Decreased density of forebrain cholinergic neurons and disintegration of the spatial organization of behavior in experimental autoimmune dementia (EAD).

Experimental autoimmune dementia (EAD) is a rat model designed to examine the potential role of anti-cholinergic neurons antibodies (Abs) in the neuropathology of Alzheimer's disease (AD) and dementia. We have previously shown that sera of AD and Down's syndrome patients contain Abs which bind specifically to the high molecular weight neurofilament protein (NF-H) of the purely cholinergic electromotor neurons of Torpedo. Production of such Abs in EAD rats by prolonged immunization with Torpedo cholinergic NF-H results in the accumulation of IgG in the septum and hippocampus of the immunized rats and in memory deficits. In the present study, we examined immunohistochemically whether the anti-cholinergic NF-H immune response of the EAD rats affects their brain cholinergic neurons. In addition, since dementia is associated with severe deterioration in the spatio-temporal organization of behavior, we examined whether EAD rats also mimic this important feature of dementia. The results obtained show that production in EAD rats of anti-cholinergic NF-H Abs similar to those found in AD patients results in a marked decrease in the density of forebrain cholinergic neurons and in derangements in the spatio-temporal organization of their behavior. These findings may replicate pathogenic processes in AD and support a role for anti-cholinergic NF-H Abs in the degeneration of cholinergic neurons in the disease.

Disintegration of the spatial organization of behavior in experimental autoimmune dementia.

Experimental autoimmune dementia is a rat model designed to examine the potential role of anti-cholinergic neurons antibodies in neuronal degeneration in dementia and Alzheimer's disease. We have previously shown that sera of patients with Alzheimer's disease contain antibodies which bind specifically to the high molecular weight neurofilament protein of the purely cholinergic electromotor neurons of Torpedo. Production of such antibodies in experimental autoimmune dementia rats by prolonged immunization with the Torpedo cholinergic high molecular weight neurofilament subunit results in accumulation of antibodies in the septum and hippocampus of the immunized rats, in a marked decrease in the density of forebrain cholinergic neurons, and in memory deficits. In the present study we characterized the open-field behavior of experimental autoimmune dementia rats, and examined whether, like in dementia, the spatiotemporal organization of their behavior is impaired. The results obtained revealed that experimental autoimmune dementia rats travel shorter distances; explore a smaller part of the open-field; and perform less round-trips to the key location--the home base--in reference to which their behavior is normally organized. The shrinkage of the explored space and the reduced number of round trips are independent of the amount of locomotion and represent a deterioration in the organization of behavior in time and space. These behavioral changes are specific to the anti-cholinergic immune response of experimental autoimmune dementia rats as they are not observed in rats which were immunized with chemically heterogeneous high molecular weight neurofilament subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased density of forebrain cholinergic neurons in experimental autoimmune dementia.

Sera of Alzheimer's disease and Down's syndrome patients contain antibodies which bind specifically to the high molecular weight neurofilament protein of Torpedo cholinergic neurons. We have recently shown that prolonged immunization of rats with this antigen results in the accumulation of IgG in neurons in the septum and hippocampus of the immunized rats and in cognitive impairments. This animal model is termed experimental autoimmune dementia. In the present study we examined whether the anti-cholinergic high molecular weight neurofilament subunit immune response of the experimental autoimmune dementia rats affects forebrain cholinergic neurons. This was performed immunohistochemically utilizing a monoclonal antibody to nerve growth factor receptor, a specific marker of cholinergic neurons in the forebrain. The results obtained revealed significant decreases in the density of cholinergic neurons in the medial septal nucleus and diagonal band of the experimental autoimmune dementia rats. These decreases are specific to the anti-cholinergic high molecular weight neurofilament subunit immune response of the experimental autoimmune dementia rats and are not observed in control rats which were immunized with chemically heterogeneous high molecular weight neurofilament subunit. The decrease in density of forebrain cholinergic neurons in experimental autoimmune dementia rats may mimic pathogenic processes in Alzheimer's disease and supports a role for anti-cholinergic high molecular weight neurofilament subunit antibodies in the degeneration of cholinergic neurons in the disease.

Monoclonal antibodies to the heavy neurofilament subunit (NF-H) of Torpedo cholinergic neurons.

Previous studies from our laboratory suggest that Alzheimer's disease sera contain a repertoire of antibodies to the heavy neurofilament subunit (NF-H) and that a subpopulation of these antibodies bind specifically to epitopes highly enriched in NF-H isolated from the purely cholinergic electromotor neurons of Torpedo. In the present study, we prepared and characterized monoclonal antibodies (MAbs) that bind to epitopes specifically enriched in Torpedo cholinergic neurons. This was performed by a differential enzyme-linked immunosorbent assay (ELISA) in which MAbs were selected that bind to epitopes much more abundant in the NF-H protein of Torpedo cholinergic neurons than in NF-H from the chemically heterogeneous Torpedo spinal cord. This yielded four MAbs, three of which (TC4, TC8, and TC21) were found to be specific to NF-H and one (TC15) that reacts with both NF-H and the medium-size neurofilament subunit NF-M. Dephosphorylation abolishes the binding of MAbs TC4 and TC15 to Torpedo cholinergic NF-H, partially reduces that of MAb TC21 and has no effect on the binding of MAb TC8. This suggests that the antigenic sites specific to Torpedo cholinergic NF-H contain phosphorylated as well as non phosphorylated epitopes. All the MAbs cross-react with rat brain NF-H.

Anti-neuronal antibodies similar to those found in Alzheimer's disease induce memory dysfunction in rats.

Although the etiology and pathogenesis of the cholinergic degeneration in Alzheimer's disease are not known, several reports implicate immunological mechanisms. Recently we have shown that sera of Alzheimer's disease patients contain antibodies which bind specifically to the heavy molecular weight neurofilament protein of Torpedo cholinergic neurons. In the present study we investigated the possibility that such antibodies play a role in neuronal degeneration by examining the behavioral and cellular effects of immunizing rats with the heavy neurofilament protein of Torpedo cholinergic neurons. The immunized rats developed antibodies which were specific to the heavy neurofilament protein of Torpedo cholinergic neurons and which cross-reacted with rat brain neurofilaments. Immunohistochemical studies revealed the accumulation of antibodies in the perikarya and neurites of neurons in the septum and hippocampus of the cholinergic neurofilament immunized rats and in white matter tracts in their forebrains. No such staining was seen in adjuvant immunized control rats. Behavioral tests revealed that rats immunized with the heavy cholinergic neurofilament protein performed significantly worse than controls in a T-maze alternation test and that their performance deteriorated profoundly after the introduction of a 20-s delay in the paradigm, indicating a deficit in short term memory. In contrast, both groups performed similarly in a T-maze discrimination test, indicating that long term reference memory was not affected by immunization with the heavy cholinergic neurofilament protein. Further experiments revealed that the rats immunized with the heavy cholinergic neurofilament protein were also deficient in a reversal of choice paradigm in a position discrimination test.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of H1 and H2 histamine receptor antagonists on positive feed-back effect of estrogen on LH in prepubertal female rats.

The aim of the present study was to determine whether histaminergic central mechanisms which exert a well known effect on gonadotrophin secretion are involved in the development of the positive feed-back effect of estrogen-progesterone (E-P) on LH secretion that normally occurs in female rats about 20-22 days old. The administration of histamine H2 (cimetidine and ranitidine) or H1 (diphenhydramine) receptor blocking agents did not modify the onset of the LH release response to E-P. Nevertheless cimetidine, ranitidine and diphenhydramine potentiated the LH release induced by ovarian steroids at 23 days of age. These results appear to indicate that histaminergic pathways are involved in the magnitude of the LH response to E-P in prepubertal female rats rather than in the maturation of this mechanism.

Effect of naloxone on the development of the positive feed-back action of oestrogen-progesterone on LH secretion in rats.

The purpose of this study was to examine the role of opiate peptides in the development of the positive feedback effect of ovarian hormones (Oe-P) on the LH secretion that matures in female rats at about the age of 20-22 days. Oe-P administration at the age of 14 days induced a significant decrease of LH levels. A single injection of naloxone (5 mg/kg) induced a significant release of LH. This release was completely blocked by Oe-P administration. At the age of 20 days, Oe-P did not induce any significant change of LH levels, whereas naloxone increased the serum LH concentration. On the other hand, injection of Oe-P into naloxone-treated rats induced a significant rise in LH that was significantly higher than that observed with naloxone alone (P less than 0.025). Oe-P administration induced a positive feedback effect on LH at the age of 25 days. At this age, naloxone also increased LH levels and a significant potentiation of the LH release in response to Oe-P was observed in a group treated with naloxone. These results indicate that naloxone advances the development of the positive feedback mechanism of ovarian hormones on LH secretion and potentiates this mechanism after its maturation. On this basis it is proposed that the probable inhibitory effect of opiates on the onset of the positive feedback mechanism is related to the well-known participation of the opiate system in the onset of puberty.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of sexual differentiation of the hypothalamus in the differential effect of the serotoninergic system on LH in prepubertal male and female rats.

5-hydroxytryptophan (5-HTP), a serotonin precursor, has been shown to induce LH release in female but not in male rats at 16, 18 and 20 days of age. The purpose of this study was to investigate the role of neonatal gonadal hormones on 5-HTP-induced LH release in male and female rats of 16 and 20 days of age. The following groups of rats were studied control females, androgenized females, control males, and castrated males. Androgenization and castration were performed within 48 h of birth. 5-HTP administration increased LH concentration in normal females and castrated males at 16 and 20 days of age. The LH release response to 5-HTP was significantly lower at 20 than at 16 days of age in both groups. Neonatal androgenization abolished the LH release response in prepubertal female rats. These results indicate that neonatal exposure to androgen is responsible for the sex difference in the LH response to 5-HTP, which may be associated with a regulatory action of serotonin on the cyclic hypothalamic control of gonadotrophin secretion.

Sexual differences in the effect of serotonin on LH secretion in rats.

The effect of 5-hydroxytryptophan (5-HTP), a serotonin (5-HT) precursor, on luteinizing hormone (LH) secretion was studied in prepubertal male and female rats of different ages. In females 5-HTP stimulated LH release on days 16, 18 and 20 but not in older rats (26, 30, 35 days of age). No effects of 5-HTP on LH levels were observed in males. The positive feed-back mechanism of oestrogen-progesterone (E-P), that normally matures in the female between 20 and 26 days, was inhibited by 5-HTP in all the ages studied during prepuberty (26, 30 and 35 days old). On the other hand, in adult ovariectomized rats, 5-HTP administration not only decreased the high LH levels induced by ovariectomy, but the LH release response to E-P as well. These results indicate that there are sexual differences in the effect of 5-HT on LH in prepubertal rats younger than 26 days old. Administration of p-chloroamphetamine (PCA) a serotonin neurotoxin selective for serotoninergic neurons that depletes 5-HT levels in the brain, induced a significant increase in the LH release response to LRH in females, but had no effect in males. These results, besides suggesting a probable pituitary participation in the 5-HT action on LH secretion in the female, appear to indicate the existence of sexual differences in the effect of 5-HT in adult rats.

Effect of the serotoninergic system on luteinizing hormone secretion in prepubertal female rats.

The effect of 5-hydroxytryptophan (5-HTP), a serotonin precursor, and p-chloroamphetamine (PCA), a serotonin neurotoxin, selective for serotoninergic neurons, that depletes serotonin (5-HT) levels in brain, on the luteinizing hormone (LH) release response to estrogen-progesterone (E-P) was studied in prepubertal female rats of different ages. E-P decreased LH levels on days 16, 18 and 20, increasing the levels of the pituitary hormone at day 26 of age. Destruction of the serotoninergic system advanced the onset of the positive feed-back mechanism, since the rats pretreated with PCA showed at day 20 an LH release by E-P administration while in the controls of the same age the ovarian hormones decreased the LH concentration. On the other hand, PCA potentiated the positive feed-back mechanism of E-P on LH in 26-day-old rats, while at this age the LH release response to E-P was significantly reduced by the administration of 5-HTP. These results suggest that the serotoninergic system has an inhibitory effect on the development of the positive feed-back of ovarian steroids on LH secretion, that could be representative of a regulatory participation of serotonin in the onset of puberty. 5-HTP stimulated LH release on days 16, 18 and 20, but did not modify the LH concentration of day 26. Since between 20 and 26 days of age the positive feed-back mechanism matures, the possibility arise that the modification in the effect of serotonin on LH release on day 26 is connected with the physiological changes in the gonadotropin control that occur after day 20 in the female rat.

Pinealectomy advances the time of development of steroid feedback on luteinizing hormone release in immature female rats.

The effect of pinealectomy (Px) on the development of steroid positive feedback on luteinizing hormone (LH) release was examined in female rats subjected to surgery at 10 days of age. Estradiol-progesterone injection decreased serum LH in sham Px or intact controls younger than 20 days, while a significant LH release was found at day 22; Px rats showed a steroid-induced LH depression only at day 16, a positive feedback being detectable at day 20, 2 days earlier than in sham Px or intact rats. Daily injections of 10-50 micrograms melatonin to intact rats disrupted the LH negative feedback response at day 20, and diminished steroid-induced LH release at day 24.

Failure of melatonin to increase serum prolactin levels in ovariectomized rats subjected to superior cervical ganglionectomy or pinelaectomy.

The effects of melatonin on serum prolactin levels were examined in ovariectomized rats primed with oestradiol and progesterone, and subjected to bilateral superior cervical ganglionectomy or pinealectomy. Ganglionectomy resulted in a significant depression of the serum prolactin concentrations, as well as in impairment of the prolactin release evoked by administration of steroid. Treatment with melatonin increased serum prolactin in control but not in ganglionectomized rats. Injection of melatonin protentiated the steroid-induced release of prolactin in control rats; this effect of melatonin was not detected in ganglionectomized rats. Pinealectomy did not affect basal prolactin levels, nor impair the release of prolactin evoked by steroid treatment; however, it was effective in blocking the melatonin-induced release of prolactin in vehicle-treated rats, as well as the potentiation of steroid-induced prolactin release by melatonin. Intracranial surgery by itself increased prolactin release. These results suggest that systemically administered melatonin needs an intact pineal gland to augment serum prolactin levels.

Effect of melatonin and superior cervical ganglioectomy on luteinizing hormone on release induced by estradiol-progesterone in castrated rats.

The effect of melatonin on luteinizing hormone (LH) relase was studied in castrated female rats injected with estradiol and progesterone. Melatonin administered s.c. twice daily for 6 days exerted a biphasic, dose-related effect on LH release, the lowest dose (125 micrograms/100 g body weight) being stimulatory and the highest (250 micrograms/100 g body weight) inhibitory. Superior cervical ganglionectomy negated the inhibitory effect of the high metatonin dose on steroid-induced LH release. Rather melatonin injection to ganglionectomized rats resulted in significantly higher serum LH values than those of animals injected with estradiol and progesterone alone. Melatonin treatment failed to modify postcastration serum LH levels.

Effect of estradiol on aminoacid incorporation into proteins of different hypothalamic areas in prepuberal rats.

Estradiol in vitro produces a significant increase in the incorporation of 3H-leucine into proteins of the anterior hypothalamic area in prepuberal female rats, 15 and 20 days old, but not in younger animals. The ovarian hormone induced no changes in the protein synthetic activity of middle and posterior hypothalamus and cerebral cortex in prepuberal female rats of different ages. Estradiol did not modify the protein synthesis of the hypothalamus and cerebral cortex in prepuberal male rats.

Effect of castration upon hypothalamic luteinizing hormone releasing factor (LH-RF).

The effect of castration upon hypothalamic LH-RF synthesis and content was determined in mature male rats. In each single experiment 20 hypothalami from both normal and 60-day-castrated rats were bisected into symmetrical portions. For the determination of LH-RF synthesis 20 hypothalamic halves were incubated for 120 min. After incubation the tissue was homogenized in medium containing 1 N acetic acid, and centrifuged; 1.0 ml of the supernatant was used to test LH-RF activity. In the other 20 halves, hypothalamic LH-RF content was determined: the samples were homogenized without incubation in 1 N acetic acid, centrifuged, and LH-RF activity was tested in 1.0 ml of the supernatant. LH-RF activity was determined in vivo in ovariectomized rats pre-treated with estrogen-progesterone by measuring, by radioimmunoassay, the LH modifications in serum after the i.v. administration of the extracts. Hypothalamic LH-RF content was significantly higher in control than in castrated rats (p is less than 0.001). After incubation there were no changes in the LH-releasing potency of control hypothalami compared with non-incubated tissue, whereas a significant increase (616%) was found after the incubation of hypothalami from castrated rats. On the other hand, after incubation LH-RF activity was higher (p is less than 0.02) in castrated than in control hypothalami. These results suggest that castration stimulates the release and synthesis of hypothalamic LH-RF in male rats.