Anatomical analysis of frontal cortex sites at which carbachol induces motor seizures in the rat.

High amplitude spiking representative of seizures, accompanied by an unusual motor behavior pattern of rearing and forelimbic clonus resembling "boxing," was elicited by microinjection of the cholinergic agonist, carbachol, 4 micrograms, into the medial prefrontal cortex of the rat. A rating scale devised to score the behavior revealed a motor pattern elicited by carbachol from the medial anterior cortex which was similar to that described by Racine for electrical stimulation of the amygdala. Topographical analysis of the areas surrounding the medial anterior cortex region revealed that the motor manifestations of seizures were elicited over a wide region of the anterior cortex, with scores significantly lower at carbachol microinjection sites greater than 1 mm rostral, 2 and 3 mm caudal, and 2 mm lateral to the standard medial prefrontal cortex site. Unilateral microinjection of carbachol yielded motor seizures primarily from the contralateral forepaw, suggesting involvement of a crossed pathway. Retrograde tracing with fast blue dye, combined with immunostaining for choline acetyltransferase and NADPH-diaphorase, found that the cholinergic neurons innervating the standard microinjection site were the dorsolateral tegmental cells, as previously reported, which have been shown to also contain substance P and corticotropin releasing factor. In addition, cholinergic neurons of the nucleus basalis of Meynert region were found to innervate the standard microinjection site. These findings implicate cholinergic innervation of the rostral cortex in classical limbic seizures.

Substance P antagonists block carbachol-induced "boxing" behavior at a site of coexistence in the rat prefrontal cortex.

A cholinergic projection from the dorsolateral tegmentum to the medial anterior cortex has previously been shown to contain substance P and corticotropin releasing factor. Behavioral analysis of acetylcholine, substance P and corticotropin releasing factor microinjected into the medial anterior cortex revealed a seizure-related "boxing" behavior elicited by carbachol, which was potentiated by coinjection with substance P and antagonized by coinjection with corticotropin releasing factor. We now report that two antagonists of substance P receptors, [D-Pro2, D-Phe7, D-Trp9]-substance P and [D-Pro2, D-Trp7,9]-substance P, attenuate "boxing" behavior when coinjected with carbachol. Neither antagonist produced observable behavioral effects when microinjected alone. An analog of substance P, [pGlu,5, MePhe,8 Sar9]-substance P (5-11) potentiated carbachol-induced "boxing" at doses similar to naturally-occurring substance P. Monoclonal and polyclonal antisera against substance P were not effective antagonists of carbachol-induced "boxing." The ability of substance P antagonists to block carbachol-induced "boxing" has two major implications: (1) endogenous substance P may be modulating endogenous acetylcholine in the tegmental-cortical pathway; and (2) substance P antagonists may provide a new avenue for the development of antiepileptic drugs.

Ventral tegmental oxytocin induces grooming.

Bilateral microinjection of oxytocin (OXY) into the ventral tegmental area (VTA) of rat brain produced a significant increase in grooming behaviors at doses from 100 pg to 400 ng. Sites in the caudal region of the VTA were sensitive to lower doses of OXY than sites in the rostral region of the VTA. The time course of action of OXY in the grooming paradigm indicated onset beginning immediately after injection, and termination at 60-75 minutes after injection. Comparison of OXY-induced grooming in male, female, and ovariectomized, estrogen-treated female rats showed no differences in potency for OXY among these groups, suggesting that the grooming effects of OXY are not regulated by sex steroids. Analysis of locomotor activity in rats microinjected with OXY 200 ng bilaterally into the caudal VTA revealed that OXY had no effect on ambulatory locomotion, suggesting that this peptide may activate neurons within the VTA which mediate grooming but not locomotion. The OXY receptor antagonist, [Pen1, pMePhe2, Thr4, Orn8]-OT, blocked OXY-induced grooming when both were simultaneously microinjected into the VTA. The dopamine D-2 receptor antagonist, haloperidol, and the D-1 receptor antagonist, SCH 23390, when microinjected into the VTA five minutes before microinjection of OXY into the VTA, did not block OXY-induced grooming, suggesting that OXY is not working through a dopamine autoreceptor on the VTA neurons. Systemic pretreatment with haloperidol and SCH 23390 effectively blocked grooming induced by OXY in the VTA, suggesting that OXY is directly stimulating OXY receptors on VTA neurons to release dopamine at postsynaptic sites regulating grooming behaviors.

Site of action of anorectic drugs: glucoprivic- versus food deprivation-induced feeding.

Feeding induced by 2-deoxyglucose was compared with feeding induced by food deprivation in terms of antagonism by anorectic drugs and of anatomical site of action. Glucoprivic feeding was completely blocked by microinjection of amphetamine, fenfluramine, and mazindol into the paraventricular nucleus of the hypothalamus (PVN). Deprivation-induced feeding was not blocked by amphetamine, fenfluramine, or mazindol microinjected into the PVN. Neither the feeding induced by 2-deoxyglucose nor its reversal by amphetamine were blocked by pretreatment with the beta-adrenergic antagonist, propranolol. Amphetamine and fenfluramine blocked both glucoprivic- and deprivation-induced feeding when microinjected into the perifornical region of the lateral hypothalamus. These data suggest that food consumption induced by 2-deoxyglucose treatment can be antagonized by anorectic drugs acting at recognition sites present in several hypothalamic nuclei, while deprivation-induced feeding acts through different receptor mechanisms which may be specific to the perifornical region of the lateral hypothalamus.

Regulation of [3H]mazindol binding to subhypothalamic areas: involvement in glucoprivic feeding.

The distribution of low-affinity sodium-sensitive binding sites of [3H]mazindol were studied in rat hypothalamic nuclei. Using microdissection methods, it was demonstrated that the highest level of [3H]mazindol binding is localized to the paraventricular nucleus (PVN) and the lowest binding is observed in the lateral hypothalamus. Following food deprivation, a significant decrease in [3H]mazindol binding in the PVN and ventromedial hypothalamus (VHM) were observed. Refeeding food-deprived rats resulted in restoration of the level of binding in the PVN, and this was correlated with changes in blood glucose levels. Thus, changes in the binding of [3H]mazindol in the PVN may reflect local changes in glucose levels. In related studies, the involvement of the PVN in the regulation of food deprivation or 2-deoxyglucose (2-DG)-induced food intake was studied. Application of amphetamine (20 micrograms) into the PVN had no effect on food deprivation induced feeding, but significantly inhibited 2-DG induced (glucoprivic) feeding. The PVN may play an important role in the glucostatic regulation of feeding and in mediating the anorectic action of amphetamine and related anorectic drugs on glucoprivic feeding.

Cholinergic induction of seizures in the rat prefrontal cortex.

Intracerebral microinjection of the cholinergic agonist, carbachol, into the medial prefrontal cortex of the rat, induced a profound behavioral syndrome consisting of repetitive, stereotyped forepaw treading in an upright posture. Electroencephalographic analysis revealed multiple bursts of sharp waves, 200-300 microV, accompanying the carbachol-elicited motor behavior. Pretreatment with intraperitoneal doses of three anticonvulsant drugs, clonazepam, diazepam, and pentobarbital, blocked the manifestation of the motor behavior. These observations suggest that activation of cholinergically innervated regions of the rat medial prefrontal cortex induces an atypical form of seizures.

Antagonists of central and peripheral behavioral actions of cholecystokinin octapeptide.

Pharmacological studies on the behavioral functions of sulfated cholecystokinin (CCK) in the gut and in the brain require potent, specific antagonists to CCK. Compounds identified as competitive antagonists at the peripheral receptors for CCK were tested for their ability to block the behavioral effects of CCK administered centrally and peripherally. Behavioral effects of CCK (8.8 X 10-10 mmol) administered centrally into the nucleus accumbens, i.e., potentiation of dopamine-induced hyperlocomotion in rats, were effectively blocked by pretreatment with proglumide (6 X 10(-5) mmol of nucleus accumbens), by benzotript (3 X 10(-5) mmol of nucleus accumbens) and by rabbit antiserum raised against CCK (0.2 microliter/nucleus accumbens), but not by CCK26-33 (1.7 X 10(-7) mmol) or unsulfated CCK26-33 (1.9 X 10(-6) mmol). The behavioral effects of peripherally administered CCK, i.e. reduced food consumption and reduced exploratory behaviors in mice, were blocked effectively by pretreatment with proglumide (0.3-0.9 mmol/kg), and by benzotript (0.03 mmol/kg), but not by CCK30-33 (0.003 mmol/kg). None of the compounds administered peripherally significantly affected food consumption or exploratory behaviors when given alone. Furthermore, none of the compounds significantly affected locomotion when administered alone into the nucleus accumbens, or significantly affected dopamine-induced hyperlocomotion when given into the nucleus accumbens before dopamine. Benzotript, proglumide and a CCK antibody appear to act as specific antagonists of the behavioral effects of CCK at both the peripheral gastrointestinal site and at the central nucleus accumbens site. Neither unsulfated CCK26-33 or CCK30-33 were effective as antagonists of peripheral or central behavioral effects of CCK. However, whereas benzotript and proglumide may be useful as pharmacologically specific antagonists, the high doses required suggest that more potent CCK antagonists are required for investigating the behavioral functions of endogenous CCK.

Cholecystokinin potentiates dopamine-mediated behaviors: evidence for modulation specific to a site of coexistence.

Cholecystokinin coexists with dopamine in mesolimbic neurons in mammalian brain. When injected directly into the nucleus accumbens, cholecystokinin (CCK) potentiated dopamine (DA)-induced hyperlocomotion and apomorphine-induced stereotypy. These effects were not mimicked by nonsulfated CCK, but were blocked by proglumide, a putative CCK antagonist, as well as by antisera raised against sulfated CCK. CCK alone had no effect on locomotion or sterotypy, indicating that this peptide acts primarily as a modulator of DA-mediated behaviors in the mesolimbic pathway. In addition, CCK did not potentiate DA-induced hyperlocomotion or apomorphine-induced stereotypy when injected into the caudate nucleus, where CCK and DA are localized in separate neurons in rats. Facilitation of DA-mediated behaviors by CCK may represent a functional interaction specific to the neuromodulator-neurotransmitter coexistence phenomenon.

A lysosomal storage disorder in the BALB/c mouse: bone marrow transplantation.

The morphological and biochemical consequences of transplanting affected bone marrow from donor BALB/c mice with a lysosomal storage disorder (BALB/c LSD) into normal recipient mice were studied. Bone marrow was removed from normal BALB/c and BALB/c LSD mice and transfused into normal BALB/c recipient mice four hours after the mice received 850 rads of irradiation. Tissues of the recipient mice were examined 240 days later. This study revealed that the defective cells that constituted the visceral lesions of BALB/c LSD could be transplanted to normal BALB/c mice by the use of bone marrow from affected BALB/c LSD homozygote; that the defective cells of BALB/c LSD proliferated and disseminated throughout the mononuclear phagocytic system of the recipient; that there were increases in cholesterol, sphingolipids, and cystine with decreases in sphingomyelinase and glucocerebrosidase activity in tissues of the recipients; and that the recipients survived substantially longer than BALB/c LSD homozygotes and their lifespan was compromised mainly by the secondary effects of irradiation. These lesions, although not as extensive as in homozygous BALB/c LSD, paralleled the lesions which develop in BALB/c LSD. Since the recipient mice were not compromised by the short life span (70 days) of the BALB/c LSD mice, they may be used to study the long-term chronic effects of these metabolic lesions.