Basal forebrain efferents to the medial dorsal thalamic nucleus in the rhesus monkey.

Thalamic efferent connections of the basal forebrain (BF); medial septal nucleus (MS), vertical limb of the diagonal band (VDB), horizontal limb of the diagonal band (HDB), nucleus basalis (NB), and ventral pallidum (VP) were investigated in twelve rhesus monkeys. In five animals, injections of radioactively labeled amino acids were placed in the BF. In four animals, the injections involved different divisions of the NB, HDB, and the most ventral part of the VDB. In those four cases, labeled fibers in the medial forebrain bundle were observed traveling caudally towards the hypothalamus where some turned dorsally to enter the inferior thalamic peduncle. These fibers terminated in the ventral half of the magnocellular part of the medial dorsal thalamic nucleus (MDmc). In a fifth case, the amino acid injection involved most of the MS and the VDB. Labeled fibers traveled caudally from the injection site and entered the stria medullaris. These fibers then traveled caudally before turning ventrally to terminate in the dorsal half of MDmc. To determine which of the diverse neuronal types in the BF gives rise to these thalamic projections, in two monkeys injections of horseradish peroxidase (HRP) were placed into MDmc. Labeled neurons were observed throughout the full extent of the NB, the VDB, the MS, and part of the VP. In order to determine the extent of the cholinergic input to MDmc from the BF, one of the HRP cases was processed for the simultaneous visualization of HRP, and acetylcholinesterase (AChE), the hydrolytic enzyme for acetylcholine, and a second case was processed for simultaneous visualization of HRP, and choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine. We observed that 30-50% of the HRP-labeled neurons were putatively cholinergic. In order to determine if the NB projection to MD is a collateral of the NB projection to orbital frontal cortex, one fluorescent retrograde tracer was injected into the orbital frontal cortex and one into MD. This case showed that approximately 5% of the BF neurons that project to MDmc also project to the orbital frontal cortex. These results confirm a significant subcortical projection by which the cholinergic system of the basal forebrain may influence higher cortical functions through the thalamus.

Identification and quantification of kynurenic acid in human brain tissue.

Serial ion-exchange and high-performance liquid chromatography separations were employed for the tissue extraction and purification of kynurenic acid (KYNA). Subsequently, the compound isolated from postmortem human brain tissue was unequivocally identified as KYNA by nuclear magnetic resonance and mass spectrometric analyses. Regional distribution analyses revealed the highest concentration of KYNA (1.58 +/- 0.43 pmol/mg tissue) in the caudate nucleus with lower levels in the thalamus, globus pallidus, hippocampus, parietal cortex and frontal cortex. Of the brain structures examined, the lowest concentration of KYNA (0.14 +/- 0.02 pmol/mg tissue) was found in the cerebellum.

Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: I. Effects of cell density and of an adult mammalian striatal-derived neuronotrophic factor (SDNF).

The use of CNS cultures for detection and quantification of neuronotrophic activity in the CNS has been analyzed. In particular the development, i.e., neurotransmitter uptake characteristics, and survival of dopaminergic and GABAergic neurons in fetal mouse (E13)-dissociated mesencephalic cells cultured in serum-free, hormone-supplemented medium have been assessed as a function of culture time and cell density. At all times, more than 98% of the cells were classified as neurons on the basis of immunocytochemical criteria. Results indicate that the increase of cell density in vitro significantly enhances specific high-affinity dopamine uptake per dopaminergic cell and cell survival. This effect is not limited to the dopaminergic cells and suggests that the development of neurotransmitter-related traits and cell survival are influenced by cell density-derived trophic signals. The above-mentioned cultures and parameters have also been used to detect neuronotrophic activity in adult mammalian brain extracts or more purified preparations. In particular, bovine striatal extracts contain activity capable of increasing high-affinity neurotransmitter uptake parameters and cell survival of at least the dopaminergic and GABAergic neurons present in the culture system. The neuronotrophic activity from bovine striatum has been partially purified and is associated with a fraction whose main component is a basic protein of approximately 14 kDa.

Regional differences in resting extracellular potassium levels of rat brain.

The extracellular potassium concentration in the cerebral cortex of the mammalian brain has been repeatedly reported to be approximately 3.0 mM. We have made detailed measurements with potassium-selective microelectrodes and have found significantly lower extracellular potassium concentrations in unstimulated rat brain caudate and thalamus (1.9-2.5 mM) when compared to cortex and cerebral spinal fluid (3.0-3.5 mM). These regional differences may be caused by variations in spontaneous activity of neurons, regional permeability differences in endothelial cells of brain capillaries to potassium, or caused by variations in uptake by glia.

5,5-Diphenylhydantoin antagonizes neurochemical and behavioral effects of p,p'-DDT but not of chlordecone.

Rats were given 75 mg/kg of 5,5-diphenylhydantoin (phenytoin) or vehicle 30 min prior to 75 mg/kg of 1,1,1-trichloro-bis(p-chlorophenyl)ethane (p,p'-DDT) (p.o.) or chlordecone (i.p.) and tremor was measured 12 h later. Rats were then killed, and regional brain levels of biogenic amines and their acid metabolites and amino acids were determined. Pretreatment with phenytoin significantly attenuated the tremor produced by p,p'-DDT but enhanced that produced by chlordecone. p,p'-DDT had significant effects on the levels of aspartate, glutamate, 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), whereas chlordecone increased glycine, 5-HIAA, and MHPG levels. Pretreatment with phenytoin blocked p,p'-DDT-induced increases of aspartate in the brainstem and spinal cord, 5-HIAA in the hippocampus, and MHPG in the brainstem and hypothalamus. Phenytoin significantly enhanced chlordecone-induced increases of MHPG in the brainstem. These data indicate that organochlorine-induced increases in noradrenergic activity in the brainstem and spinal cord may be directly related to the tremorigenic effects of these chemicals.

[CRF-like immunoreactivities in mammalian brains examined with a radioimmunoassay for CRF].

A sensitive and useful radioimmunoassay for CRF was established using synthetic ovine CRF. CRF-like immunoreactivities of hypothalami in the bovine, human, rat, guinea pig, and mouse could be detected with this radioimmunoassay. A variety of CRF-like immunoreactivities of hypothalami in the mammalian brains led us to discuss a putative species difference of amino-acid structure in CRF. In the bovine, CRF-like immunoreactivities of the frontal cortex, caudate nucleus, putamen, and hippocampus could be detected in addition to CRF-like immunoreactivity of hypothalamus. These observations suggested a wide distribution of CRF in the central nervous system, and gave room for discussion about a physiological role of extrahypothalamic CRF. Gel chromatographic studies revealed a high-molecular-weight form of CRF-like immunoreactivity in the bovine frontal cortical profile that was not found in the hypothalamic profile. The results show a multiplicity of CRF-like immunoreactivity in the bovine brain.

High-performance liquid chromatography of biogenic amines and metabolites in brain, cerebrospinal fluid, urine and plasma.

A method for high-performance liquid chromatographic separation and electrochemical detection of biogenic amines and metabolites in a variety of biological matrices is described. The method employs either homogenization, precipitation or dilution followed by direct injection of the samples and permits the chromatographic resolution of dopamine, norepinephrine, epinephrine, serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in brain; 3-methoxy-4-hydroxyphenylglycol, DOPAC, 5-HIAA and HVA in cerebrospinal fluid; 5-HIAA, HVA and 5-HT in plasma; and 5-HIAA and HVA in urine. Alterations in chromatographic conditions, voltammetry and in vivo pharmacological manipulations are employed to verify the identity of the putative neurotransmitter and metabolite peaks in the biological samples.

Lateralized decrease in self-stimulation induced by haloperidol in rats with unilateral 6-hydroxydopamine lesions.

Rats with bilateral hypothalamic electrode placements which generated similar self-stimulation rate-intensity functions were subjected to unilateral injections of 6-hydroxydopamine (6-OHDA) into A9 and A10 areas. Following 12 weeks of postoperative recovery which was bilaterally symmetrical the rats were administered 0.1 mg/kg haloperidol. In sham- and vehicle-injected control rats the haloperidol produced bilaterally symmetrical decreases in self-stimulation. In the rats with 6-OHDA lesions the haloperidol effect was asymmetric with a much greater decrease in self-stimulation evident for electrode placements in the dopamine deficient hemisphere than for electrodes in the non-lesion hemisphere. Biochemical evaluation of the lesions indicated that dopamine was severely depleted in limbic and striatal forebrain areas. The combined use of a lesion with a pharmacological blockade of a neurotransmitter system appears to be an effective technique to distinguish reward versus performance effects of the transmitter on self-stimulation.

Effects of nicotine on exploratory behavior in rats: correlation with regional brain monoamine levels.

The results of a dose-response study of the effects of nicotine on exploratory behavior in male rats is reported. Nicotine at 0.5 mg/kg elevated locomotor activity without significantly changing other parameters of exploration. Low-dose nicotine (0.2 mg/kg) did not produce any effect on exploration measures, while high-dose nicotine (0.8 mg/kg) produced a state of ataxia in animals and decreased most exploration measures in general. Additional, nicotine at high doses seems to reduce the animal's state of fear/anxiety, while at low dose the drug seems to increase the animal's level of curiosity in a novel environment. Biochemically, nicotine has been found to accelerate dopamine synthesis and norepinephrine turnover, and to decrease serotonin turnover. More importantly, the amino acid precursors tyrosine and tryptophan were found to be the neurochemical measures most related to the behavioral changes produced by nicotine.

Neurotensin immunoreactivity in post-mortem brain is increased in Down's syndrome but not in Alzheimer-type dementia.

Neurotensin immunoreactivity and choline acetyltransferase (ChAT) activity were measured in post-mortem brain from 10 cases of Down's syndrome (7 aged 53-63 years, one aged 27 years, one aged 16 months and one aged 10 months), 6 cases of Alzheimer-type dementia (ATD) and 19 control subjects (13 aged 40-88 years and 6 aged 9-18 months). Neurotensin concentrations in anterior and basal hypothalamus, amygdala, septal area, caudate nucleus and temporal cortex were unaltered in ATD. The concentrations of neurotensin were significantly increased in the caudate nucleus, temporal cortex and frontal cortex in the cases of Down's syndrome aged 53-63 years with the neuropathological features of ATD, and were also increased in the cerebral cortex of the 27-year-old, which did not have the neuropathological features of ATD, and in two infant Down's cases. ChAT activity was reduced in the ATD and the 53-63-year-old cases of Down's syndrome, but not in the 27-year or 10-month-old Down's cases. The increased neurotensin concentrations appear to be a feature of Down's syndrome not related to the presence of plaques and neurofibrillary tangles or to a deficit in ChAT activity.

Electroacupuncture alters catecholamines in brain regions of rats.

Electroacupuncture (EA) stimulation mediated the release of [3H]norepinephrine (NE) from synaptosomes prelabeled with [3H]NE. The pulse release of [3H]NE by EA stimulation was dependent on the presence of Ca2+. Treatment of rats with EA for 30 min at 4 Hz did not significantly alter the dopamine (DA) content in hypothalamus, cerebellum, pons, midbrain, and cerebral cortex regions, but the DA level was decreased by 20% in caudate nucleus. The NE level was found to increase by 43% in caudate nucleus and 38% in hypothalamus. The results indicate that only certain neuronal pathways are affected by the EA treatment, and that NE and DA may respond differently to such stimulation.

Modulation of anterior pituitary dopamine receptors by estradiol 17-beta: dose-response relationship.

Exposure of ovariectomized rats to estradiol-17-beta for 48-96 hr resulted in a dose-dependent reduction in the number but not the Kd of D-2 dopamine receptors of the anterior pituitary. No influence of estrogen was observed on dopamine or muscarinic acetylcholine receptors of caudate or hypothalamus. The dose-response relationship observed suggested that the influence of estrogen is directly on the pituitary, not secondary to the alteration of dopaminergic systems in the hypothalamus.

Determination of dopamine, norepinephrine, serotonin and their major metabolic products in rat brain by reverse-phase ion-pair high performance liquid chromatography with electrochemical detection.

A method is described for the separation and quantitation of catecholamines, serotonin, and their major metabolites with use of reverse-phase, ion-pair liquid chromatography with electrochemical detection. This method employs columns packed with a microparticulate C-18 resin, octyl sodium sulfate as the ion-pairing agent, and isocratic elution with a citrate-phosphate buffer containing methanol. Conditions are described for the separation of norepinephrine, normetanephrine, 3-methoxy-4-hydroxyphenylglycol, epinephrine, metanephrine, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindole-3-acetic acid and for their quantitation in extracts of rat brain tissue.

Effect of phencyclidine (PCP) on blood pressure and catecholamine levels in discrete brain nuclei.

PCP (10 mg/kg, s.c.) causes hypertension that is associated with decrease or tendency to decrease the levels of epinephrine and norepinephrine in the hypothalamus and the brainstem regions. If this represents a decreased catecholaminergic activity, then this may enhance sympathetic activity and contribute to the PCP-induced hypertension.

Copper distribution in the normal human brain.

Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated.

Measurement of enkephalin peptides in canine brain regions, teeth, and cerebrospinal fluid with high-performance liquid chromatography and mass spectrometry.

Endogenous enkephalin pentapeptides are measured with unambiguous molecular specificity in canine and human tissue and fluid extracts. Both field desorption and fast atom bombardment mass spectrometry have been used to produce a protonated molecular ion of the peptide high-performance liquid chromatography fraction. The protonated molecular ion is subjected to collision-activated dissociation processes and a linked-field scan (B/E) selects a unique amino acid sequence-determining ion for monitoring and measurement. Stable isotope-incorporated peptide internal standards are used for quantification. Endogenous enkephalins are measured in hypothalamus, cerebrospinal fluid, pituitary, caudate nucleus, and tooth pulp extracts. Part-per-billion levels of endogenous peptide are measured.

Distribution and characterization of synenkephalin immunoreactivity in the bovine brain and pituitary.

The distribution of synenkephalin, the N-terminal fragment of proenkephalin, was studied in various parts of the bovine brain (globus pallidus, caudate nucleus, hypothalamus) and in the posterior pituitary by the use of a radioimmunoassay. The distribution of synenkephalin-immunoreactivity (IR) was compared to the distribution of Met-enkephalin-IR. Gel exclusion chromatography was used to examine the molecular forms of the immunoreactivities present in the tissues. The distribution of synenkephalin-IR was similar to the distribution of Met-enkephalin-IR, with a molar ratio of Met-enkephalin/synenkephalin ranging between 2.7 and 5.9. In all regions tested except the hypothalamus the synenkephalin-IR was present as a single species. However, in the hypothalamus a small amount of IR material (3% of the total synenkephalin-IR) was detected in fractions where larger Met-enkephalin-containing peptides eluted. Based on the concordance between the molar ratio of Met-enkephalin to synenkephalin found in the tissues and the molar ratio present in the sequence of adrenal proenkephalin, it is concluded that the brain and adrenal glands utilize a similar precursor for enkephalin biosynthesis.

An automated method for the determination of biogenic amines and their metabolites by high-performance liquid chromatography.

An automated high-performance liquid chromatographic method has been developed that allows for the determination of a number of compounds related to catechol- and indoleamine metabolism. The compounds that can be measured include L-DOPA, dopamine, dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine, norepinephrine, 3-methoxy-4-hydroxyphenylglycol, dihydroxyphenylglycol, vanilmandelic acid, epinephrine, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol. Dihydroxybenzylamine is used as an internal standard. Although dihydroxyphenylglycol and vanilmandelic acid could be detected and quantified, they could not be separated from each other. The method is completely automated and is sensitive enough to detect amounts as low as 500 fmol. Up to 200 samples a week can be analyzed in the automated mode. Using this method, analyses of brain tissue can be accomplished with no need for a cleanup procedure. The value of this procedure lies in its ability to simultaneously determine various amines and metabolites from small tissue samples in the same animals and with automation to analyze a relatively large number of samples a day with little attention by a technician.

Somatostatin and dementia in Parkinson's disease.

The concentrations of somatostatin in the cortex, hippocampus and caudate nucleus of subjects with Parkinson's disease were determined by radioimmunoassay. Somatostatin levels in the frontal cortex were significantly reduced in Parkinsonian subjects who were slightly or severely demented compared to controls and to non-demented Parkinsonians. Significant reductions were also observed in the hippocampus and entorhinal cortex of severely demented subjects.