Brain angiotensin receptor subtypes in the control of physiological and behavioral responses

The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models.

Regulation of muscarinic acetylcholine receptor signaling

The evoked expression of the immediate early gene (IEG)-encoded proteins c-Fos and Krox-24 was used to monitor spinal visceronociceptive processing that results from cyclophosphamide cystitis in behaving rats. Animals received a single dose of 100 mg/kg i.p. of cyclophosphamide and survived for 30 min to 5 h. Longer survival times were not considered because of ethical considerations. Cyclophosphamide-injected animals developed characteristic behavioral signs in parallel with development of bladder lesions and spinal evoked expression of IEG-encoded proteins. Histological examination of the urinary bladder was used to evaluate the degree of cystitis and as a criterion for selection of groups of animals to be quantitatively analyzed. Controls consisted of freely behaving animals including control (uninjected), sham (saline-injected) or diuretic (furosemide-injected) animals. Behavioral modifications consisted of lacrimation, piloerection, assumption of a peculiar “rounded-back” posture, which was accompanied by head immobility and various brief “crises” (tail hyperextension, abdominal retractions, licking of the lower abdomen, backward withdrawal movements). Abnormal behaviors, which first appeared (lacrimation, piloerection) at the end of postinjection hour 1, progressively increased in severity (rounded-back posture) over the following 90 min to reach a plateau at about postinjection hour 2; the rounded-back posture was maintained up to time of death. Histological modifications of bladder tissue were assessed using a 4-grade scale in a blind setting. The 1st grade consisted of control or sham animals with no bladder lesion; 2nd grade, animals with simple chorionic edema; 3rd grade, animals with chorionic edema associated with mucosal abrasion, fibrin deposit, and onset of polymorphonuclear leukocyte infiltration; 4th grade, animals with complete cystitis corresponding to an increase in severity and spread of all the signs of cystitis described above plus petechial hemorrhage. Simple chorionic edema was observed from 30 min to 3 h post-injection, but with a progressive increase in severity over time. Edema accompanied by epithelial abrasion was observed for animals that survived 3–4 h postinjection; complete inflammation was observed in animals that survived 4–5 h postinjection. The study of c-Fos- and Krox-24-encoded protein expression demonstrated that few lumbosacral spinal areas were specifically involved in the processing of visceral inputs in response to bladder stimulation. These areas were the parasympathetic column (SPN), the dorsal gray commissure (DGC as the caudal extent of lamina X), and superficial layers of the dorsal horn. Differential basal and evoked labeling between c-Fos and Krox-24 allowed a functional distinction between these spinal subregions: Krox-24 was an indicator of the first signs of inflammation (simple chorionic edema); c-Fos was an indicator of more pronounced impairments. DGC, which displayed a high level of basal Krox-24 expression and in which both Krox-24 and c-Fos were evoked in relation to disease evolution, probably codes inputs from both physiological (progressive bladder filling) and all types of pathological (inflammatory processes, overdistension) conditions. SPN, which, except for basal Krox-24 expression, responded like DGC and contains preganglionic motor cells, probably codes inputs involved in eliciting motor activity that results in bladder emptying as needed from natural filling and/or pathological situations. Laminae I and II, which responded like DGC and SPN with respect to Krox-24 expression, may code inputs from physiopathological changes of the bladder, though lamina I would be primarily involved in pain processing as it was the only region in which c-Fos expression increased during abrasion and complete inflammation. The cyclophosphamide-cystitis model has the following unique features compared with other visceral models: first, the stimulus is a “pure” visceral one, confined to one viscus (bladder; no somatic stimulation is induced by either introducing a stimulating device or from surgical wounds, and no anesthesia is required); second, it is the exact replica of a human disease, and its evolution can be efficently monitored through behavioral and histological observations; third, it can be used in behaving animals without departing from ethical rules.

Cyclophosphamide cystitis as a model of visceral pain in rats: model elaboration and spinal structures involved as revealed by the expression of c-Fos and Krox-24 proteins

We have examined several aspects of neurotransmitter function in the brains of mice carrying a deletion mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). During the first 6 weeks of postnatal development, dopamine levels in whole-brain extracts from the mutant mice (HPRT-) failed to increase at rates comparable to normal animals, resulting in 40% lower dopamine levels throughout adulthood. Regional analysis in adult animals showed the caudoputamen to be the most severely affected region, with dopamine deficits of 48-64%. Dopamine levels in other regions were normal or less severely affected. The decrease in dopamine was accompanied by a decrease in tyrosine hydroxylase (TH) activity, the rate-limiting step in dopamine synthesis. Kinetic analysis of TH extracted from the caudoputamen of normal and HPRT- mice demonstrated a 45% decrease in Vmax with an increased affinity for the tetrahydropterin cofactor in the mutants. Labeling of midbrain dopamine neurons using TH immunohistochemistry revealed no obvious deficits in the number of midbrain dopamine neurons, but quantitative autoradiographic studies revealed significant reductions in the binding of 3H-N-[1-(2-benzo(beta)thiophenyl)cyclohexyl]piperidine (3H-BTCP) to dopamine uptake sites in the forebrain of the mutants. In contrast to these abnormalities of the dopamine systems in the mutant mice, other neurotransmitter systems appeared relatively unaffected. Norepinephrine, 5-HT, tryptophan hydroxylase, and glutamic acid decarboxylase were present at normal levels in the brains of the mutants. ChAT activity was slightly lower than normal in the caudoputamen of the mutant animals, but was normal in all other brain regions examined. These results indicate that HPRT deficiency is associated with a relatively specific deficit in basal ganglia dopamine systems that emerges during the first 2 months of postnatal development.

https://www.jneurosci.org/content/jneuro/14/3/1164.full.pdf

Dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease

The rough handling with repeated saline administration (1.2 ml/kg s.c. for 7 days) enhanced cortical c-fos mRNA expression in the rat brain after a single saline stimulation (1.2 ml/kg s.c.) due to increasing baseline c-fos mRNA levels, whereas the gentle handling with repeated saline administration declined c-fos mRNA expression after a single injection due to decreasing the baseline of c-fos mRNA levels. These two types of handling with the repeated injection led to diametrically opposite results on c-fos mRNA expression after a single stimulation. Neither two types of handling with repeated saline injections affected the net increment of c-fos mRNA induction after a single stimulation, therefore, the effects of handling with repeated treatment on c-fos mRNA expression might be independent of the effects of a single saline stimulation. The present study suggests that c-fos mRNA induction after a single stimulation might be affected by the types or intensities of handling and that care must be taken to estimate c-fos mRNA induction.

Opposite effects of rough and gentle handling with repeated saline administration on c-fos mRNA expression in the rat brain.

Post-ischemic administration of bifemelane hydrochloride prohibits ischemia-induced depletion of the muscarinic M1-receptor and its mRNA in the gerbil hippocampus

Ischemia-induced changes in α-tubulin and β-actin mRNA in the gerbil brain and effects of bifemelane hydrochloride

To clarify the interactions between dopamine receptors and muscarinic cholinergic receptors by which neurotransmitters may affect genetic responses, we studied the effects of the muscarinic cholinergic agonist, carbachol, and the muscarinic cholinergic antagonist, trihexyphenidyl, on levodopa-induced c-fos messenger RNA (mRNA) expression in rat striatum. Animals were administered levodopa (levodopa with one-tenth dosage of carbidopa), carbachol or thrihexyphenidyl alone or administered in combination as levodopa (100 mg/kg) + carbachol, or levodopa + trihexyphenidyl given as a single bolus. Levodopa given alone increase the expression of c-fos mRNA. Although carbachol or trihexyphenidyl alone was ineffective in inducing c-fos mRNA, the combination of levodopa and carbachol (⩾ 0.1 mg/kg) significantly suppressed the induction of c-fos mRNA as compared with levodopa given alone. The combined administration of levodopa and trihexyphenidyl showed a trend toward an additive effect on the induction of c-fos mRNA vs levodopa alone. These findings suggest that the muscarinic cholinergic system may modulate the levodopa-induced c-fos mRNA expression which then regulates the expression of other mRNAs.

H. Chou

Papers

Opposite effects of rough and gentle handling with repeated saline administration on c-fos mRNA expression in the rat brain.

Post-ischemic administration of bifemelane hydrochloride prohibits ischemia-induced depletion of the muscarinic M1-receptor and its mRNA in the gerbil hippocampus

Ischemia-induced changes in α-tubulin and β-actin mRNA in the gerbil brain and effects of bifemelane hydrochloride

Muscarinic cholinergic receptor-mediated modulation on striatal c-fos mRNA expression induced by levodopa in rat brain.

Rapid response of striatal muscarinic M1-receptor mRNA to muscarinic cholinergic agents in rat brain.