Rebecca K. Zoltoski

Knockout of the intermediate filament protein CP49 destabilises the lens fibre cell cytoskeleton and decreases lens optical quality, but does not induce cataract

In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.

Chapter 46: Cholinergic receptor subtypes and REM sleep in animals and normal controls

Publisher Summary Acetylcholine, in conjunction with other neurotransmitter systems, plays a very important role in the regulation of circadian and sleepwake states. To briefly recapitulate, several current basic concepts about the regulation of sleepwake states include: (a) REM sleep, or at least its phasic events (eye movements and PGO spikes), are promoted by cholinergic neurons originating within the peribrachial regions [LDTPPT]; (b) REM sleep may be inhibited by noradrenergic and serotonergic neurons in the locus coeruleus and dorsal raphe, respectively; (c) stages 3 and 4 (Delta) sleep are inhibited by cholinergic terminals from basal forebrain to cortex and from LDT/PPT to thalamus; (d) Delta sleep is modulated by the complex serotonergic mechanisms, for example, it is increased by pharmacological antagonists of 5HT, receptors, although the mechanism and neuroanatomical site at which this effect occurs is unknown. Given the importance of mACHR mediation of components of REM sleep, it is unfortunate that so little is known about the distribution of the various subtypes of mACHRs in brainstem areas, which regulate REM sleep. mACHR subtypes have been identified by molecular, biological, and pharmacological methods. Using pirenzepine, a selective ligand for the M1 mACHR subtype, investigators have shown that M1 receptor subtypes predominate in the hippocampus and in the cortex, whereas the M2 receptor subtypes predominate in the septum, thalamus and brainstem areas. It is mentioned in this chapter that, the role of nicotinic receptors in the regulation of sleep remains poorly understood. Recent developments in the localization of these receptors will stimulate new studies designed to discuss these issues.

The relative effects of selective M1 muscarinic antagonists on rapid eye movement sleep

Three muscarinic antagonists, scopolamine, trihexyphenidyl and biperiden were systemically administered (0, 0.5, 1, 2 and 4 mg/kg) in rats. Scopolamine increased wakefulness and deceased sleep, both slow wave and REM. Trihexyphenidyl increased wakefulness and decreased REM sleep while biperiden decreased REM sleep selectively. The rank order REM-suppressing effect was roughly scopolamine and trihexyphenidyl having a greater suppressing effect than biperiden. These results suggest that the regulation of the sleep-wake cycle is at least partially controlled by the M1 muscarinic receptor.

Biochemical Pharmacology of Sleep

Publisher Summary This chapter describes the biochemical pharmacology of sleep. Various theoretical models have been proposed to account for the regulation of sleep and wakefulness. One model that takes into account both circadian and homeostatic considerations is the two-process model developed by Borbely and associates. The first, process S, is a homeostatic process—the longer the organism is awake, the greater will be its propensity to sleep and to have more intense sleep. The second, process C, reflects an oscillatory process that determines the threshold of sensitivity to the homeostatic factor and thus affects the propensity for sleep and waking. Process C is hypothesized to entrain or be entrained with other oscillators responsible for biologic rhythms, such as temperature, cortisol secretion, and rapid eye movement (REM) sleep. The current widely accepted paradigm is that REM sleep is promoted by cholinergic neurons that originate in the brain stem, most probably the lateral dorsal tegmental nucleus and pedunculopontine tegmental group, and that it is inhibited by noradrenergic and serotonergic neurons located in the locus ceruleus and dorsal raphe, respectively. Cholinergic neurons exert a widespread and crucial role in the orchestration of REM sleep, through their projections to medial pontine reticular formation, medulla, and forebrain areas in the thalamus and basal forebrain.

The expression of m1-m3 muscarinic receptor mRNAs in rat brain following REM sleep deprivation.

We used in situ hybridization histochemistry to study the effects of REM sleep deprivation on ml-m3 muscarinic receptor mRNA expression in the rat brain. REM sleep deprivation for 72 h did not affect ml receptor mRNA expression. However, we found significantly increased m3 receptor mRNA expression in the pontine nuclei and nucleus accumbens-bed nucleus of the stria terminalis region of REM sleep-deprived rats compared with controls. Paradoxically, we found significantly decreased m2 receptor mRNA expression in the pontine nuclei of REM sleep-derived rats vs controls. The present findings implicate these structures in the cholinergic effector pathways of REM sleep, although the type and magnitude of the effects of these structures on REM sleep may vary with different receptor subtypes.

Connexin23 deletion does not affect lens transparency

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23-null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the co-expressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency.

Physiological and Optical Alterations Precede the Appearance of Cataracts in Cx46fs380 Mice

Purpose Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexin46, Cx46. Lenses from Cx46fs380 mice develop cataracts that are first observed at ∼2 months in homozygotes and at ≥4 months in heterozygotes. The present studies were conducted to determine whether Cx46fs380 mouse lenses exhibited abnormalities before there are detectable cataracts. Methods Lenses from wild-type and Cx46fs380 mice were studied at 1 to 3 months of age. Connexin levels were determined by immunoblotting. Gap junctional coupling was calculated from intracellular impedance studies of intact lenses. Optical quality and refractive properties were assessed by laser scanning and by photographing a 200-mesh electron microscopy grid through wild-type and Cx46fs380 mouse lenses. Results Connexin46 and connexin50 levels were severely reduced in mutant lenses. Gap junctional coupling was decreased in differentiating and mature fibers from Cx46fs380 lenses; in homozygotes, the mature fibers had no detectable coupling. Homozygous lenses were slightly smaller and had reduced focal lengths. Heterozygous and homozygous lenses significantly distorted the electron microscopy grid pattern as compared with wild-type lenses. Conclusions Before cataract appearance, Cx46fs380 lenses have decreased gap junctional conductance (at least in heterozygotes) and alterations in refractive properties (heterozygotes and homozygotes). The decreased focal distance of Cx46fs380 homozygous lenses is consistent with an increase in refractive index due to changes in cellular composition. These data suggest that Cx46fs380 lenses undergo a sequence of changes before the appearance of cataracts: low levels of connexins, decreased gap junction coupling, alterations in lens cell homeostasis, and changes in refractive index.

Central alpha-1-adrenergic control of vasopressin secretion in newborn lambs.

ABSTRACT: The hypothesis that central α-1 adrenoceptors are inhibitory to the hypotension-induced secretion of vasopressin was tested by subjecting lambs that were instrumented for a long term to varying degrees of hypotension after intracerebroventricular injections of prazosin or placebo. Eight lambs in the 1st wk of life treated with intracerebroventricular injections of placebo had their mean arterial blood pressures decreased 14 and 21% by i.v. infusion of nitroprusside. Arginine vasopressin levels rose to 7.3 ± 2.4 pmol/L only with the greater degree of hypotension. When the Iambs were treated with intracerebroventricular injections of 1 μg/kg of prazosin, the blood pressures were decreased 13 and 23%, and the vasopressin levels were 15.4 ± 16.6 and 27.5 ± 20.3 pmol/L, respectively. A relationship was shown between the degree of hypotension and the plasma arginine vasopressin levels with both the placebo and prazosin, the slope being much steeper for the prazosin treatment (—1.11) than for the placebo treatment (—0.31). Plasma renin activity was increased a similar amount in both groups, and there was no change in plasma cortisol levels. We conclude that α-1 adrenoceptors in the brain are inhibitory to the secretion of arginine vasopressin. These results differ from observations in adult rats and dogs and may be accounted for by developmental or species differences.