Histochemical Study of Aldehyde Dehydrogenase in the Rat CNS

A quantitative histochemical method was developed to determine aldehyde dehydrogenase (EC 1.2.1.3; ALDH) activity in the CNS. The distribution of ALDH activity in all rat brain and spinal cord regions is described. Among the CNS neuron structures, high enzyme activity was found in receptor and effector neurons, whereas low activity was noted in perikarya of the majority of intermediate neurons, including all aminergic neurons. A positive correlation was demonstrated between the distribution of ALDH activity among rat CNS microregions (our own data) and the density of dopaminergic terminals, dopamine content, and monoamine oxidase activity (literature data) among the same microregions. They may reflect a spatial linkage between ALDH and the predicted sites of natural aldehyde production. Lower enzyme activity was found in phylogenetically younger brain structures. It may explain the differential resistance of CNS structures to ethanol (acetaldehyde). Among the barrier CNS structures, moderate ALDH activity was found in capillaries and surrounding astrocytes and high activity was noted in ependimocytes covering the brain cavities and those of the vascular plexus. This provides realization of the function of ALDH as a brain metabolic barrier for aldehydes.     

Structural changes in the rat cerebral cortex induced by alcohol in combination with aldehyde dehydrogenase inhibitors

The cerebral parietal cortex in rats subjected to an acute (single) and subacute (for 5 days) ethanol effect in combination with aldehyde dehydrogenase (AldDG) (enzymes classification 1.2.1.3 AldDG) inhibitors--disulfiram and cyanamide--has been investigated histochemically and electron microscopically. The inhibitors mentioned produce an essential decrease of AldDG activity in the cerebral cortex; it remains in some structures even 6 days after their single administration. Against the background of AldDG inhibitors alcohol produces more noticeable structural disorders in the cerebral cortex, they are possibly connected with accumulation of a highly toxic ethanol metabolite-acetaldehyde--in blood and with its easy penetration into the brain. This demonstrates an important role of AldDG in protection of the brain from alcoholic (aldehydic) lesions, as well as a peculiar danger for the brain of ethanol in combination with inhibitors of this enzyme.     

Localization of alcohol and aldehyde dehydrogenases in the human spinal cord and brain

Location of aldehyde dehydrogenase (AldDG) and alcohol dehydrogenase (ADG) has been studied in 38 nuclei of the human brain. Neurons with a high AldDG activity predominate in the nucleus of the descending root of the trigeminal nerve, motor nuclei of the craniocerebral nerves (trigeminal, facial, abducent, blocking, sublingual, supraspinal), motor nuclei of the anterior horns of the spinal cord, lateral vestibular nucleus, posterior nucleus of the vagus nerve, pedunculopontine nucleus, superior salivary nucleus, and in the nucleus of Westphal-Edinger-Jacobovich. Neurons with a moderate AldDG activity predominate in the superior olivary complex, nucleus of the lateral loop, parabrachial (pigmented) mesencephalic nucleus and reticular lateral nucleus. A low enzymatic activity is specific for neurons of the pons proper, inferior vestibular nucleus, trapezoid body of the inferior olivary complex, dentate nucleus of the cerebellum, reticular nucleus of the tegmen of Bekhterev's pons and posterior nucleus of Gudden's suture. A high ADG activity is revealed in piriform neurons of the cerebellar cortex. Functional importance of ADG and AldDG activity in the brain is discussed.     

Systems principle for separating out neuron groups as relatively independent working units

The respiratory center has been studied as an example of the neural center organization. This organization is presented by a number of cellular populations, each of them consisting of several neuronal groups (components of the populations) of various types. These groups are considered as relatively autonomic sets of various neuronal categories, where 1-5 large efferent (phase) neurons are present as a central link. Analyzing the spatial arrangement and functional interrelations of the neurons in the group, it is possible to conclude that the groups revealed (respirons) are functional units of neuronal activity. Applying the theory of functional system (P. K. Anokhin) for analyzing connections between the neurons in the group and the afferent impulsation that gets into action sphere of the group, it is possible to formulate certain criteria on integrity and a relative functional independence of the neuronal groups as working units of neuronal activity, in which the reticular component of the groups as widely represented in all parts of the CNS, a suggestion is made that the respirons are the natural invariant of the structure when the cerebral function is reorganized.     

The distribution of cytochrome-oxidase activity in rabbit brain

1. The cytochrome-oxidase activity in eleven structures of the rabbit brain has been investigated. 2. Kinetic data suggest that the same enzyme occurs in all brain structures but that the activity on a tissue-weight basis differs from structure to structure, being in general higher in the phylogenetically newer structures. 3. When the kinetic data are related to cell density (as measured by DNA concentration) all the neuron-containing structures of the cerebrum and brain stem show the same activity but the corpus callosum and the cerebellum show much less. 4. A special study was made of the distribution of activity and cell density within the diencephalon. 5. The results suggest that all neurons in the cerebrum and brain stem have a similar cytochrome-oxidase activity, which is about 80 times that of glia, and that the cerebellum is relatively richer in glia or that it contains neurons with a much lower cytochrome-oxidase activity, or both.     

Various neurochemical aspects of the action of the tetrapeptide tuftsin on limbic system structures (histochemical and biochemical study)

Tetrapeptide tuftsin action on the activity of succinate dehydrogenase (SDG), malate dehydrogenase (MDG) and monoamine-oxidase (MAO) in microstructures of the neocortex, hippocampus and hypothalamus (supraoptic and paraventricular nuclei, retrochiasmic zone) has been investigated by means of histochemical methods. Simultaneously, pyruvate-, malate-, glutamate-, alfaketoglutamate-, succinate- and lactate-dehydrogenase activity in the neocortex and in the structures of the limbic system has been studied biochemically. SDG and MDG activity increases in neurons and glycocytes of all the hypothalamic formations mentioned. Changes in the activity of dehydrogenases in the hippocampus and neocortex under the same stimulation are less pronounced. MAO activity also increases in the nerve terminals converging on the bodies and dendrites of hypothalamic neurons and in the preterminal fibers of the neocortex, Tuftsin increases oxidative-reducing processes in various structures of the brain, but at the same time it possesses a predominant influence on the limbic system structures.     

Neuronal correlates of corticalization in mammals: a theory

The cerebral cortex of mammals has been found to be uniformly organized, and to be composed of elementary processing units or modules having an essentially constant number of neurons. In the present paper the hypothesis is put forward that the relative proportion of local circuit neurons (LCNs) within a module reflects the evolutionary level of corticalization of a mammal. The modules, in turn, are interconnected so as to form basic neuronal networks or columns with a species-specific width varying from 90 to 310 microns. A mathematical formalism is presented from which the hypothetical ratio between LCNs and projection neurons, as well as the size of the cortical column and the number of modular units that it contains, can be calculated.     

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.     

Synaptic relationship between somatostatin- and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats

J. Neurochem. (2012) 122, 923-933. ABSTRACT: The pre-B?tzinger complex (pre-B?tC) in the ventrolateral medulla oblongata is critical for the generation of respiratory rhythm in mammals. Somatostatin (SST) and neurokinin 1 receptor (NK1R) immunoreactivity have been used as markers of the pre-B?tC. SST immunoreactivity almost completely overlaps with small fusiform NK1R-immunoreactive (ir) neurons, the presumed rhythmogenic neurons, but not with large multipolar NK1R-ir neurons. Understanding the neurochemical characteristics, especially the synaptic relationship of SST/NK1R-ir neurons within the pre-B?tC network is essential in providing cellular and structural bases for understanding their physiological significance. This work has not been documented so far. We found that SST immunoreactivity was highly expressed in terminals, somas, and primary dendrites in the pre-B?tC. Besides the small fusiform neurons, a small population of medium-sized NK1R-ir neurons also colocalized with SST. Large NK1R-ir neurons were not SST-ir, but received somatostatinergic inputs. SST-ir terminals were glutamatergic or GABAergic, and synapsed with NK1R-ir neurons. Most of synapses between them were of the symmetric type, indicating their inhibitory nature. Asymmetric synapses were evident between SST-ir terminals and NK1R-ir dendrites, strongly suggesting an excitatory innervation from the presumed rhythmogenic neurons as these neurons are glutamatergic. We speculate that SST-mediated excitatory and inhibitory synaptic transmission onto NK1R-ir rhythmogenic and follower neurons synchronizes their activity to contribute to respiratory rhythmogenesis and control.     

Insects and Vertebrates: Analogous Structures in Higher Integrative Centers of the Brain

This work deals with studies on anatomical relationships, neuronal composition, and some synaptic connections that exist in the central complex (CC) in the supraesophageal ganglion in larva of dragonfly g. Aeschna. It has been shown that CC contains protocerebral bridge of an elongated and slightly curved cylindrical shape, fan-shaped and ellipsoid bodies of a bean-like shape and two small roundish noduli. There were revealed (stained) neurons providing both internal connections of CC and its connections with other CNS regions. Connections with tritocerebrum, the higher center of the autonomic nervous system, and subesophageal ganglion, an intermediate relay between supraesophageal ganglion and truncal brain, have been established. The existence of connections of CC with nuclei of abdominal nervous chain is suggested. Connection of ocelli with the CC has been traced. Unipolar neurons of the same type have been revealed, each of them giving collaterals to protocerebral bridge and ending as bushy terminals that form the main part of glomerule in the fan-shaped and ellipsoid bodies. Glomeruli are arranged in rows, in which cross connections have been found. It has been established that the structure of neuropils of the fan-shaped and ellipsoid bodies represent a shielding structure described in the cerebral cortex, midbrain cortex, and cerebellar cortex of vertebrates. Thus, in insects, like in vertebrates, the shielding structures developed not only in optic centers, but also in structures performing higher integrative functions. A possible functional role of the central complex is discussed.     

Immunocytochemical studies on the central nervous system of the earthworm, Lumbricus terrestris

There are numerous aldehyde fuchsin (AF)-positive, neurosecretory cells of medium size (A cells) and a small number of large, AF-negative neurons (B cells) in the cortical layer of the cerebral ganglion. In the subesophageal ganglion, symmetrical groups of AF-positive cells lie ventrally. The peroxidase--antiperoxidase (PAP) method was used for the immunocytochemical study of substance P and ACTH in these ganglia. In addition, the presence of L-enkephalin and alpha endorphin could be confirmed. Using rabbit antibodies to substance P we found small immunoreactive neurons among negative A and B cells in the cerebral ganglion. The processes of these immunoreactive cells could be traced to the subcortical synaptic neuropil. With antibodies to ACTH, activity was visible in perikarya similar in size to A neurons. A part of the nerve terminals of the synaptic zone, some of the B neurons and further several nerve cells of the subesophageal ganglion reacted positively. Successive demonstration of substance P and ACTH on the same section showed that the two materials occurred in different cell types. Using antiopsin antibody in an indirect immunocytochemical test we observed strong reaction in numerous medium-sized perikarya and in nerve fibres of the synaptic zone of the cerebral ganglion, further in some neurons of the subesophageal and abdominal ganglia. In contrast to this result, the photoreceptor cells of the prostomium and cerebral ganglion were negative. Presumably, substance P is present in a perikaryon type hitherto unrecognized while ACTH and antiopsin reactions seem to be located first of all in A cells.     

Co-Expression of Glutamic Acid Decarboxylase Isoform 67, Membrane Nicotinic Acetylcholine Receptors,and Connexin 36 in Ischemia-Resistant Hippocampal Interneurons

As is known, hippocampal pyramidal neurons are highly sensitive to cerebral ischemia, while some other hippocampal neurons (particularly, interneurons) survive and keep their functional activity under these conditions for a longer time. We studied interneurons of the rat hippocampal organotypic culture after 30-min-long oxygen-glucose deprivation (OGD) using immunohistochemical approaches. Four and 24 h after OGD, the somata of interneurons with no signs of degeneration (revealed by propidium iodide, PI, staining) were immunopositive to antibodies against glutamic acid decarboxylase isoform 67 (GAD67) and to an extracellular domain of a7 nicotinic acetylcholine receptor (nAChR) but negative with respect to choline acetyltransferase (ChAT). GAD67/nAChR-positive interneurons were abundant within all layers of the hippocampal CA1-CA4 zones and also in the dentate gyrus. Co-localized GAD67/nAChR immunopositivity was also observed on numerous punctuate terminals close to the somata of pyramidal neurons stained by PI. After OGD followed by incubation with a blocker of gap junctions, carbenoxolone, only single PI-stained units were revealed in the pyramidal layer. In experiments with connexin 36 cyan fluorescent protein (Cx36-CFP) on gene-reporter mice, we have found that the combination of GAD67/nAChR immunopositivity and ChAT negativity in the hippocampus is specific for the interneuronal somata expressing Cx36-CFP, a component of electrotonic gap contacts in the neuronal networks. Our results indicate that OGD-resistant hippocampal interneurons display co-localization of GAD67, a7 nAChR, and Cx36-CFP. By these neurochemical features, OGD-resistant neurons can be classified as inhibitory GABA-ergic acetylcholine-sensitive interneurons able to couple electrotonically with other hippocampal units through Cx36-CFP-containing gap junctions. The existence of hippocampal interneurons coexpressing the above factors shows that further investigations towards elucidation of cooperative endogenic mechanisms responsible for cerebral neuroresistance are expedient.     

Properties of neurons of the tectal portion of the visual system of the axolotl Ambystoma mexicanum]

In the tectum opticum of the adult neotenic A. mexicanum, responses of single neuronal units to diffuse illumination and moving visual stimuli have been investigated. Of 111 unites investigated, 27 are presented by tectal neurons, their maximum distribution being observed at a depth of 500-600 mu. In superficial layers 9 ipsi-elements were found; their receptive fields are located in the antero-dorsal part of the visual field, at both sides of the body axis. Among the units identified as the terminals of visual fibers, 70% have receptive fields of 5-10 degrees, being localized in general more close to the surface as compared to the units with the receptive field diameter of 40 and more degrees (11%). Visual neurons and ganglionic retinal cells with axons terminating in the tectum, exhibit poor specificity to the size of a stimulus within 5-30 degrees and do not react to stimuli of 2 degrees.     

Morphofunctional changes in the adrenergic innervation of the major cerebral arteries during bilateral electrostimulation of the locus coeruleus

The intramural adrenergic nervous apparatus of cerebral arteries was studied in adult rabbits after 3-10 sessions of electrical stimulation of locus coeruleus. The activity of nerve structures was determined by estimating the density of adrenergic perivascular plexuses and by semi-quantitative cytophotometry of changes in the catecholamine content of nerve varicosities. The stimulation was followed by a 28.2 +/- 1.5% increase in adrenergic innervation density. while catecholamine content in perivascular nerve structures displayed a tendency to decrease. The problem of central effects on cerebral blood flow autoregulation is discussed.     

Development of the adrenergic innervation in the myocardium and coronary arteries of the dog

The development of the adrenergic cardiac innervation was studied in premature dog fetuses, puppies and adult dogs by means of the formalin-induced fluorescence technique. A point-counting technique was used to evaluate the density of innervation. Two types of fluorescent profiles can be observed in the heart during development: (1) sprouting axons, and (2) beaded terminals. The axonal fluorescence disappears in adult neurons. A different morphology and a different time course of development enable to study separately the innervation of the myocardium (cardiomotor innervation) and that of the vessels (vasomotor innervation). The late prenatal innervation is very poor (0.1 hit). The first but very scant cardiomotor terminals appear in this period. A mature cardiomotor innervation is found in 4-month-old puppies [1.5 +/- 0.3 (SD) hits]. The vasomotor innervation is shifted to the right. The development of beaded vascular terminals begins and matures 1-2 weeks later. The growing fluorescent axons reveal that the myocardium is supplied by axons of the cardiac plexus and of the perivascular nerves; the vascular wall, on the other hand, is supplied by the perivascular nerves only. The developmental, spatial and morphological differences in innervation suggest that two different types of neurons exist in the sympathetic ganglia: (1) neurons innervating the vessels (coronaromotor neurons), and (2) neurons innervating the myocardium (cardiomotor neurons).     

Synaptic connections of the neurokinin 1 receptor-like immunoreactive neurons in the rat medullary dorsal horn

The synaptic connections between neurokinin 1 (NK1) receptor-like immunoreactive (LI) neurons and γ-aminobutyric acid (GABA)-, glycine (Gly)-, serotonin (5-HT)- or dopamine-β-hydroxylase (DBH, a specific marker for norepinephrinergic neuronal structures)-LI axon terminals in the rat medullary dorsal horn (MDH) were examined under electron microscope by using a pre-embedding immunohistochemical double-staining technique. NK1 receptor-LI neurons were observed principally in laminae I and III, only a few of them were found in lamina II of the MDH. GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were densely encountered in laminae I and II, and sparsely in lamina III of the MDH. Some of these GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were observed to make principally symmetric synapses with NK1 receptor-LI neuronal cell bodies and dendritic processes in laminae I, II and III of the MDH. The present results suggest that neurons expressing NK1 receptor within the MDH might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons located in the MDH and 5-HT- or norepinephrine (NE)-containing descending fibers originated from structures in the brainstem.     

A light and electron microscopic study of betaine/GABA transporter distribution in the monkey cerebral neocortex and hippocampus

The present study aimed to elucidate the distribution of betaine/γ-aminobutyric acid (GABA) transporter-1 (BGT-1) in the normal monkey cerebral neocortex and hippocampus by immunoperoxidase and Immunogold labelling. BGT-1 was observed in pyramidal neurons in the cerebral neocortex and the CA fields of the hippocampus. Large numbers of small diameter dendrites or dendritic spines were observed in the neuropil. These made asymmetrical synaptic contacts with unlabelled axon terminals containing small round vesicles, characteristic of glutamatergic terminals. BGT-1 label was observed in an extra-perisynaptic region, away from the post-synaptic density. Immunoreactivity was not observed in portions of dendrites that formed symmetrical synapses, axon terminals, or glial cells. The distribution of BGT-1 on dendritic spines, rather than at GABAergic axon terminals, suggests that the transporter is unlikely to play a major role in terminating the action of GABA at a synapse. Instead, the osmolyte betaine is more likely to be the physiological substrate of BGT-1 in the brain, and the presence of the transporter in pyramidal neurons suggests that these neurons utilize betaine to maintain osmolarity.     

Evidence for the existence of monoamine neurons in the central nervous system

Summary With the help of the highly specific and sensitive fluorescence method of Falck and Hillarp together with the histochemical and pharmacological criteria for the specificity of the fluorescence reaction convincing evidence has been obtained that the fine, varicose nerve fibres observed in a vast number of regions in the mammalian central nervous system (mouse, hamster, rat, guineapig, rabbit, cat), which exhibit a green or yellow fluorescence, contain primary catecholamines and 5-HT respectively. Strong support has been given for the view that CA fibres showing a rapid recovery after administration of -MMT contain DA, while those showing a slow recovery contain NA.There is little doubt that the monoamine-containing fibres in the brain represent the terminal ramifications of axons belonging to specific monoamine neurons and that they are true synaptic terminals. They seem to make their contacts via the varicosities which have extremely high concentrations of amines and in all probability represent the presynaptic structures, specialized for synthesis, storage and release of the amines. The central monoamine terminals thus have the same characteristic appearance as the adrenergic synaptic terminals in the peripheral nervous system.All the data strongly support the view that the specific central neurons giving rise to the terminals are monoaminergic, i.e. function by releasing their amines from the synaptic terminals. Consequently, DA, NA and 5-HT seem to be central neurotransmitters.Not only the median eminence but also the nuc. caudatus putamen, tuberculum olfactorium, nuc. accumbens and the small circumscribed areas medial to nuc. accumbens contain very fine (partly sublightmicroscopical) CA terminals. These areas react to treatment with reserpine, nialamide-dopa and -MMT in the same way and since the nuc. caudatus putamen and tuberculum olfactorium are known to have a high DA content it seems likely that abundant DA terminals are accumulated in these special areas.The Following Abbreviations are Used CA Catecholamine - DA Dopamine - dopa 3.4-Dihydroxy-phenylalanin - NA Noradrenaline - A Adrenaline - 5-HT 5-Hydroxytryptamine - -MMT -Methyl-meta-tyrosine - MAO Monoamine oxidase For generous supplies of drugs the author is indebted to the following companies: Swedish Ciba, Stockholm, Sweden (reserpine); Swedish Pfizer, Stockholm, Sweden (nialamide); Abbott Research Laboratories, Chicago, USA. (MO 911). This study has been supported by a Public Health Service Grant (NB 02854-04) from the National Institute of Neurological Diseases and Blindness and by grants from the Knut and Alice Wallenberg Foundation, and the Swedish Medical Research Council.     

Effect of actinomycin D on the ultrastructure of the hippocampal cortex

Ultrastructure of the CA1 zone in the rat dorsal hippocampus has been investigated after injection of actinomycin D into the cerebral lateral ventricles. Actinomycin D possesses a wider spectrum of action as it was previously thought. The data obtained make it possible to suppose that certain cerebral disturbances (in particular, memory), produced with actinomycin D, can be dependent on or stipulated by: decreased DNA synthesis in the neuronal nuclei, disorder of RNA synthesis in neurons and astrocytes, damage of the protein synthesis apparatus only in neurons with a dense granular endoplasmic reticulum, decreasing contents of functionally active neurons, possessing a loose granular endoplasmic reticulum, decreasing production of energy by mitochondria of synapses, neurons and astrocytes.