Neurochemical Methods Research Articles

Neurochemical evidence that [Ca2+]o antagonizes the effect of neomycin on acetylcholine release from mouse hemidiaphragm preparation: an attempt to assess the margin to safety.

Although the neuromuscular junction is the most thoroughly studied synapse of any type and has become the model of our understanding of synaptic transmission, some questions remain unanswered; e.g. there has been no direct assessment of the size of margin of safety. In this study the [Ca2+]o-dependent, quantally released acetylcholine measured by a neurochemical method, and the contraction of the mouse hemidiaphragm in response to phrenic nerve stimulation were recorded, and the effect of neomycin was studied. It was found that a much higher concentration of neomycin was needed to depress contractions, than to reduce acetylcholine release to the same extent, and that there was an inverse correlation between [Ca2+]o and the inhibitory effect of neomycin on acetylcholine release. Ninety percent of the release of acetylcholine had to be reduced by neomycin before any reduction in muscle responses could be seen. This indicates that the margin of safety is about 10. In conclusion, at the neuromuscular junction any reduction in ACh release, whatever the mechanism, first produces a reduction in the margin of safety. The nondepolarizing neuromuscular blocking agents block primarily the nicotinic receptors located on the postjunctional site. Many receptors have to be blocked before a reduction of muscle response is observed. This is probably the reason why unexpected clinical problems (e.g. recurarization) have been described when a patients has been treated with antibiotics, even though the dose of muscle relaxant injected was relatively low.

Age-related changes of the noradrenergic and acetylcholinesterase reactive nerve fibres innervating the Pigeon Bursa of Fabricius

Age-dependent changes in the innervation of the pigeon (Columba livia, L.) bursa of Fabricius, from hatching to 120 days of age, were studied by fluorescence-histochemical and neurochemical methods for demonstrating noradrenergic and acetylcholinesterase (AChE)-reactive nerve fibres respectively. The distribution of both nerve fibre types was largely perivascular. Furthermore, a few isolated nerve fiber profiles were observed beneath the bursal epithelium, in the interfollicular septa and in the follicular cortex. No nerve fibre profiles reaching the medulla of the lymphoid follicles were observed. In addition to nerve fibres, AChE reactive neuron-like cells were encountered within the capsule and interfollicular septa. AChE reactivity was also found in dendritic-like cells localized in the cortical and cortico-medullary border. No changes in the density of perivascular noradrenergic innervation were noticeable during the ages studied, whereas the density of AChE-reactive fibres supplying vessels reached the adult pattern at 30 days, and then remained unvaried. The density of non-perivascular nerve fiber profiles, specially the AChE reactive type, increased until 30 days, remained unchanged until 75 days and then increased with aging (90-120 days). The interrelationship between the autonomic nervous system and the immune system is discussed.

Behavioral and neurochemical changes induced by aging in dopaminergic systems of male and female rats

Changes induced by aging in dopaminergic activity of male and female rats were compared by behavioral and neurochemical methods. Young (3 months) and old (23 months) rats were used. Aging decreased animal activity in the open field and increased apomorphine-induced stereotyped behavior. No differences in open field data were observed between males and females. Young and aged female rats had higher striatal DA and HVA levels than males; aging induced a decrease in both striatal DA and HVA levels in males, but not in females. No changes in HVA/DA ratios were observed among the different groups. These results show that aging reduces nigrostriatal activity as well as nigrostriatal DA levels. Furthermore, they indicate that time course events related to aging differ between males and females.

The role of endogenous opioids in the guinea pig dentate gyrus: A brief review

Research in this lab has been focused on the role of the opioid neuropeptides in the regulation of synaptic transmission in the mammalian brain. We ask how specific opioid peptides function in the control of neuronal excitability in the rodent hippocampus by defining 1) the physiological conditions under which the peptides are released, 2) the postsynaptic cell types on which they act, 3) the specific receptors and signal-transduction mechanisms mediating their actions, and 4) the specific physiological consequences of their action on the functioning of the neural circuit. Electrophysiological, anatomical and neurochemical methods are used to define the pre- and postsynaptic features of the neuropeptide synapse

Mental tiredness in the Lundby study: incidence and course over 25 years

The Lundby study is a prospective, epidemiological survey of mental health in a total population. When evaluating the material of 2550 individuals followed from 1947 to 1972, we found a large group with mental tiredness as the dominating symptom. Next to depression this was the most frequent diagnosis in the population. According to our concept of Tiredness, one third of the women ran a lifetime risk of developing a first-ever episode of Tiredness, while the risk for men was one fifth. The incidence of Tiredness is described together with the total frequency of episodes. The course of mental illness starting with a first episode of Tiredness within the 25-year investigation period shows that about one third of the men and half of the women relapsed into Tiredness or other mental illnesses, not infrequently with a serious outcome. However, most of the episodes of Tiredness lasted less than 2 years and were of milder impairment. We emphasize the importance of further investigations by means of epidemiological, clinical and neurochemical methods as regards the heterogeneous syndrome of mental fatigue.

Synaptic inhibition in primary and secondary chronic epileptic foci induced by intrahippocampal tetanus toxin in the rat.

1. Injecting twelve mouse minimum lethal doses of tetanus toxin into one hippocampus of a rat leads to the development of chronic epileptic foci in both hippocampi. These generate intermittent epileptic discharges for 6-8 weeks. Here we compare GABAergic inhibition, 10-18 days after injection, in slices prepared from the injected and contralateral hippocampi (respectively the primary and the secondary or 'mirror' foci), using both neurochemical and electrophysiological methods. 2. Epileptic activity was recorded from slices of both hippocampi from all tetanus toxin-injected rats. Evoked epileptic discharges were similar on the two sides, but spontaneous epileptic discharges were more common contralaterally. 3. Ca(2+)-dependent, K(+)-stimulated (synaptic) release of radiolabelled GABA was depressed in slices from the injected hippocampus, compared with vehicle-injected controls. In contrast, slices from the contralateral hippocampus had normal levels of Ca(2+)-dependent, K(+)-stimulated GABA release, even though adjacent slices were epileptogenic. 4. Intracellular recordings revealed that both fast and slow stimulus-evoked inhibitory postsynaptic potentials (IPSPs) were abolished in CA3 pyramidal cells in the primary focus. In the secondary focus, however, fast IPSPs were seen in seven of twenty-five cells, and slow IPSPs were seen in all cells if the stimulus was strong enough. 5. Monosynaptic IPSPs were isolated pharmacologically by blocking glutamatergic excitatory postsynaptic potentials (EPSPs) with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphopentanoic acid (AP-5). No monosynaptic IPSPs were uncovered in cells from the primary focus at any stimulus strength. Monosynaptic IPSPs were evoked in all cells from both the secondary focus and control slices. The estimated conductances of monosynaptic fast IPSPs were similar in cells from the secondary focus and from the controls, although the former required twice the stimulus strength. 6. Slow IPSPs were found in the secondary focus and in controls, but not in the primary focus. They were sensitive to 3-amino-2-(4-chlorophenyl)-2-hydroxy-propylsulphonic acid (2-OH saclofen). The estimated conductances of slow IPSPs evoked by weak stimuli in the secondary focus were much smaller than in the controls. However, stimuli that could trigger epileptic discharges in the secondary focus, evoked 2-OH saclofen-sensitive slow IPSPs with estimated conductances approaching the controls. This marked increase in the slow IPSP did not occur when EPSPs, and epileptic bursts, were blocked with CNQX and AP-5, suggesting that a strong barrage of excitation is needed to generate full-sized slow IPSPs in the secondary focus.(ABSTRACT TRUNCATED AT 400 WORDS)

QEEG profiles of different benzodiazepine ligands

Alcohol-nontolerant (ANT) rats, produced by selective breeding for high sensitivity to motor-impairing effects of ethanol, have a point mutation in the cerebellar γ-aminobutyric acid type A (GABAA) receptor α6 subunit, which has been proposed to underlie enhanced sensitivity to benzodiazepine agonists as well. We compared ANT and alcohol-tolerant (AT) rats using behavioral and neurochemical methods to assess the significance of α6- and nonα6-containing GABAA receptor subtypes. Motor performance in a tilting plane test was largely unaffected by a type I benzodiazepine receptor-preferring agonist, zolpidem [1–10 mg/kg, intraperitoneally (IP)], partial benzodiazepine agonists bretazenil and ZG-63 (both at 40 mg/kg, IP), and a novel broad-spectrum anticonvulsant loreclezole (40 mg/kg, IP) in both ANT and AT rats. In contrast, diazepam (10 mg/kg, IP) impaired performance of the ANT but not AT animals. These data, supported by results from brain regional autoradiography of [3H]Ro15-4513 and membrane binding of [3H]ZG-63 and [35S]TBPS as influenced by these ligands, strongly suggest that only ligands with full agonist actions on mutant (ANT) but not wild-type (AT) α6-containing GABAA receptors are able to produce motor impairment in the ANT rats.

Endorphins, Opiates and Behavioural Processes

Overview of the Endogenous Opioid Systems: Anatomical, Biochemical and Functional Issues Physiological Dependence on Opioids Opioid Reinforcement Processes Tolerance, Endorphins and Other Aspects of Opiate Drug Discrimination Environmentally-Induced Analgesia: Situational Factors, Mechanisms and Significance Endorphins, Opiates and Food Intake Evidence for Opioid Involvement in Controls of Drinking and Water Balance The Role of Opioid Mechanisms in Social Interaction and Attachment Endorphins and Sexual Behaviour Endorphins, Exploration and Activity Opioid Modulation of Learning and Memory Endogenous Opioid Systems and Neurobehavioral Development Neurochemical Methods in the Study of the Role of Endorphins in Psychiatric Disorders Narcotic Antogonist and Opioid Treatment in Psychiatry.

Selective sparing of NADPH‐diaphorase–somatostatin–neuropeptide Y neurons in ischemic gerbil striatum

In the present study we investigated the relative vulnerability of neuronal subsets in the striatum to ischemia that was induced by bilateral transient ligation of the common carotid arteries in gerbils. After 4 days of survival, brains were evaluated using histochemical methods (NADPH-diaphorase and silver degeneration procedures) and neurochemical methods with radioimmunoassays for somatostatin-, neuropeptide Y-, and substance P-like immunoreactivity and measurements of amino acids using high-pressure liquid chromatography with electrochemical detection. NADPH-diaphorase-positive neurons were strikingly preserved in the ischemic dorsolateral portion of the striatum, in which there was severe neuronal loss. There was no significant depletion of NADPH-diaphorase-positive neurons in the striatum or cerebral cortex. Concentrations of neuropeptide Y-like and somatostatin-like immunoreactivity were unchanged despite a significant 25% depletion of substance P-like immunoreactivity and gamma-aminobutyric acid. Ischemic brain damage may be mediated by a neurotoxic effect of glutamate acting at the N-methyl-D-aspartate (NMDA) receptor. Previous studies of NMDA excitotoxin lesions in rat striatum have shown a sparing of neurons containing NADPH-diaphorase, somatostatin, and neuropeptide Y. The similar sparing of these neurons following ischemic lesions in gerbil striatum provides further evidence that NMDA receptor activation may play a role in ischemic injury.

Atrial natriuretic factor in sympathetic ganglia of the rat: Dependence on cholinergic innervation

Atrial natriuretic factor is detectable in the peripheral autonomic ganglia of the rat by radioimmunoassay and immunohistochemistry. In the present study, surgical and neurochemical methods were used to evaluate the source of this peptide in sympathetic ganglia. Decentralization of the ganglia and/or central administration of colchicine diminished the atrial natriuretic factor content in para- and prevertebral ganglia. Axotomy did not affect levels of ganglionic atrial natriuretic factor. A messenger ribonucleic acid species hybridizing with rat atrial natriuretic factor complementary deoxyribonucleic acid was not found within the total ribonucleic acid extracted from superior cervical ganglia. These results indicate a direct dependence of ganglionic atrial natriuretic factor on cholinergic innervation.

Sprouting of striatal serotonin nerve terminals following selective lesions of nigro-striatal dopamine neurons in neonatal rat

The effects of neonatal intracisternal 6-hydroxydopamine (6-OHDA; 50 μg) treatment on striatal serotonin (5-HT) nerve terminals in rat have been characterized using histo- and neurochemical methods. The 6-OHDA lesion caused a 60% reduction of striatal dopamine (DA) concentration when analyzed in the adult stage, while 5-HT levels were increased by about 40% and 3H-5-HT uptake in vitro was increased by about 60%. Using computerized image analysis, a marked increase in 5-HT-like immunoreactive terminal density was found in both rostral (+200%) and caudal (+50%) striatum. Pretreatment with the DA uptake blocker amfolenic acid completely counteracted the 6-OHDA-induced alterations in both DA and 5-HT neurons in the striatum, while pretreatment with the noradrenaline uptake blocker desipramine had no significant effects. Regional analysis of 5-HT levels in the CNS after neonatal 6-OHDA treatment or the combined desipramine + 6-OHDA treatment showed no significant effect in any of the brain areas analyzed, apart from the observed 5-HT increase in striatum. It was furthermore observed that the striatal 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was decreased, while the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio was increased following the 6-OHDA lesion, indicating compensatory mechanisms in turnover of transmitters. These alterations were completely reversed after pretreatment with amfolenic acid. The present results support the view that the 5-HT hyperinnervation following neonatal 6-OHDA treatment is a collateral sprouting response induced by lesioning of the striatal DA neurons.

Concepts in the validation of neurochemical methods: The proper generation and use of statistics

Nearly every advance made in the study of neurotransmission has resulted from the development of new analytical methods or from the application of such methods to neurochemical problems in new ways. Each investigator places extremely high dependence on the laboratory method from which the data are gathered. It is therefore vital that these methods be proven valid when first selected and when used throughout the experiments being conducted. The process of method validation has flourished and been refined in the field of laboratory medicine. Methods are primarily validated by their accuracy and reproducibility in determining the analyte of interest in the tissue(s) to be studied. It is important that standards of performance be established that will allow objective decisions to be made when methods are tested for these characteristics. Performance standards for several neurotransmitters are suggested. Studies performed to collect method performance data are presented. From these data, statistics can be generated that help to estimate analytical errors. Guidelines for the proper generation and use of these statistics are discussed. Use of these validation approaches should be expanded in the whole of neurochemistry, which should enrich the data gathered within a laboratory and improve the harmonization between laboratories.

Does pipecolic acid interact with the central GABA-ergic system?

Several previous studies have suggested a strong GABA-mimetic action of the endogenous brain imino acid, L-pipecolic acid (L-PA). In the present study, these observations were evaluated using electrophysiological and neurochemical methods. In contrast to published data our electrophysiological studies on rat cortical neurones in situ showed only a weak, but bicuculline-sensitive depressant action of L-PA on cortical neurones. Furthermore, L-PA proved to have no affinity for any of the three components of the GABA-benzodiazepine-chloride channel receptor complex. However, using a modification of published methods a weak affinity for the GABA-B receptor site was demonstrated (IC50 = 1.8 X 10(-3) M). L-PA showed no anticonvulsive activity in several tests; in particular, it did not protect mice from seizures induced by inhibition of L-glutamate-1-decarboxylase (EC 4.1.1.15: GAD). L-PA had a very weak action on brain GABA levels of mice, and did not modify the rate of GABA synthesis. In conclusion, these results are not compatible with a strong in vivo interaction between L-PA and GABA-mediated inhibitory transmission.

Cholinergic, GABAergic and excitatory amino acidic neurotransmission in the goldfish vagal lobe.

Some neurotransmitter systems operating in the goldfish vagal lobe, an hypertrophied gustatory center, have been studied by means of experimental (kainic acid injection and vagal rhizotomy), neurochemical and ultrastructural methods. The use of the neurotoxin, kainic acid, revealed the existence of cholinergic and GABAergic neurons in the vagal lobe. The results of histochemical observations and biochemical assays performed after rhizotomy of sensory and motor vagal roots, suggest that the motor neurons of the vagal motor layer are cholinergic. The same experiments also indicate that the primary gustatory afferents distributing to the sensory layer of the vagal lobe are, at least in part, cholinergic. By contrast, no decrease of excitatory amino acid uptake was demonstrated following the experimental lesions. GABA is likely to play an important role in the goldfish vagal lobe, particularly in the sensory layer, where the highest level of its synthetic enzyme, glutamate decarboxylase, is recorded. The significant decrease of glutamate decarboxylase in the sensory layer after vagal rhizotomy suggests that either GABAergic primary afferents reach the vagal lobe, or that deafferentation results in a decreased GABA synthesis in intrinsic GABAergic neurons.

Possible role of histamine in brain function: neurochemical, physiological, and pharmacological indications.

Recently accumulated neurochemical, physiological, and pharmacological evidence strongly supports a role for histamine as a central neurotransmitter. Neurochemical methods, which became available within the last years, allow determination of small amounts of histamine and its metabolites in the brain and make possible future studies of central histamine regulation. The demonstration of histamine H1 and H2 receptors in the brain of several species suggests a possible role for histamine in brain function. Microelectrophysiological studies on single central neurones suggest both excitatory and depressant effects of histamine which are receptor mediated. In addition, brain histamine has been demonstrated to be subject to cyclic variations, to play a role in hormonal regulation, and to be altered by stressful conditions. Several psychotropic drugs significantly affect brain histamine regulation and elicit inhibitory effects on central histamine receptors. These findings bring new approaches and stimulus to further research on the significance of brain histamine.

Mechanism of analgesic action of mesaconitine. I. Relationship between analgesic effect and central monoamines or opiate receptors

The contribution of the central monoamines and the opiate receptor to mesaconitine (MA)-induced analgesia was investigated by means of pharmacological and neurochemical methods in which morphine (Mor) was used for comparison. The analgesic action of MA (i.c.) determined by the acetic acid-induced writhing method and the tail flick method was dose-dependent, indicating that its activity is elicited through the central nervous system. MA-induced analgesia was decreased by α-methyl-p-tyrosine (α-MT), 6-hydroxydopamine, diethyldithiocarbamate, disulfiram and reserpine, and increased by methamphetamine and norepinephrine (NE). The mode of action of MA was similar to that of Mor except for the results obtained upon combined administration with l-dopa, dopamine, α-MT or chemicals related to 5-hydroxytryptamine. In addition, MA promoted the α-MT-induced decrease in NE levels in hippocampus, medulla oblongata plus pons and spinal cord. Levallorphan did not affect the analgesic activity of MA, showing that its activity is not mediated via the opiate receptors. Consequently, it is concluded that the analgesic activity mediated by MA is closely related to response involving the central catecholaminergic system, in particular, the noradrenergic system.

On the excitotoxic properties of quinolinic acid, 2,3-piperidine dicarboxylic acids and structurally related compounds

To obtain information about the receptors which mediate the neurotoxic actions of quinolinic acid, a series of pyridine dicarboxylates and piperidine dicarboxylates and structurally related compounds were tested for their neurotoxic effects following intrastriatal or intrahippocampal infusion in the rat, and for their activity in assays of binding and uptake sites for acidic amino acids. Of the compounds tested, only cis- and trans-2,3-piperidine dicarboxylates and quinolinic acid showed pronounced neurotoxic effects. At 600 nmol, 2,6- and 3,4-pyridine dicarboxylates were weakly active and the remaining compounds were inactive in both brain regions. After injection into the striatum of the adult rat, trans-2,3-piperidine dicarboxylate, quinolinic acid and cis-2,3-piperidine dicarboxylate caused axon-sparing neuronal degeneration as assessed by light microscopic and neurochemical methods, the threshold doses being 12, 24 and 120 nmol, respectively. In the striatum of the 7-day old rat 30 nmol of trans-2,3-piperidine dicarboxylate caused extensive neuronal degeneration, but 120 nmol quinolinic acid or 600 nmol cis-2,3-piperidine dicarboxylate were inactive. Small doses of cis-2,3-piperidine dicarboxylate (60 nmol) and quinolinic acid (30 nmol) injected into the adult rat hippocampus resulted in a preferential loss of pyramidal neurons. In larger doses granule cells also degenerated. In contrast, trans-2,3-piperidine dicarboxylate was equally toxic to hippocampal neurons, regardless of the dose used. No “distant” neuronal damage was observed after the intracerebral application of any test compound. Equimolar amountsof (−)-2-amino-7-phosphonoheptanoic acid completely blocked the neurotoxic effects of quinolonic acid, cis- and trans-2,3-piperidine dicarboxylate after injection into the striatum or hippo-campus. None of the analogs tested were good inhibitors of Cl −-dependent or independent binding of L-[ 3H]glutamate, [ 3H]kainate or high-affinity, Na +-dependent uptake of L-glutamate in striatal or hippocampal tissue at 1 mM. The results indicate that the receptors mediating the neurotoxic effects of these compounds have strict structural requirements for activation. Whereas the excitotoxic characteristics of trans-2,3-piperidine dicarboxylate suggest a direct action on N- methyl- D-aspartate receptors, the properties of quinolinic acid and cis-2,3-piperidine dicarboxylate are far more complex and make categorization of their receptor-interactions difficult. Indirect mechanisms may account for the excitotoxicity of quinolinic acid and cis-2,3-piperidine dicarboxylate.

Development of catecholaminergic nerves in the spinal cord of the rat

The development of the noradrenergic and dopaminergic innervations in the spinal cord of rat was studied using fluorescence histochemical and neurochemical methods. From fetal day (FD) 16 to neonatal day (ND) 26, the cord increased in weight by 4–6 mg/day, except for the period between ND 14 and 20, when the increase was 13 mg/day. Norepinephrine was first detectable in the whole cord at ND 18, and then increased rapidly thereafter, peaking at ND 14, then declining at the end of neonatal life to the values found in the young adult spinal cord. The innervation in the intermediolateral cell column of the thoracic cord appeared to be more extensive at ND 14 than in the adult, raising the possibility of the selective destruction of a part of this noradrenergic innervation during later development. The nerve terminals in the ventral horn were first visualized clearly at birth, with a pattern similar to that of the adult. When the fetal locus coeruleus is transplanted into the transected spinal cord of the adult rat, it induces an extensive proliferation of the cut rostral axons in the ventral horn specifically. It is proposed that the transplanted fetal locus coeruleus produces a neurotrophic substance which stimulates the proliferation of the cut rostral axons derived from the locus coeruleus. Dopamine was first detectable in the cord at ND 20. Unlike noradrenergic nerves, dopaminergic nerves developed slowly throughout neonatal life. The adult innervation presumably develops slowly between ND 26 and young adulthood. In the fetus and very young neonate, DA was most concentrated in the thoracic region. Dopamine metabolism in the cord during neonatal life was a fraction of that found in the adult. It is concluded that the spinal dopaminergic and noradrenergic innervations develop with quite different time sequences. The rapid peaking of the noradrenergic innervation of ND 14 may play a significant role in the overall development and functional maturation of the cord.

Clinical neurochemistry of autism and associated disorders.

Advances in information concerning brain function in animals and advances in analytical neurochemical methods for determining extremely low levels of compounds in physiological fluids have opened great opportunities for clinical neurochemical studies of autism. Nevertheless, the behavioral deficits in autistic individuals are major obstacles to clarification of the relations between symptoms and biochemical dysfunction in the brain. The fundamental preclinical and clinical studies of serotonin, dopamine, and norepinephrine metabolism related to infantile autism are reviewed, and new studies are suggested as examples of the productive strategies that will illuminate features of the autistic syndrome in the next decade.

Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.

A procedure for occluding the stem of the proximal middle cerebral artery of the rat is described. The operation is performed under anaesthesia through a small subtemporal craniectomy. After occlusion, 3 animals were perfused with carbon block and 8 with a FAM fixative (40% formaldehyde, glacial acetic acid, and methanol). The findings were compared with sham-operated animals. Carbon black studies demonstrated an area of impaired perfusion corresponding to the territory of the occluded artery in each animal. Neuropathological studies invariably showed that there was ischaemic brain damage in the cortex and basal ganglia. The frontal cortex was involved in every animal, as was the lateral part of the neostriatum; the sensorimotor and auditory cortex were involved in most animals, whereas the occipital cortex and medial striatum were involved only infrequently. The damage produced by ischaemia could be readily distinguished from the small local lesion seen at the surgical site in sham-operated animals. The ability to produce a consistent focal ischaemic lesion in the rodent brain provides a technical approach that is sufficiently reproducible to enable investigation of the pathophysiology of ischaemia using recently developed autoradiographic and neurochemical methods.