Allan E. Johnson

Electrophysiological Actions of Oxytocin on Hypothalamic Neurons in vitro: Neuropharmacological Characterization and Effects of Ovarian Steroids

Oxytocin (OT) neurotransmission in the brain has a facilitatory effect on sexual receptivity in rats. This effect of OT is dependent on priming by ovarian steroids, estrogen and progesterone. These steroids modulate OT binding in specific brain nuclei, including the ventrolateral portion of the ventromedial hypothalamic nucleus (vlVMN). In the present study, single-unit activity was recorded from the vlVMN in hypothalamic slices to characterize the electrophysiological actions of OT. To examine the effects of ovarian steroids on OT actions, we used brain slices prepared from ovariectomized rats either treated with estrogen or not, and some slices were treated with progesterone in vitro. OT had little modulatory action on neuronal responses to other agents, but affected the activity of large numbers of vlVMN units. Of those neurons affected, 94% responded with excitation. This predominant stimulatory action of OT is consistent with its lordosis-facilitating effect, because increases in the activity of VMN neurons are generally associated with the facilitation of lordosis. Pharmacological analyses with selective OT agonists and antagonists as well as structurally related peptides showed that the excitatory action of OT is mediated by OT receptors. Estradiol modulated several aspects of OT transmission. First, it increased neuronal responsiveness to OT, especially at the lowest concentration used (0.2 nM). In addition, it caused neuronal responses to OT to correlate significantly with responses to acetylcholine and norepinephrine, which also can act on the ventromedial hypothalamus to facilitate lordosis. Finally, estradiol enhanced the excitability of laterally projecting neurons, which have been implicated in lordosis. In estrogen-pretreated slices, addition of progesterone in vitro caused little further effect on responses of individual neurons to exogenous OT. Altogether, the present electrophysiological findings are consistent with the hypothesis that estrogen potentiates OT action by increasing functional OT receptors preferentially in lordosis-relevant neurons, thereby enabling OT to efficiently facilitate female reproductive behavior.

Localization and characterization of vasopressin binding sites in the rat brain using an iodinated linear AVP antagonist

The binding characteristics and central distribution of 125I-Linear AVP antagonist, a new ligand for vasopressin binding sites, are described in the following studies. Saturation studies performed on rat brain septal membranes demonstrated that 125I-Linear AVP antagonist binds to a single class of sites with high affinity (55 pM) and limited capacity (88 fmol/mg protein). In autoradiographic studies, 125I-Linear AVP antagonist labeled brain areas known to contain vasopressin receptors without binding to neurophysins. 125I-Linear AVP antagonist also labeled sites in cortex, hypothalamus, ventral tegmental area and substantia nigra. In competition studies, 125I-Linear AVP antagonist binding was most readily blocked by AVP and a selective V1a agonist. Oxytocin and a selective V2 ligand were effective only in micromolar concentrations. A selective oxytocin agonist was virtually ineffective in blocking 125I-Linear AVP antagonist binding. In regions that contain a high density of oxytocin binding sites, however, oxytocin-displaceable binding was observed. In agreement with studies on peripheral tissues, the binding profile generated from these studies indicates that 125I-Linear AVP antagonist binds to vasopressin receptors of the V1a subtype. These results suggest that 125I-Linear AVP antagonist is a valuable ligand for the study of central AVP receptors.

Characterization of dopamine receptor binding sites in the subthalamic nucleus.

Experiments were undertaken to determine the distribution and binding profile of dopamine (DA) receptors within a key extra-striatal region of the rat basal ganglia, the subthalamic nucleus (STh). Analysis of [125I]NCQ-298 autoradiograms showed that binding sites of the D2-receptor family are abundant in the STh. Competition studies indicated that these sites were specifically of the D2 subtype. However, contrary to previously published data, [125I]SCH-23982 autoradiograms failed to reveal D1 receptor binding in the STh. These data suggest that DA acting at D2 receptors may directly modulate STh neural activity and furthermore that the antagonism of STh D2 receptor binding by neuroleptics may be involved in the expression of extrapyramidal motor disturbances.

Castration reduces vasopressin receptor binding in the hamster hypothalamus.

A recently developed ligand with very high affinity and selectivity for the vasopressin (AVP) V1a receptor subtype (i.e. [125I]Linear AVP antagonist ([125I]-LinAntag) was used to describe the distribution of AVP binding sites in the hamster brain, and to determine whether AVP receptor binding was influenced by testicular hormones in sites involved in the regulation of steroid-dependent social behaviors. These studies demonstrated [125I]LinAntag binding in regions of the hamster brain which have not been previously identified with other AVP ligands. In addition, testicular hormones were found to alter [125I]LinAntag binding in two distinct regions, the posterior lateral preoptic-anterior lateral hypothalamic continuum and the posterior ventrolateral hypothalamic nucleus and adjacent tuberal area.

Glutamate inhibits ingestive behaviour.

Male rats treated with reserpine were motionless and ingested only a few of ten consecutive intraoral injections of a 1 M solution of sucrose. While injection of apomorphine, a dopamine agonist, stimulated locomotion and stereotyped sniffing in reserpinized rats, it did not reactivate ingestive responses. The non‐competitive N‐methyl‐D‐aspartate receptor antagonist MK801, however, stimulated locomotion as well as ingestion suggesting involvement of glutamate in the suppressive effect of resperpine on ingestive responses. A series of experiments was therefore undertaken to investigate the possible physiological role of glutamate in feeding.

Cholecystokinin, dopamine D2 and N-methyl-d-aspartate binding sites in the nucleus of the solitary tract of the rat: possible relationship to ingestive behavior

Receptor autoradiography was used to investigate the distribution of brainstem binding sites for cholecystokinin, dopamine and N-methyl-D-aspartate with particular reference to the nucleus of the solitary tract of the rat, an area involved in the control of ingestive behavior. Binding sites for the A and B subtypes of the cholecystokinin receptor, labeled with [(125)I]cholecystokinin octapeptide sulfate in the presence or absence of antagonists for the devazepide (A) or L-365,260 (B) receptor, were present throughout the caudal rostral extent of the nucleus of the solitary tract, the A type predominating in the commissural, medial and gelatinous part and the B type in the lateral part. In the most rostral part of the medial nucleus of the solitary tract, both A and B receptors were present. Dopamine D2 receptors, labeled with [(125)I]NCQ-298, were found in all parts of the nucleus of the solitary tract. No binding to the dopamine D1 receptor, labeled with [(125)I]SCH-23982, was found in the brainstem. N-Methyl-D-aspartate receptors, labeled with [(3)H]dizocilpine maleate, were also present in the entire caudorostral extent of the nucleus of the solitary tract. Binding to cholecystokinin A receptors was co-distributed with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the caudal and rostral parts of the nucleus of the solitary tract, and binding to cholecystokinin B receptors overlapped with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the rostral nucleus of the solitary tract. These results are consistent with the hypothesis that cholecystokinin, dopamine and glutamate interact in the nucleus of the solitary tract in the control of ingestive behavior.

Sex differences in the regulation of oxytocin receptors by ovarian steroids in the ventromedial hypothalamus of the rat.

The facilitation of sexual receptivity by oxytocin (OT) in female rats is related to the regulation of oxytocin receptors (OTR) by ovarian steroids in the ventromedial nuclei (VMN) of the hypothalamus. In a previous study, we have shown that estradiol benzoate (EB) causes a twofold increase in OTR binding in the VMN. Progesterone (P) then modulates levels of the estrogen-induced OTR and increases the area occupied by the receptors by acting on the neuronal membrane. In the present study, we compared the effects of EB and P on OTR binding between males and females. In both sexes, EB increased the density of OTR and the area covered by the receptors at the level of the medial and caudal VMN. In estrogen-primed females, P further increased OTR levels in the medial VMN and the area covered by OTR at the level of the caudal VMN. By contrast, P did not modulate OTR binding in estrogen-primed males. Thus, the behavioral insensitivity of male rats to ovarian hormones, in particular to P, may be related to sex differences affecting the modulation of OTR binding.

Testosterone Modulates Oxytocin Binding in the Hypothalamus of Castrated Male Rats

Oxytocin (OT) binding sites are modulated by estrogens in several brain regions including the ventromedial hypothalamic nucleus (VMN) in both male and female rats. To further study steroid regulation of OT receptor binding, we examined the effect of androgen replacement in castrated male rats on OT binding with quantitative autoradiographic methods. Castrated adult male rats were treated with either 250 micrograms testosterone propionate (TP) or oil for 2 days and killed 48 h after the last injection. Brain sections through the preoptic area and VMN were labeled with 5.0 nM[3H]-OT +/- 5.0 microM unlabeled OT or 1.0 microM[Thr4,Gly7]OT and apposed to tritium-sensitive film for 7 weeks. Results of this study show that TP increased [3H]-OT binding up to 5-fold in the ventrolateral VMN and 4-fold in the bed nucleus of the stria terminalis. In addition [Thr4,Gly7]OT completely displaced [3H]-OT binding in the VMN indicating that binding in this brain region was specific to OT receptors. Because estrogens also increase OT receptor binding in male rats, it is possible that TP affects OT binding after being converted by aromatase to estradiol.

Autoradiographic studies of central α2A- and α2C-adrenoceptors in the rat using [3H]MK912 and subtype-selective drugs

Abstract In the present study we examined the distribution of α2A- and α2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between α2A- and α2C-adrenoceptors, the slices were incubated with [3H]MK912 in the presence of graded concentrations of the α2A-selective drug, BRL44408, or the α2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [3H]MK912 plus 185 nM BRL44408 selectively labeled α2C-adrenoceptors, while 0.4 nM [3H]MK912 plus 220 nM spiroxatrine selectively labeled α2A-adrenoceptors. Using this approach, α2C-adrenoceptors were detected in the striatum, while α2A-adrenoceptors predominated in the cortical layers 1–4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.

Chronic treatment with a classical neuroleptic alters excitatory amino acid and GABAergic neurotransmission in specific regions of the rat brain

The purpose of the following experiments was to describe some of the neurochemical changes that occur in the basal ganglia of rats exposed chronically to a classical neuroleptic, fluphenazine, and to relate these changes to extrapyramidal motor dysfunction. For these studies a combination of behavioural, receptor autoradiographic and in situ hybridization methods were employed. Preliminary pharmacological studies on GABA receptors showed that incubation in Tris-acetate rather than Tris-citrate buffer increased the number of binding sites labelled by [3H]muscimol by over 120% without affecting binding affinity or selectivity. The results of experiments with fluphenazine showed that treatment for six months increased the frequency of vacuous chewing movements compared to controls. In the striatum, changes in GABA transmission were observed in fluphenazine-treated rats with increases in glutamate decarboxylase mRNA levels in the caudate nucleus, dorsal shell and core of the accumbens and decreases in [3H]muscimol binding in the caudate and dorsal shell regions. These data suggest that fluphenazine treatment increased GABA transmission in specific subregions of the caudate and accumbens nuclei. In addition, glutamate decarboxylase mRNA levels were elevated in the entopeduncular nucleus of fluphenazine-treated animals. Autoradiographic analysis of excitatory amino acid binding showed that fluphenazine exposure decreased [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding in entopeduncular nucleus and in the ventrolateral thalamic nucleus and decreased [3H]dizocilpine maleate binding in the medial geniculate nucleus. These experiments show that in addition to altering GABA transmission, chronic neuroleptic exposure alters excitatory amino acid transmission in specific regions of the basal ganglia-thalamocortical motor system. The neuroleptic dependent increases in glutamate decarboxylase mRNA levels in the entopeduncular nucleus may reflect changes in neurotransmission in the indirect pathway connecting the major input and output nuclei of the basal ganglia. Changes in some of these brain regions may be related to the occurrence of extrapyramidal motor disturbances.