James A. Joseph

Conformational changes in muscarinic receptors may produce diminished cholinergic neurotransmission and memory deficits in aged rats

Both clinical and laboratory studies suggest that age-related memory deficits may be due, at least in part, to disturbances in muscarinic acetylcholine (mAChR) receptors. In order to further evaluate this premise, the present studies examined the electrophysiological responses rates of hippocampal pyramidal cells to iontophoretically applied ACh in young, middle-age and aged animals. The relationship between age and muscarinic agonist and antagonist binding in the hippocampus was also examined. In addition, possible age-related changes in receptor-effector coupling were assessed by determining calmodulin levels and the activities of phospholipid methyl-transferase I and II. Analysis of electrophysiological data showed selective age-related decrements in the ability of ACh to alter burst rate but not simple spike rate. These age-related decreases in the efficacy of ACh to increase burst rate were not paralleled by decreases in mAChR density as assessed by 3H-QNB binding, but they were temporally paralleled by age-related changes in the ability of oxotremorine to inhibit 3H-QNB binding. In the young animals, the resultant Hill coefficients derived from these analyses approached 1, while in the middle and old aged animals, the Hill coefficients deviated significantly from 1, indicating the possible existence of 2 or more receptor states with differential affinity for oxotremorine in the 2 older age groups. When carbamylcholine was used to inhibit 3H-QNB, these complex binding patterns were seen even in the young, since carbamylcholine induces conformational/orientational changes in the mAChR while oxotremorine does not.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-Related Decrements in the Muscarinic Enhancement of K(+)-Evoked Release of Endogenous Striatal Dopamine: An Indicator of Altered Cholinergic-Dopaminergic Reciprocal Inhibitory Control in Senescence

Previous experiments have indicated that the release of striatal dopamine (DA) is controlled by inhibitory DA autoreceptors which are mediated by inhibitory cholinergic heteroreceptors (HTRs). Activation of the HTRs by muscarinic or nicotine agonists potentiates the K+-evoked release of DA from the striatum. Present experiments were carried out to determine if this relationship is altered as a function of aging. Cross-cut striatal tissue slices obtained from 3 age-groups (6, 12-18 and 24 months) Wistar rats were superfused with a modified Krebs-Ringer basal release medium containing 2.5 mM KCl. After a 30-min equilibration period, a 5-min baseline fraction was collected from each chamber. The medium was then switched to one containing 30 mM KCl, and depending upon the experiment, 1 of 4 concentrations of a particular muscarinic (oxotremorine, pilocarpine, carbachol or bethanecol) or nicotinic (nicotine) agonist. In some experiments DA autoreceptor function was assessed directly with haloperidol. Six 5-min fractions were taken during depolarization. DA release was assessed using high performance liquid chromatography coupled to electrochemical detection. Results indicated that the efficacy of the muscarinic agonists was reduced in an age-dependent manner with the oldest age groups showing the smallest enhancement. The age at which the decline was seen was dependent on the muscarinic agonist that was applied. Deficits were seen as early as 12 months when full agonists (e.g. carbachol) were applied, but did not appear until 18 months when partial agonists (e.g. oxotremorine) were applied. These age-related alterations were not seen when haloperidol or nicotine were used to enhance the K+-evoked release of DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in muscarinic control of striatal dopamine autoreceptors in senescence: a deficit at the ligand-muscarinic receptor interface?

Research has indicated that the release of striatal dopamine (DA) is controlled by inhibitory DA autoreceptors which are in turn regulated by inhibitory muscarinic inhibitory cholinergic heteroreceptors (HTRs) located in close vicinity to the autoreceptors. Muscarinic activation enhances K+-evoked release of DA from striatal slices from mature but not senescent rats. Since it has been shown that age-dependent declines in Ca2+ mediated acetylcholine release can be restored by the ionophore A23187, it was of interest to determine if age-related decrements in Ca2+ mobilization might contribute to the alterations in muscarinic control of the striatal DA autoreceptors seen in senescence. Cross-cut striatal tissue slices obtained from two age-groups (6 and 24 months) of Wistar rats were superfused with a modified Krebs-Ringer medium containing 2.5 mM KCl. After a 30-min equilibration period, a 5-min baseline fraction was collected. The medium was then switched to one which contained 30 mM KCl and, depending upon the experiment, the muscarinic agonists carbachol, or oxotremorine or the Ca2+ mobilizing agents A23187 or inositoltrisphosphate (IP3) and enhancement of K+-evoked release of DA was examined. Six 5-min fractions were collected. DA release was determined by HPLC coupled to electrochemical detection. Results indicated that although deficits were seen in oxotremorine and carbachol enhancement of K+-evoked release of DA, these decrements were not observed when either A23187 or IP3 were utilized to enhance the K+-evoked release of DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Deficits in the Sensitivity of Striatal Muscarinic Receptors Induced by 56Fe Heavy-Particle Irradiation: Further 'Age-Radiation' Parallels

We had previously shown that there was a loss of sensitivity of muscarinic receptors (mAChR) to stimulation by cholinergic agonists (as assessed by examining oxotremorine enhancement of K(+)-evoked release of dopamine from neostriatal slices) in animals that had been exposed to energetic particles (56Fe, 600 MeV/n), an important component of cosmic rays. This loss of mAChR sensitivity was postulated to be the result of radiation-induced alterations in phosphoinositide-mediated signal transduction. The present experiments were undertaken as a first step toward determining the locus of these radiation-induced deficits in signal transduction by examining K+ enhancement of release of dopamine in 56Fe-exposed animals (0, 0.1, and 1.0 Gy) with agents [A23187, a potent Ca2+ ionophore, or 1,4,5-inositol trisphosphate (IP3)] that bypass the mAChR-G protein interface and by comparing the response to oxotremorine-enhanced K(+)-evoked release of dopamine. Results showed that although oxotremorine-enhanced K(+)-evoked release of dopamine was reduced significantly in the radiation groups, no radiation effects were seen when A23187 or IP3 was used to enhance K(+)-evoked release of dopamine. Since similar findings have been observed in aging, the results are discussed in terms of the parallels between aging and radiation effects in signal transduction that might exist in the neostriatum.

An assessment of the behavioral toxicity of high-energy iron particles compared to other qualities of radiation

Conditioned taste aversion was used to evaluate the behavioral toxicity of exposure to high-energy iron particles (56Fe, 600 MeV/amu) in comparison to that of gamma photons (60Co), high-energy electrons, or fission neutrons. Exposure to high-energy iron particles (5-500 cGy) produced a dose-dependent taste aversion with a maximal effect achieved with a dose of 30 cGy. Gamma photons and electrons were the least effective stimuli for producing a conditioned taste aversion, with a maximal aversion obtained only after exposure to 500 cGy, while the effectiveness of fission neutrons was intermediate to that of photons and iron particles, and a maximal aversion was obtained with a dose of 100 cGy. In the second experiment, rats with lesions of the area postrema were exposed to iron particles (30 cGy), but failed to acquire a taste aversion. The results indicate that (1) high-energy iron particles are more toxic than other qualities of radiation and (2) similar mechanisms mediate the behavioral toxicity of gamma photons and high-energy iron particles.

Behavioral and neurochemical abnormalities after exposure to low doses of high-energy iron particles.

Exposure of rats to high-energy iron particles (600 MeV/amu) has been found to alter behavior after doses as low as 10 rads. The performance of a task that measures upper body strength was significantly degraded after irradiation. In addition, an impairment in the regulation of dopamine release in the caudate nucleus (a motor center in the brain), lasting at least 6 months, was also found and correlated with the performance deficits. A general indication of behavioral toxicity and an index of nausea and emesis, the conditioned taste aversion, was also evident. The sensitivity to iron particles was 10-600 times greater than to gamma photons. These results suggest that behavioral and neurobiological damage may be a consequence of exposure to low doses of heavy particles and that this possibility should be extensively studied.

Behavioral endpoints for radiation injury

The relative behavioral effectiveness of heavy particles was evaluated. Using the taste aversion paradigm in rats, the behavioral toxicity of most types of radiation (including 20Ne and 40Ar) was similar to that of 60Co photons. Only 56Fe and 93Nb particles and fission neutrons were significantly more effective. Using emesis in ferrets as the behavioral endpoint, 56Fe particles and neutrons were again the most effective; however, 60Co photons were significantly more effective than 18 MeV electrons. These results suggest that LET does not completely predict behavioral effectiveness. Additionally, exposing rats to 10 cGy of 56Fe particles attenuated amphetamine-induced taste aversion learning. This behavior is one of a broad class of behaviors which depends on the integrity of the dopaminergic system and suggests the possibility of alterations in these behaviors following exposure to heavy particles in a space radiation environment.

Desensitization of muscarinic acetylcholine receptors: possible relation to receptor heterogeneity and phosphoinositides.

The increases in firing rates of hippocampal cells were examined following microiontophoretic application of several muscarinic cholinergic receptor agonists. The agonists studied had been pharmacologically characterized previously and divided into two classes: class A agonists (e.g. acetylcholine, carbamylcholine, and oxotremorine-M) which maximally stimulate PI turnover and reveal mAChR heterogeneity, and class B agonists (e.g. bethanecol and oxotremorine-1) which poorly stimulate PI turnover and do not alter mAChR conformation/orientation in the hippocampus. While comparable stimulatory effects on hippocampal pyramidal cell firing rates were seen with both classes of agonists during short (20 s) ejection periods, longer applications (greater than 25 s) produced class-dependent differential firing patterns. Prolonged ejection of class A agonists selectively desensitized cells to further, continued application in the same ejection period, and the firing rates declined. Class B agonists produced stimulatory responses in hippocampal cells during the entire ejection period, and DE was not observed. This desensitization effect (DE) was observed only for bursts and not for simple spikes.

Reduction of motor behavioral deficits in senescent animals via chronic prolactin administration II. Non-stereotypic behaviors

The effects of chronic prolactin administration on three non-stereotypic psychomotor behaviors (inclined screen performance, rod walking and wire hanging) were examined in senescent (24 month) Fisher 344 rats. Prolactin (150 ng/hr) was administered for 7 days via Alzet minipumps to rats which had been pretested on the three tasks. The animals were tested on days 4 and 7 following the implants and the pumps were removed after testing on day 7. In order to assess the persistence of any prolactin effects on psychomotor performance the animals were tested again on days 7 and 14 after pump removal. Control animals were implanted with Alzet pumps containing only saline and tested in a similar manner. Results demonstrated that the prolactin treated animals showed improvements on both the inclined screen and rod walking tasks but not the wire hanging test. Moreover, the improved performance seen on the inclined screen test persisted for as long as 7 days after pump removal. Results are discussed in terms of the importance of techniques which increase striatal dopaminergic responsivity on enhancing psychomotor performance in senescence.

Reduction of motor behavioral deficits in senescent animals via chronic prolactin administration. I. Rotational behavior.

The effects of chronic prolactin administration on amphetamine or dopamine (DA) induced rotational behavior was examined in mature (6 month) and senescent (24 month) Wistar rats which were unilaterally lesioned in the left substantia nigra with 6-hydroxydopamine. Prolactin (150 ng/hr) was administered for 7 days via subcutaneously implanted Alzet minipumps. Amphetamine (AMPH) (0, 10 micrograms) or DA (0, 25 micrograms) was administered through cannula which had been implanted into the right (intact) striatum. Both DA-active agents were given prior to pump implantation and on day 4 of prolactin administration. The AMPH was dissolved in saline (1 microliter; pH, 5.5-6.0), while DA was dissolved in N2 bubbled distilled H2O (1 microliter; pH, 5.5-6.0) and the animals were pretreated with nialamide (50 mg/kg) intraperitoneally 1 hr before DA or DA-vehicle injection. The order of drug administration was counterbalanced within the age groups. Results showed that both groups of animals exhibited higher rotational behavior scores following prolactin treatment. In fact, there was a trend toward greater enhancement of rotational behavior in the senescent animals following prolactin treatment than that seen in mature animals. These results parallel those reported previously wherein it was found that striatal DA receptor concentrations (as assessed with [3H]spiperone binding) were higher in prolactin treated mature and senescent animals than in their respective controls. The findings suggest that there is a relationship between increases in the density of striatal DA receptors and improvement in motor performance tasks in senescent animals.