Effects of thyrotropin releasing hormone and its analogues on unconsciousness following head injury in mice.

We examined the effects of thyrotropin releasing hormone (TRH) and its analogues (DN-1417: gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate; MK-771: L-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide; TSII-37: H-Lys-Gln-His-Pro-Gly-Ser-OH) on arousal in head injured mice, an animal model of unconsciousness. TRH, DN-1417, MK-771 and TSII-37 were injected 10 min before the head injury. TRH, DN-1417 and MK-771 caused dose-dependent decreases in the time required for recovery of the righting reflex time and in the time from the head injury to the onset of spontaneous movement. TSII-37 had no effect, when compared with the control group. In terms of the minimum effective dose, TRH and DN-1417 were of similar potency, but MK-771 was about 30-fold stronger than TRH. Measurement of the cross-reactivities of these TRH analogues by radiolabeled receptor assay suggest that the structure-binding relationship is proportional to the structure-activity relationship.     

Metrifonate effects on acetylcholine and biogenic amines in rat cortex

The effect of systemic and local administration of metrifonate (MTF), a long-acting cholinesterase inhibitor (ChEl) on extracellular levels of acetylcholine (ACh), norepinephrine (NE), dopamine (DA) and serotonin (5-HT) was investigated in the rat cortex by using transcortical microdialysis. Metrifonate (20, 40, and 80 mg/kg, s.c.) increased ACh levels in a dose-dependent manner above the baseline. Two consecutive administrations (80 mg/kg) enhanced ACh levels producing two similar patterns of elevation. A significant increase in NE was also seen at 80 mg/kg. Systemic administration (20 mg/kg) of MTF produced a significant increase of DA levels. Local cortical perfusion of MTF through the probe caused a significant but slow increase of ACh as well as an increase of NE levels. Compared to NE, the elevation of DA was more rapid and more longlasting. The cortical levels of 5-HT were not modified by MTF given by either route. These results support the concept of MTF being a potential drug for treatment of Alzheimer disease (AD).     

Effects of MK-771, A TRH analog,on pentobarbital-induced alterations of cholinergic parameters in discrete regions of rat brain

MK-771 (l-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide) was administered intraventricularly to conscious and pentobarbital-narcotized rats. In the conscious rats MK-771 did not affect the regional levels of acetylcholine (ACh) or the rate of sodium-dependent high-affinity choline uptake (HACU). MK-771 was found to antagonize pentobarbital-induced elevations of ACh levels in the cortex, hippocampus and striatum. MK-771 also reversed the depressant effects of pentobarbital on the HACU of the cortex and hippocampus. Striatal HACU was unaltered by the administration of pentobarbital or the combination of pentobarbital and MK-771.     

5-Fluoronicotine,noranhydroecgonine, and pyridyl-methylpyrrolidine release acetylcholine and biogenic amines in rat cortex in vivo

The effects of nicotinic receptor agonists 5-fluoronicotine, noranhydroecgonine and pyridyl-methylpyrrolidine on the cortical release of acetylcholine (ACh), norepinephrine (NE), dopamine (DA) and serotonin (5-HT) were investigated with microdialysis in rat. 5-Fluoronicotine significantly elevated ACh to 76% above basal values and DA to 69% above baseline. Pyridyl-methylpyrrolidine significantly increased the release of ACh to 39% above basal values and NE to 63% above baseline. Noranhydroecgonine significantly elevated NE to 64% above basal values and DA to 147% above baseline. 5-Fluoronicotine did not affect NE release; pyridylmethylpyrrolidine did not alter DA release; and noranhydroecgonine did not significantly elevate ACh release. None of these agonists increased the release of 5-HT. All responses were blocked by prior administration of mecamylamine, a nicotinic receptor antagonist. The distinctive neurotransmitter-related profiles for the three agonists are suggestive of activity at subtypes of nicotinic receptors, an effect that may be related to the structural diversity of these compounds.     

Potential anti-depressive effects of thyrotropin releasing hormone (TRH) and its analogues

The anti-depressive effects of thyrotropin releasing hormone (TRH) and its analogues (DN-1417: gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate; MK-771: L-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide) were examined in behavioral despair rats, an animal model of depression. TRH, DN-1417, MK-771, amitriptyline and diazepam were injected three times after the first forced swimming. One hr after the last injection, a 5-min swimming test was performed. Experimental animals were placed in a Hall's type open-field apparatus immediately before and after the 5-min test, and their locomotor activities were determined. No significant difference was noted in the locomotor activity immediately before the 5-min test among any group. In the 5-min swimming test, TRH, DN-1417 and MK-771 caused a dose-dependent decrease in immobility, showing an anti-depressive effect similar to amitriptyline. Diazepam showed no difference compared with the control group. After the swimming test, locomotor activity remarkably decreased in the control rats, while decreased locomotor activity was partially prevented in the TRH, DN-1417, MK-771 and amitriptyline treated rats which exhibited active movement not only during the swimming period but also after it. In terms of the minimum effective dose, TRH and DN-1417 seemed to be of similar potency, while MK-771 was 40-fold stronger than TRH. An examination of a possible correlation between the cross-reactivity of TRH analogues in a radioreceptor assay and the effects of the analogues on despair rats suggested that the structure-binding relationship was proportional to the structure-activity relationship.     

Effects of thyrotropin-releasing hormone (TRH) and MK-771 on schedule-controlled behavior of squirrel monkeys, rabbits and pigeons

The effects of TRH (0.001-10.0 mg/kg) and a more potent TRH analog, MK-771 (0.001-5.6 mg/kg), were studied on comparable schedule-controlled performances of squirrel monkeys, rabbits and pigeons. Responding was maintained in the presence of different stimuli by a multiple fixed-ratio (FR), fixed-interval (FI) schedule of food presentation (monkeys and pigeons) or 0.25% saccharin solution (rabbits). Generally, TRH and MK-771 produced decreases in responding under both schedules and in all three species. TRH and MK-771 were roughly equipotent in the squirrel monkey, whereas in the pigeon and rabbit MK-771 was approximately 20 times more potent than TRH in decreasing responding to 50 percent of control levels. The duration of action of doses of TRH and MK-771 that reduced responding to 50 percent of control was approximately 3 hr in the squirrel monkey; recovery of performance occurred twice as fast under the FR schedules. With the pigeon, TRH effects that produced 50 percent decreases in responding lasted over 6 hours, whereas behaviorally comparable doses of MK-771 lasted about 4 hours. With few exceptions, TRH and MK-771 appear to produce similar effects of schedule-controlled behavioral performances of the squirrel monkey, rabbit and pigeon. Compared to the effects of other behaviorally-active substances under these procedures, TRH and MK-771 exert a distinctive array of effects.     

Effects of local and repeated systemic administration of (−)nicotine on extracellular levels of acetylcholine,norepinephrine, dopamine,and serotonin in rat cortex

Systemically administered (–)nicotine (0.2–1.2 mg/kg, s.c.) significantly increased the release of acetylcholine (ACh), norepinephrine (NE) and dopamine (DA) in rat cortex. The lowest dose of (–)nicotine examined (0.2 mg/kg, s.c) also significantly elevated extracellular serotonin (5-HT) levels, and the maximal increases of extracellular ACh (122% at 90 min post injection) and DA levels (249% at 120 min post-injection) were observed following this dose. In contrast, the maximal increase of NE release (157% at 30 min post-injection) was observed following the highest dose of (–)nicotine injected (1.2 mg/kg, s.c.). This higher dose consistently produced generalized seizures. Repeating the (–)nicotine (0.58 mg/kg, s.c.) injection four hours after the first administration significantly elevated extracellular NE levels and also appeared to increase DA and CCh release. In addition, extracellular ACh and DA levels increased significantly in the dialysate after (–)nicotine was administered directly to the neocortex through the microdialysis probe membrane. Norepinephrine levels appeared to be elevated in the cortex following local administration as well.     

Effects of MK-771 on the isolated amphibian spinal cord: comparison with thyrotropin-releasing hormone.

Topically applied MK-771 (pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide), a novel thyrotropin-releasing hormone (TRH) analog, was found to be equipotent with TRH in depolarizing the ventral roots of the isolated, hemisected amphibian (Bufo marinus) spinal cord. The 3-methyl-histidyl analog of TRH was approximately 10 times more potent than MK-771 and TRH. MK-771 is known to be equiactive with TRH in their actions on the pituitary gland. Taken together these findings suggest that the previously observed enhanced potency of systemically administered MK-771 over TRH in in vivo central nervous system (CNS) test paradigms is not likely to be due to a difference in the agonist requirements of CNS as compared with pituitary receptors for TRH.     

A microdialysis study of the effects of the nicotinic agonist RJR-2403 on cortical release of acetylcholine and biogenic amines

Transcortical dialysis was employed to investigate the effects of subcutaneous (s.c.) injections of RJR-2403 (1.2–7.2 μmol/kg) on extracellular levels of acetylcholine (ACh), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat. Systemic administration of RJR-2403 produced a 90% increase of cortical extracellular ACh levels that persisted for up to 90 minutes after injection. Norepinephrine and DA release were increased 124% and 131% above basal values, respectively. Serotonin (5-HT) levels in the dialysate were also significantly elevated by RJR-2403 (3.6 μmol/kg, s.c.) 70% above baseline at 90 minutes post-injection. Comparison of these responses to those of (−)nicotine from a previous study reveals little difference between the two compounds in their ability to influence cortical neurotransmitter release following systemic administration.     

Neurochemical asymmetries in the albino rat's cortex, striatum, and nucleus accumbens

The concentrations of dopamine (DA), norepinephrine (NE), serotonin (5-HT), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) were measured in the right and left cortex, striatum, and nucleus accumbens of adult Purdue-Wistar rats. There was more DA in the right cortex and accumbens and a greater concentration of NE in the left striatum. There is more 5-HT in the left striatum and right accumbens, more 5-HIAA in the left cortex, as well as a greater 5-HT turnover in the left accumbens. These results are considered in the light of previous findings concerning the relationship of neurochemical asymmetries and behavioral lateralization.     

Increase of tonic parasympathetic outflow to the pupil produced by an analog of TRH (MK-771)

MK-771, a potent analog of thyrotropin-releasing, hormone, was found to cause a long-lasting, dose-dependent antagonism of the centrally induced pupillary dilation produced by clonidine in anesthetized cats. MK-771 had little direct effect on the dilated iris in cats treated with mecamylamine. Both MK-771 and TRH selectively antagonized the pupillary dilation, but not the contractions of the nicitating membrane induced by peripheral sympathetic nerve stimulation. MK-771 was found to increase nerve activity in the short ciliary nerve. TRH, although studied less extensively, produced similar effects. This action of MK-771 was not blocked by prior sectioning of the spinal cord, by elimination of the tonic light reflex pathway, or by treatment with scopolamine or atropine. These findings suggest that the pupillary effects of MK-771 (and TRH) are related to the ability to increase the parasympathetic tone to the iris.     

Stress-Related Changes in Cerebral Catecholamine and Indoleamine Metabolism: Lack of Effect of Adrenalectomy and Corticosterone

The concentrations of catecholamine and indoleamine metabolites were measured in intact and adrenalectomized mice to determine whether adrenal hormones mediate or modulate the stress-induced responses. Thirty minutes of footshock resulted in significant increases of the ratios of the dopamine (DA) catabolite, dihydroxyphenylacetic acid (DOPAC), to DA in prefrontal cortex, nucleus accumbens, striatum, hypothalamus, and brainstem, and of homovanillic (HVA)/DA ratios in nucleus accumbens, striatum, amygdala, and hypothalamus. Ratios of 3-methoxy-4-hydroxyphenylethyleneglycol to norepinephrine (NE) were also increased in prefrontal cortex, nucleus accumbens, septum, amygdala, hypothalamus, hippocampus, and brainstem. The concentration of NE was decreased in amygdala. 5-Hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT, serotonin) ratios and free tryptophan were also increased in every brain region. Very similar data were obtained from mice restrained for 30 min. Adrenalectomy resulted in increased HVA/DA ratios in prefrontal cortex and striatum, and 5-HIAA/5-HT in septum. The stress-related changes were largely similar in adrenalectomized mice. Significant interactions between adrenalectomy and footshock treatment occurred in prefrontal cortical DOPAC/DA and hypothalamic NE which was depleted only in adrenalectomized mice, suggesting tendencies for these measures to be more responsive in adrenalectomized mice. Corticosterone administration (0.5-2.0 mg/kg s.c.) which resulted in plasma concentrations in the physiological range did not alter the concentrations of the cerebral metabolites measured in any region. We conclude that adrenal hormones do not mediate cerebral catecholamine or indoleamine metabolism in stress, although adrenalectomy may affect HVA and 5-HIAA metabolism, and there was a tendency for catecholamines to be more sensitive to stress in adrenalectomized animals.     

Effects of hypothalamic releasing hormones and biogenic amines on identified neurones in the circumoesophageal ganglia of the water snail (Planorbis corneus)

1. The effect of locally applied releasing hormones, thyrotropin-releasing hormone (TRH) and luteinizing-hormone-releasing hormone (LHRH) and the putative neurotransmitters, acetylcholine (ACh) and dopamine (DA), on the neuronal excitability of identified invertebrate giant dopaminergic neurone (GDN) and serotoninergic neurone (5-HT) (Planorbis corneus) were investigated by intracellular recording in vitro. 2. The membrane potential of GDN was of the order of -60 to -70 mV. The microiontophoretically applied substances produced membrane depolarization as well as spike activation. Their order of efficacy was as follows: TRH greater than ACh greater than DA greater than LHRH. 3. The effects of the tested TRH, ACh, LHRH and DA on serotoninergic neurones were less pronounced. 4. During ACh depolarization the membrane resistance of GDN was found to be strongly reduced, whereas TRH produced only a small reduction in membrane resistance. 5. Dihydro-beta-erythroidin (DHE) added to the bath solution reversibly blocked ACh depolarization without influencing TRH depolarization. Concentrations of atropine sulfate required to block the ACh depolarization were higher by at least 100 order of magnitude. 6. These effects are discussed in relation to the immunoreactive TRH detected earlier in the central nervous system of invertebrates and vertebrates. The results are consistent with the postulate that TRH acts as a neuromodulator and/or neurotransmitter on invertebrate and vertebrate neurones.     

Substance P antagonist-induced spinal cord vasoconstriction: Effects of thyrotropin-releasing hormone and substance P agonists

Local spinal cord vasomotor effects of 3 substance P (SP) antagonists were studied in the rat following intrathecal (IT) administration. Each SP antagonist (3.3 nmol) increased spinal cord vascular resistance and reduced blood flow. A LH-RH antagonist analog (10 nmol) of similar molecular weight and which also contained multiple D-Trp residues did not cause spinal cord vasoconstriction. The vasoconstrictor action of the SP antagonist, [D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹]-SP ([D-Arg]-SP) was unaffected by pretreatment with a stable SP receptor agonist (5 nmol IT). Given evidence for a cerebral vasodilator action of TRH agonists, the effects of TRH (IV) and a stable TRH analog (MK-771, IT) on [D-Arg]-SP-induced vasoconstriction were also assessed. Neither TRH nor MK-771 prevented the [D-Arg]-SP-induced vasoconstriction. However, TRH (IV) but not MK-771 (IT) partially opposed [D-Arg]-SP-induced reduction in thoracic spinal cord blood flow. Thus, SP antagonists cause spinal cord vasoconstriction by a non-SP receptor mediated phenomenon. In addition, the attenuation of SP-antagonist-induced neuropathological changes previously reported with IV. TRH administration is likely due to less severe consequences of vasoconstriction in the presence of a higher initial baseline blood flow rather than direct prevention of the vasoconstriction.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

TRH analog MK-771 reverses neurochemical and learning deficits in medial septal-lesioned rats

Microinjection of ibotenic acid into medial septum of rats decreased choline acetyltransferase (CAT) and high-affinity choline uptake (HACU) activities in hippocampus and retarded the learning of a spatial memory task in the radial-arm maze. Administration of MK-771, a stable TRH analog, to such animals restored HACU activity in hippocampus to normal levels. Daily treatment of rats with MK-771 prior to maze running also restored the animals' learning ability. MK-771 did not enhance hippocampal HACU activity or maze performance in sham-lesioned rats. These results suggest that MK-771 reversed the ibotenic acid-induced memory deficit by restoring septohippocampal cholinergic function. MK-771 and other TRH analogs may represent novel agents for improving memory deficits produced by cholinergic insufficiency in Alzheimer's disease.     

Analysis of γ-Aminobutyric AcidA Receptor Subunits in the Mouse Cochlea by Means of the Polymerase Chain Reaction

Abstract: Unlike 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces consistent decreases in levels of striatal dopamine (DA) with considerably smaller and more variable effects on mouse brain levels of serotonin (5-HT) and norepinephrine (NE), a novel amine-substituted MPTP analogue, 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH₂-MPTP), administered in a standard mouse dosing paradigm for MPTP (20 mg/kg X 4) did not affect striatal DA but led to marked reductions (60–70%) in levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and NE measured in frontal cortex and hippocampus 1 week after treatment. Another 2'-substituted MPTP analogue, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine, affected cortical and hippocampal 5-HT, 5-HIAA, and NE only minimally, while markedly reducing the DA content in striatum (90%), thus indicating that the substituent (-NH₂ versus -CH₃) at the 2'position is important for the differential effects of these MPTP analogues. In a replication study with a 3-week end point, hippocampal and cortical 5-HT, 5-HIAA, and NE levels remained depressed with no indication of recovery. These results suggest that 2'-NH₂-MPTP may be a novel, regionally selective neurotoxin for serotonergic and norad-renergic nerve terminals.     

Correlation of behavioral and neurochemical effects of acute administration of cocaine in rats.

Effects of cocaine were investigated on spontaneous motor activity (SMA) and stereotypy as well as on the concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and acetylcholine (ACh) in the discrete brain areas, such as the caudate nucleus (CN), diencephalon-midbrain (DM) and pons-medulla (PM) in rats up to 90–120 min following its injection in single doses (15–20 mg/kg, i.p.). After cocaine administration, the SMA was increased usually reaching its peak between 10–20 min, and then decreased gradually. Stereotypy and its components gradually increased to their maximum at about 50–60 min and remained at that level during rest of 120 min sessions. NE levels slightly increased in the DM and PM at 10 min post-drug after which they were decreased at 20 min. DA levels in the CN and DM were increased markedly at 20 min post-drug and decreased at 40 min. 5-HT levels in DM and PM decreased gradually up to 20 min, then began to increase. ACh level in the CN was gradually increased at 40 min and then decreased. It appears that cocaine-induced hyperactivity and stereotypy followed release of NE and DA after their accumulation in the respective brain areas.     

Effects of thyrotropin-releasing hormone (TRH) and MK-771 on behavior of squirrel monkeys controlled by noxious stimuli

The effects of TRH (0.1-30 mg/kg) and an enzyme-resistant analogue, MK-771 (0.1-10 mg/kg), were characterized in squirrel monkeys on responding maintained in the presence of different visual stimuli by a multiple 3-min fixed-interval (FI), 30-response fixed-ratio (FR) schedule of stimulus-shock termination or by a multiple 5-min FI schedule of food or shock presentation. Under the termination schedule, the first response at the end of 3 min in the FI component or the completion of the 30-response requirement in the FR component terminated the visual stimulus in the presence of which shocks occurred (escape schedule). Under the schedule of food or shock presentation, the first response at the end of the 5-min FI produced food in the presence of red stimulus lights or shock in the presence of white lights. TRH and MK-771 produced large, dose-related increases in responding maintained under the FR stimulus-shock termination schedule whereas these peptides produced smaller increases or did not affect responding under the FI schedule. TRH and MK-771 also produced marked increases in responding maintained by shock presentation at doses that did not alter or decreased food-maintained responding in the same subject. Thus, performances maintained by noxious stimuli are uniquely sensitive to the rate-increasing effects of TRH and MK-771. These findings suggest that the behavioral effects of the neuropeptides, TRH and MK-771, can depend on the specific consequences of behavior and, as such, the effects of these substances are determined by many of the same variables that determine the effects of other behaviorally-active drugs.     

Dysregulation of the Norepinephrine Transporter Sustains Cortical Hypodopaminergia and Schizophrenia-Like Behaviors in Neuronal Rictor Null Mice

The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multimeric signaling unit that phosphorylates protein kinase B/Akt following hormonal and growth factor stimulation. Defective Akt phosphorylation at the mTORC2-catalyzed Ser473 site has been linked to schizophrenia. While human imaging and animal studies implicate a fundamental role for Akt signaling in prefrontal dopaminergic networks, the molecular mechanisms linking Akt phosphorylation to specific schizophrenia-related neurotransmission abnormalities have not yet been described. Importantly, current understanding of schizophrenia suggests that cortical decreases in DA neurotransmission and content, defined here as cortical hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder. We sought to identify a mechanism linking aberrant Akt signaling to these hallmarks of schizophrenia. We used conditional gene targeting in mice to eliminate the mTORC2 regulatory protein rictor in neurons, leading to impairments in neuronal Akt Ser473 phosphorylation. Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated behavior. In addition, they show reduced prefrontal dopamine (DA) content, elevated cortical norepinephrine (NE), unaltered cortical serotonin (5-HT), and enhanced expression of the NE transporter (NET). In the cortex, NET takes up both extracellular NE and DA. Thus, we propose that amplified NET function in rictor KO mice enhances accumulation of both NE and DA within the noradrenergic neuron. This phenomenon leads to conversion of DA to NE and ultimately supports both increased NE tissue content as well as a decrease in DA. In support of this hypothesis, NET blockade in rictor KO mice reversed cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in NET expression, which we show is ultimately influenced by Akt phosphorylation status. These data illuminate a molecular link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific mechanism for cortical hypodopaminergia and hypofunction. Additionally, our findings identify Akt as a novel modulator of monoamine homeostasis in the cortex.