduced (24.1%); however, elevated quantity of SWS2 can be attributed to an increase in occurrence duration (81%). Reduced PS quantity results from a decrease in occurrence frequency (46%). In comparison, paroxetine increased SWS2 occurrence frequency (50%), with decreased frequency (27%) and duration (21%) of PS. The data for SB-243213-A in the present study is consistent with that following ritanserin supporting 5-HT(2C) receptor subtype mediation of this response. The similar effect of SB-243213-A to paroxetine with regard to PS quantity provides further evidence that 5-HT(2C) receptor antagonists maybe beneficial in the treatment of depression/anxiety.

A behavioural characterisation of the FVB/N mouse strain

The use of transgenic models in scientific research has made an enormous contribution to our understanding of the causes and symptoms of many diseases, including neurodegenerative conditions such as Alzheimers Disease (AD) and Parkinsons Disease (PD). In the creation of transgenic models of neurodegenerative disease, effects of the background strain of the animal on the resulting genotype must be taken into consideration. This is particularly true for behavioural studies in which the background strain of the mouse may mask the phenotype of the genetic manipulation. Here, the behaviour of two mouse strains used in transgenic models, FVB/N and C57BL6/J, were compared. Studies of circadian wheel activity, cognition and aggression revealed considerable phenotypic differences between strains. These data also indicate that the FVB/N strain is not appropriate as a background strain in the behavioural assessment of transgenic mouse models.

Cortical spreading depression in the feline brain following sustained and transient stimuli studied using diffusion-weighted imaging

Cortical spreading depression (CSD) was induced by transient (10 min) applications of KCl in agar upon the cortical surface of α‐chloralose anaesthetised cats. Its features were compared with CSD resulting from sustained applications of crystalline KCl through a mapping of the apparent diffusion coefficient (ADC) using diffusion‐weighted echo planar imaging (DWI) over a poststimulus period of 60–100 min. Individual CSD events were computationally detected with the aid of Savitzky‐Golay smoothing applied to critically sampled data derived from regions of interest (ROIs) made up of 2 × 2 pixel matrices. The latter were consistently placed at three selected sites on the suprasylvian gyrus (SG) and six sites on the marginal gyrus (MG). The CSD events thus detected were then quantitatively characterised for each ROI using the original time series. Both stimuli consistently elicited similar spreading patterns of initial, primary CSD events that propagated over the SG and marginal MG and were restricted to the hemispheres on which the stimuli were applied. There followed secondary events over smaller extents of cortical surface. Sustained stimuli elicited primary and secondary CSD events with similar amplitudes of ADC deflection that were distributed around a single mean. The ADC deflections were also conserved in peak amplitude throughout the course of their propagation. The initial primary event showed a poststimulus latency of 1.1 ± 0.1 min. Successive secondary events followed at longer, but uniform, time intervals of around 10 min. Primary and secondary CSDs showed significantly different velocities of conduction (3.32 ± 0.43 mm min−1vs. 2.11 ± 0.21 mm min−1, respectively; n= 5) across the cerebral hemisphere. In contrast, transient stimuli produced significantly fewer numbers of CSD events (3.8 ± 0.5 events per animal, n= 5) than did sustained stimuli (7.4 ± 0.5 events per animal, mean ±s.e.m., n= 5, P= 0.002). The peak ADC deflection of their primary CSD events declined by ≈30 % as they propagated from their initiation site to the interhemispheric boundary. The primary CSD event following a transient stimulus showed a latency of 1.4 ± 0.1 min. It was followed by successive and smaller secondary ADC deflections that were separated by progressively longer time intervals. Conduction velocities of secondary events were similar to those of primary events. Conduction velocities of both primary and secondary events were slower than their counterparts following a sustained stimulus. ADC changes associated with CSD thus persist at times well after stimulus withdrawal and vary markedly with the nature of the initiating stimulus even in brain regions remote from the stimulus site.

A delayed class of BOLD waveforms associated with spreading depression in the feline cerebral cortex can be detected and characterised using independent component analysis (ICA).

An application of independent component analysis to blood oxygenation level- dependent MRI (BOLD-MRI) results was used to detect cerebrovascular changes that followed the initiation of cortical spreading depression (CSD) in feline brain. The cortical images were obtained from a horizontal plane at 28 s intervals before, and for 1.4-1.75 h after, KCl dissolved in agar (KCl/agar) had been directly applied to the left suprasylvian gyrus of 13 anesthetized cats for 10 min. It successfully resolved, for the first time, a novel class of prolonged, and delayed, biphasic BOLD waveforms. These were larger in amplitude ( approximately 20%), longer lasting and more delayed in onset (13-33 min) than the brief propagating (90 s) BOLD increase ( approximately 4%) already known to be associated with CSD on earlier occasions. Furthermore, such changes occurred in localized regions on the hemisphere ipsilateral to the site of stimulus application in 4 out of 5 control subjects rather than themselves generating propagating waves. Finally, the biphasic waveforms were consistently abolished in the 4 experimental animals studied following the i.v. administration of sumatriptan (0.3 mg kg(-1)), an antimigraine 5-HT(1B/1D) agonist, 15 min before the application of the transient stimulus. They were abolished in 2 out of 4 animals following the intraperitoneal (i.p.) administration of SB-220453 (tonabersat: 10 mg kg(-1), 90 min before stimulus application), a novel anticonvulsant that has recently been reported to inhibit CSD. ICA has thus been successful in detecting a novel localized, as opposed to propagating, signal of potential physiological significance hidden in complex BOLD- MRI data, whose sensitivity to sumatriptan may relate it to the cerebrovascular changes reported in the headache phase of migraine.