Biomedical subjects

B Ulfhake

Publications and source records attributed to B Ulfhake.

At least 37 records · Page 2Linked to original sources

PubMed1997

Fluorescence lifetime measurements in confocal microscopy of neurons labeled with multiple fluorophores.

In order to resolve multiple fluorophores by their lifetimes in discrete tissue domains, the labeling intensity must be sufficiently strong and the intensity-difference between the labels must not be too large, the rate of fading should be similar for all fluorophores, and the lifetimes of the fluorophores should be sufficiently discrete. We could readily distinguish Cyanine-3.18 (Cy-3), Lissamine Rhodamine (LRSC), and Texas Red when they were not colocalized in tissue profiles. Colocalization of Cy-3 and LRSC, as well as Cy3 and Texas Red, could also be distinguished, while the combination of LRSC and Texas Red was more difficult. We have used fluorescence lifetime recordings in confocal microscopy to detect different neuropeptides in neurons. We demonstrate that somatostatin and galanin are colocalized in axon profiles of the spinal cord dorsal horn.

Animals↗

PubMed1996

Neuropeptides and neurotrophin receptor mRNAs in primary sensory neurons of aged rats.

Neuropeptides and neurotrophin receptors are regulated in primary sensory neurons in response to axonal injury, and axonal lesions are characteristic stigmata of aging primary sensory neurons. We have therefore examined the expression of neuropeptides and neurotrophin receptor mRNAs in 30-month-old (median survival age) Sprague-Dawley rats to see if similar adaptive mechanisms operate in senescence. The content of neuropeptides was examined with immunohistochemistry (IHC) and in situ hybridization (ISH), and the cellular mRNA expression of neurotrophin receptors was studied with ISH. All of the aged rats had symptoms of hind limb incapacity (posterior paralysis), but fore limbs did not seem affected. The size-distribution of neuronal profiles in cervical and lumbar dorsal root ganglia (DRGs) was similar in aged and young adult (2-3 months old) rats. In aged rats, the DRG neurons showed an increase in both immunolabelling and mRNA content of neuropeptide tyrosine (NPY), as well as an increased cellular expression of galanin mRNA. In the same animals, there were decreased cellular levels of calcitonin gene-related peptide (CGRP; IHC and ISH) and substance P (SP; IHC and ISH), while the difference in neuronal somatostatin (IHC and ISH) was small. The distribution of neuropeptide immunoreactivities in the dorsal horn of the corresponding spinal cord segments revealed a decreased labelling for CGRP-, SP-, and somatostatin-like immunoreactivities (LI) in the aged rats at both cervical and lumbar levels. NPY- and galanin-LI had a similar distribution in aged and young adult rats. NPY-immunoreactive fibers were also encountered in the dorsal column of aged but not young adult rats. ISH revealed that most of the primary sensory neurons express mRNA for the p75 low-affinity neurotrophin receptor (p75-LANR) and that there was no discernible difference between young adult and aged rats. The labelling intensity for mRNA encoding high-affinity tyrosine kinase receptors (TrkA, TrkB, and TrkC) was decreased in aged rat DRG neurons, while the percentage of neuronal profiles expressing mRNA for TrkA/B/C was similar in young adult and aged rats. The changed pattern of neuropeptide expression in primary sensory neurons of aged rats resembled that seen in young adult rats subjected to axonal injury of peripheral sensory nerves and may, thus, indicate aging-related lesions of sensory fibers. Since NPY is primarily present in large and galanin in small DRG neurons, the stronger effect on NPY as compared to galanin expression may indicate that aging preferentially affects neurons associated with mechanoreception (A alpha and A beta fibers) as compared to nociceptive units (A delta and C fibers). Furthermore, the observed changes in neuropeptide expression were most pronounced in lumbar DRGs, that harbors the sensory neurons supplying the affected hindlimbs of the rats.

Adaptation, Physiological↗

PubMed1996

Qualitative and quantitative analysis of glycine- and GABA-immunoreactive nerve terminals on motoneuron cell bodies in the cat spinal cord: a postembedding electron microscopic study.

The distribution of glycine- and gamma-aminobutyric acid (GABA)-like immunoreactivity (LI) in nerve terminals on the cell soma of motoneurons in the aldehyde-fixed cat L7 spinal cord was examined using postembedding immunogold histochemistry in serial ultrathin sections. Quantitative examination of 405 terminals on eight neurons of alpha-motoneuron size in the L7 motor nuclei from one animal was performed. A majority of the terminals (69%) were immunoreactive to glycine and/or GABA. These terminals contained flat or oval synaptic vesicles, thus classifying them as F type or as C type in one case. In no case was a type-F terminal unlabeled for both glycine and GABA. Most of the immunolabeled terminals were immunoreactive to glycine only (62.5%), whereas 35.4% contained both glycine- and GABA-LI. A very small number of immunolabeled terminals (2%) were immunoreactive to GABA only. In those terminals, where glycine- and GABA-LI coexisted, the gold particle density for each amino acid was only half of that seen in boutons containing only one of the two amino acids. The involvement of glycine and GABA in postsynaptic inhibition of spinal alpha-motoneurons is discussed, with particular reference to the possibility that these two inhibitory amino acids may be coreleased from a significant proportion of the nerve terminals impinging on the cell bodies.

Animals↗

PubMed1996

Decreased expression of TrkB and TrkC mRNAs in spinal motoneurons of aged rats.

Several studies have indicated that a decrease in availability and/or responsiveness to neurotrophin(s) may be of importance in ageing and disease-related neurodegeneration. Using in situ hybridization we have studied the mRNA expression of the full-length neurotrophin receptors TrkB and TrkC in spinal cord motoneurons of aged rats (30 months old) with symptoms of hindlimb incapacity and in young adult rats (2-3 months old). The labelling intensity for TrkB of the individual cell profile was decreased by 25% (P < 0.001) in both the cervical and lumbar motor nuclei of aged rats. In thoracic motoneurons of aged and young adult rats the difference in expression of TrkB mRNA was smaller (down by 15%; P < 0.05). The labelling for TrkC mRNA was much weaker than that for TrkB in both aged and young adult rats, but TrkC mRNA expression also seemed to decrease. Comparison of the different motor nuclei along the spinal cord axis revealed that the motoneurons of the L6/S1 nuclei showed the strongest hybridization signal for the two Trk receptors in both aged and young adult rats. The possibility that a decrease in TrkB mRNA may contribute to age-related motor disturbances is discussed.

Aging↗

PubMed1995

Ia boutons to CCN neurones and motoneurones are enriched with glutamate-like immunoreactivity.

The monosynaptic connection between muscle spindle Ia afferents and motoneurones is one of the most extensively studied reflex pathways in the central nervous system. Physiological evidence has pointed to glutamate as the fast transmitter in this synaptic connection. In this study of the spinal cord segment C2, we used dual labelling with cholera toxin subunit B-horseradish peroxidase (B-HRP) and postembedding immunohistochemistry to show that group Ia primary afferent boutons synapsing on retrogradely labelled central cervical nucleus (CCN) neurones and motoneurones are enriched in glutamate-like immunoreactivity (LI). All double-labelled Ia terminals studied could be classified as S-type (CCN neurones and motoneurones) or M-type (motoneurones) boutons.

Afferent Pathways↗

PubMed1995

Increase in alpha-CGRP and GAP-43 in aged motoneurons: a study of peptides, growth factors, and ChAT mRNA in the lumbar spinal cord of senescent rats with symptoms of hindlimb incapacities.

Sprague-Dawley rats develop progressive motor dysfunctions during the third year of life. We use this as a model to examine possible neuronal mechanism(s) that may cause motor impairments occuring during aging. In this study we have used indirect immunofluorescence histochemistry (IF) and in situ hybridization histochemistry (ISH) to study quantitatively and qualitatively the staining pattern and mRNA expression of calcitonin gene-related peptide (alpha-CGRP), growth-associated protein 43 (GAP-43), and acidic fibroblast growth factor (aFGF) in spinal lumbar motoneurons of young adult (2-3 months) and aged (30 months) Sprague-Dawley rats. In addition, mRNAs encoding choline acetyltransferase (ChAT), beta-CGRP, and cholecystokinin (CCK) were analyzed. All aged rats used in this study disclosed symptoms of hindlimb incapacity, ranging from mild weight-bearing insufficiency to paralysis of the hind limbs. The symptoms were confined to the musculature of the hindlimb and hip regions. Only a small number (approximately 15%) of the large motoneurons that innervate the hindlimb muscles were lost in those aged rats that had clinical symptoms of hindlimb motor incapacities. The remaining motoneurons expressed ChAT mRNA at levels similar to those of young adult rats. The vast majority of these motoneurons showed increased mRNA levels for alpha-CGRP and GAP-43. Aged motoneurons contained more CGRP like immunoreactivity (LI), but the number of immunoreactive neurons was smaller than in adult rats. GAP-43-LI could be detected in motoneurons in aged, but not in adult, rats. GAP-43-LI was always colocalized with CGRP-LI in aged motoneurons. Studies of individual aged rats revealed that the increase of GAP-43 mRNA-positive cell bodies occurred in cases with the most severe clinical symptoms, whereas the increase in alpha-CGRP was even evident in rats with mild symptoms. No alterations in content of aFGF-LI or aFGF mRNA could be detected in the aged rat, and the content of CCK and beta-CGRP mRNAs was also normal. The usefulness of this rat model for studies of neuromuscular aging and possible functional roles for GAP-43 and CGRP in plastic and regenerative processes during aging are discussed.

Aging↗

PubMed1995

Improved fluorophore separation with IMS confocal microscopy.

Some of the most extensively used fluorophores for multicolour fluorescence labelling of tissue, such as Lucifer yellow, fluorescein, Cyanine-3.18 and rhodamine, all have the disadvantage of being difficult to separate spectrally due to their broad and overlapping emission spectra. With the intensity-modulated multiple-beam scanning (IMS) technique, cross-talk between labels can be reduced to a fraction of that seen using conventional excitation and detection technique. Thus, cross-talk to the 'red' channel decreased with about one order of magnitude for the 'green' fluorophores Lucifer yellow and FITC. For the tested 'red' fluorophores, the amount of cross-talk to the green channel was reduced by 60-90%.

Animals↗

PubMed1995

Increased expression of serotonin transporter messenger RNA in raphe neurons of the aged rat.

The action of serotonin (5-hydroxytryptamine; 5HT) in the nervous system is terminated by reuptake of the neurotransmitter into the presynaptic nerve terminal via a plasma membrane-bound transporter protein. Recently, cDNAs encoding serotonin transporter protein were cloned. Using in situ hybridization we have studied the expression of serotonin transporter mRNA in the bulbospinal 5HT system of adult (2-3 months) and aged (30 months) rats. In adult rats, serotonin transporter mRNA labelled neurons were detected in the nucleus raphe obscurus, pallidus and magnus. In all aged rats, the optical density of the serotonin transporter mRNA labelling of individual cell profiles was increased by 20-30% (P < 0.001) when compared with adult rats. Five of the six aged rats studied had various degrees of hindlimb motor dysfunctions. In rats with the most pronounced signs of hindlimb motor dysfunction the number of labelled neurons was increased by 25-75% (P < 0.01) when compared with young adult rats. It is concluded that an increased serotonin transporter gene expression is present in the bulbospinal 5HT system of aged animals.

Aging↗

PubMed1995

Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope.

We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be individually identified in discrete tissue domains by confocal microscopy. A further advantage of lifetime recordings is that fluorophores that emit light within the same wavelength band can be used and chromatic aberrations are therefore circumvented, thereby improving the spatial accuracy in imaging of multiple fluorophores. Low and high pH, respectively, tended to influence fluorophore emission spectra and fluorescence lifetime. IgG conjugation of the fluorophores tended to shift the spectra towards longer wavelengths and to change the fluorescence lifetimes. The IgG-conjugated form of the fluorophores may, when applied to tissue specimens, change the emission spectrum and lifetime. In addition, different tissue embedding procedures may influence fluorescence lifetime. These observations emphasize the importance of spectral and lifetime characterization of fluorescent probes within the chemical context in which they will be used experimentally. Changes in spectra and fluorescence lifetimes may be a useful tool to gain information about the chemical environment of the fluorophores.

Animals↗

PubMed1994

Quantitative and qualitative aspects on the distribution of 5-HT and its coexistence with substance P and TRH in cat ventral medullary neurons.

By use of the indirect immunofluorescence technique the distributions of 5-hydroxytryptamine (5-HT)-, substance P- and thyrotropin-releasing hormone (TRH)-immunoreactive (IR) neurons have been studied in the midline raphe nuclei and nucleus reticularis lateralis of the caudal brainstem (levels P18.3-P8.5; according to Berman (1968), in the cat, after treatment with colchicine. In addition, by use of the double-labelling technique, the coexistence between 5-HT-, substance P- and TRH-like immunoreactivity (LI) in these neurons was analysed. The results show that cell bodies in the midline raphe nuclei and nucleus reticularis lateralis contain 5-HT-, substance P- and TRH-LI. 5-HT-IR cells were more abundant than peptidergic neurons in all areas analysed. Quantitative estimations indicated that the total number of 5-HT-IR cells in the regions studied was about 17 x 10(3), while the corresponding numbers for substance P- and TRH-IR cells were 11 x 10(3) and 12 x 10(3), respectively. From double-labelled sections it was concluded that the vast majority of peptidergic cells also contained 5-HT-LI (87-100%). However, a subpopulation of 5-HT-IR neurons lacked peptide-LI (10-55%). The degree of coexistence varied with the brainstem level, in that neurons at more rostral locations showed a lower incidence of coexistence between 5-HT and peptide(s). The presence of all three compounds in one and the same cell body could also be demonstrated. In summary, 5-HT-, substance P- and TRH-IR cell bodies were encountered in medullary nuclei known to contain neurons with projection to the spinal cord. A high degree of coexistence between the compounds was demonstrated in these nuclei. The obtained results fit well with earlier studies on the patterns of distribution and peptide colocalization of 5-HT fibres in the spinal cord. The existence of biochemically distinct neuronal subpopulations within the 5-HT bulbospinal pathway is discussed.

Animals↗

PubMed1994

Serotoninergic, peptidergic and GABAergic innervation of the ventrolateral and dorsolateral motor nuclei in the cat S1/S2 segments: an immunofluorescence study.

Indirect single- and double-staining immunofluorescence techniques were used to study the serotoninergic, peptidergic and GABAergic innervation of the ventrolateral (Onuf's nucleus) and dorsolateral (innervating intrinsic foot sole muscles) nuclei, located in the S1/S2 segments of the cat spinal cord. The relative density of 5-hydroxytryptamine-, thyrotropin-releasing hormone-, substance P- and gamma-aminobutyric acid-immunoreactive axonal varicosities was similar in both nuclei. The highest relative density was recorded for varicosities immunoreactive to gamma-aminobutyric acid, while those immunoreactive to 5-hydroxytryptamine or thyrotropin-releasing hormone yielded the lowest values. The density of enkephalin-immunoreactive varicosities was higher in the ventrolateral than in the dorsolateral nucleus. Calcitonin gene-related peptide-like immunoreactivity could be seen in neurons of the ventrolateral and dorsolateral nuclei. Occasionally, calcitonin gene-related peptide-immunoreactive axonal fibers were also encountered in these nuclei. Virtually all thyrotropin-releasing hormone-immunoreactive varicosities in the ventrolateral and dorsolateral nuclei also contained 5-hydroxytryptamine-like immunoreactivity, while a somewhat smaller number of them were co-localized with substance P. About 5-10% of the 5-hydroxytryptamine-immunoreactive varicosities were devoid of peptide-like immunoreactivity, and the number of 5-hydroxytryptamine-immunoreactive varicosities lacking thyrotropin-releasing hormone-like immunoreactivity was higher in the dorsolateral than in the ventrolateral nucleus. Finally, the free fraction of substance P-immunoreactive varicosities, i.e., those lacking both 5-hydroxytryptamine and thyrotropin-releasing hormone, was about 39% in the ventrolateral and 26% in the dorsolateral nucleus. Spinal cord transection at the lower thoracic level induced a depletion of 5-hydroxytryptamine and thyrotropin-releasing hormone-immunoreactive fibers from the ventrolateral and dorsolateral nuclei, indicating an exclusive supraspinal origin for these fibers. A reduction in substance P-like immunoreactivity following spinal cord transection alone or spinal cord transection combined with unilateral dorsal rhizotomy was also detected in both nuclei, suggesting a dual origin for substance P-immunoreactive fibers, i.e., both supra- and intraspinal. The decrease in number of substance P-immunoreactive fibers was however smaller than expected from the analysis of the fraction of substance P-immunoreactive fibers co-localized with 5-hydroxytryptamine, indicating thus that the experimental lesions may have triggered a sprouting of substance P-immunoreactive axons originating from spinal cord sources. The distribution of gamma-aminobutyric acid in the ventrolateral and dorsolateral nuclei was not affected by the different lesion paradigms. It is therefore assumed that these inputs are intrinsic to the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Enkephalin-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal innervation of the ventrolateral dendritic bundle in the cat sacral spinal cord: an ultrastructural study.

The distribution and synaptic arrangement of thyrotropin-releasing hormone-, substance P- and enkephalin-immunoreactive axonal boutons have been studied in the ventrolateral nucleus (Onuf's nucleus) of the upper sacral spinal cord segments in the cat. For this purpose, the peroxidase-antiperoxidase immunohistochemical technique was used. Immunoreactive axonal boutons were traced in complete series of sections in order to reveal synaptic contacts with the bundled dendrites of the ventrolateral nucleus. As judged from the cross-sectional diameter of the postsynaptic dendrites, the distribution of immunoreactive boutons was non-random. Enkephalin-immunoreactive axonal boutons, presumed to be mostly of segmental origin, displayed a rather restricted distribution to mainly (> 80%) medium-to-large dendrites. Thyrotropin-releasing hormone-immunoreactive boutons, that derive from supraspinal levels, were also found to impinge on medium-to-large dendrites (> 80%), indicating a proximal location within the dendritic trees. The skewness toward large postsynaptic dendrites was even more marked for thyrotropin-releasing hormone- than for enkephalin-immunoreactive boutons. Substance P-immunoreactive boutons, that are of either supraspinal or spinal origin, showed a more even distribution throughout the dendritic trees, including both thin distal branches and thick proximal dendrites. In view of the well-known fact that virtually all thyrotropin-releasing hormone-immunoreactive boutons in the ventral horn co-contain substance P (and serotonin) it was assumed that substance P-immunoreactive boutons in synaptic contact with the finest-calibre dendrites as well as most of those with a very proximal juxtasomatic location on the dendritic trees were of segmental origin, while those impinging on medium-to-large dendrites could be of either spinal or supraspinal origin. Fine-calibre dendrites (< 1 micron) represent about 25% of the dendritic branches in the ventrolateral nucleus, but receive, with the exception of substance P (8%), very little (< 3%) peptidergic or GABAergic (Ramírez-León and Ulfhake, 1993) input, although the degree of dendritic membrane covering by bouton profiles in the ventrolateral nucleus does not seem to vary much with the calibre of the postsynaptic dendrite (Ramírez-León and Ulfhake, 1993). Both substance P- and enkephalin-immunoreactive axonal boutons established synaptic contact with more than one dendrite. Furthermore, one and the same bouton could be found in contact with two dendrites that were coupled to each other by a dendro-dendritic contact of desmosomal or puncta adherentia type.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

GAP-43, aFGF, CCK and alpha- and beta-CGRP in rat spinal motoneurons subjected to axotomy and/or dorsal root severance.

The mRNA levels for growth-associated protein 43 (GAP-43), acidic fibroblast growth factor (aFGF), alpha- and beta-calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and choline acetyltransferase (ChAT) in rat lumbar spinal motoneurons were studied by in situ hybridization 1, 5 and 21 days and 20 weeks following unilateral peripheral nerve sectioning, ventral rhizotomy or dorsal rhizotomy. Furthermore, CGRP- and aFGF-like immunoreactivities in the ventral horn were studied using immunohistochemistry. One to 21 days after axotomy, GAP-43 and alpha-CGRP mRNAs increased in lesioned motoneurons, while the aFGF mRNA levels were marginally higher in motoneurons on the lesion side as compared to the control side. beta-CGRP, CCK and ChAT mRNA levels, on the other hand, decreased during the short-term response (1-21 days) to axotomy. After ventral rhizotomy, but not peripheral axotomy, there was complete disappearance of aFGF-like immunoreactivity in the ventral root proximal to the lesion. In animals subjected to long-term survival (20 weeks) after peripheral axotomy, the expression of all studied substances had returned to normal levels. Unilateral dorsal rhizotomy did not induce any substantial short- or long-term shifts in the cellular expression of the GAP-43, aFGF, CGRP and CCK peptides or their mRNAs in motoneurons of lesioned segments. These results indicate that peptides/proteins in motoneurons are expressed differentially after axotomy. Whereas alpha-CGRP and GAP-43 are up-regulated, CCK and beta-CGRP become down-regulated and aFGF is largely unaffected.

Animals↗

PubMed1993

The serotoninergic bulbospinal system and brainstem-spinal cord content of serotonin-, TRH-, and substance P-like immunoreactivity in the aged rat with special reference to the spinal cord motor nucleus.

The 5-hydroxytryptamine (5HT) containing bulbospinal pathway was studied with immunohistochemical (IF) and chemical techniques in 2-3 and 30 months old male Sprague-Dawley rats. The coexisting neuropeptides substance P (SP), thyrotropin-releasing hormone (TRH) and galanin were also analysed. Furthermore, the expression of mRNA encoding aromatic L-amino acid decarboxylase (AADC), prepro-TRH, and preprotachykinin (prepro-SP) was analysed with in situ hybridization (ISH) in the midline raphé nuclei inthe lower brainstem. The results showed a decreased number of axonal 5HT fibers with a normal morphology in the ventral horn of the aged rat lumbosacral spinal cord, and several 5HT immunoreactive (IR) fibers with an aberrant morphology, suggestive of axonal degeneration, were intermingled. This was evident in both the dorsal and ventral horn of the spinal cord. The 5HT-IR fibers with an aberrant morphology usually also contained TRH-and/or SP- and/or galanin-like immunoreactivity (LI) in the ventral horn. These signs of degeneration were clearly less evident in the thoracic and cervical spinal cord segments. Moreover, these changes varied between aged litter-mates. This was in agreement with behavioural signs of motor disturbances, present in about 40% of the aged rats and which in all cases were confined to the hindlimbs. Chemical analyses disclosed significantly lower levels of TRH-LI and, in particular, SP-LI in both the ventral and dorsal quandrants of the spinal cord in the aged rat compared to young adults. The differences were largest in the lumbar regions of the spinal cord. Corresponding analysis of 5HT and 5-hydroxyindoleacetic acid (5HIAA) in the same tissue specimens revealed largely unaltered levels of 5HT and a slight increase in 5HIAA, indicating the possibility of an increased 5HT turnover in the aged rat spinal cord. Neurons in nucleus raphé obscurus and nucleus raphé pallidus were immunoreactive to 5HT, and after pretreatment with colchicine to TRH-, SP-, and galanin-LI as well. There was no obvious difference in number of labeled cells, or labeling intensity, between colchicine-treated young adult and aged rats, although, in the corresponding region of medulla oblongata, chemical analysis disclosed significantly lower levels of 5HT, TRH, and, in particular, SP in untreated aged rats. In contrast, in situ hybridization analysis revealed increased mRNA levels encoding prepro-TRH and prepro-SP in old rats, while mRNA content encoding AADC mRNA was similar in young adult and aged rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1993

GABA-like immunoreactive innervation and dendro-dendritic contacts in the ventrolateral dendritic bundle in the cat S1 spinal cord segment: an electron microscopic study.

The motoneurons (MNs) in the ventrolateral nucleus (VLN) of the upper sacral spinal cord segments in the cat supply the external sphincters and the ischiocavernosii muscles. The dendrites of the MNs in the VLN are arranged into rostro-caudally oriented bundles (ventrolateral dendritic bundle, VLB). In this study we describe the distribution and synaptic arrangement of gamma-aminobutyric acid-immunoreactive (GABA-IR) axonal bouton profiles innervating the VLB. This was accomplished using the peroxidase-antiperoxidase technique and a polyclonal antibody raised against glutaraldehyde-conjugated GABA. The VLN receives an extensive innervation of GABA-IR axonal bouton profiles that surround both cell bodies and dendrites. Twenty-five per cent of the total number of vesicle-containing axonal profiles in the VLN neuropil were estimated to be GABA-IR. On cell bodies in the alpha-motoneuron size-range, the membrane covering of GABA-IR bouton profiles was about 18% and they constituted about 29% of the total membrane covering of axonal bouton profiles. Quantitative analysis of GABA-IR bouton profiles on dendrites revealed membrane covering figures rather similar to those on the cell bodies. They were not randomly distributed within the dendritic arborisations. Instead, they were very infrequent (2.5% of the covering) on small calibre dendrites (< 1 microns) as compared to larger dendrites (> 1 microns, 14-18.5% of the covering), although the total membrane covering of axonal bouton profiles was rather similar for all dendrites (42-52%). The data on membrane covering by GABA-IR boutons presented here may be low estimates due to technical limitations, indicating that the GABAergic input to this region might be even more extensive. A frequent finding was that one and the same GABA-IR bouton made synaptic contact with two to three adjacent dendrites. This type of synaptic arrangement among the VLN MNs indicates a divergence of the GABAergic input at the terminal level. In addition, the postsynaptic dendrites involved in such arrangements often disclosed dendro-dendritic contacts. In total, 44% of the bundled dendrites in the VLN disclosed direct dendro-dendritic contact regions. These contacts were most often of the puncta adherentia type, while desmosome-type contacts were less frequent. None of the dendro-dendritic contacts studies had the characteristics of a gap junction. Taken together, the present results indicate that GABA may be a transmitter substance in a large fraction of the synaptic input to the VLN MNs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Immunocytochemical localization of amino acid neurotransmitter candidates in the ventral horn of the cat spinal cord: a light microscopic study.

The distribution of immunoreactivities to six amino acids, possibly related to synaptic function, was investigated in the motor nucleus of the cat L7 spinal cord (laminae VII and IX) using a postembedding peroxidase-antiperoxidase technique. Consecutive 0.5 micron transverse sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of gamma-aminobutyric acid (GABA), glycine, aspartate, glutamate, homocysteate, and taurine. This method allowed localization of the different immunoreactivities in individual cell profiles. The results showed that all these amino acids, except homocysteate, could be clearly detected in either neuronal or glial elements in the ventral horn. In cell bodies of neurons in lamina VII, immunoreactivity was observed for aspartate, glutamate, GABA, and glycine. Adjacent section analysis revealed that combinations of immunoreactivity for glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate and glutamate/aspartate, respectively, may occur in one and the same cell. In the motor nuclei (lamina IX), immunoreactivity to amino acids was observed in two types of neuron. Large cells, probably representing alpha-motoneurons, were harboring immunoreactivity to both glutamate and aspartate, while a few small neurons in this area displayed a colocalization of glycine, glutamate, and aspartate. Dendrites and axons in the motor nuclei contained glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate, and glutamate/aspartate immunoreactivities. In both laminae VII and IX, taurine-like immunoreactivity was absent in neuronal cell bodies, but highly concentrated in perivascular cells and small cells with a morphology resembling that of glial cells. A punctate immunolabeling, in all probability representing labeling of nerve terminals, could be demonstrated in the ventral horn for GABA, glycine, and glutamate, but not with certainty for aspartate or taurine. A quantitative estimate of the covering of cell bodies of alpha-motoneuron size by immunoreactive puncta revealed that glycine immunoreactive terminal-like structures were most abundant (covering 26-42% of the somatic membrane), while glutamate immunoreactive terminals were seen least frequently (5-9% covering). GABA-immunoreactive terminals covered from 10 to 24% of the soma surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

PubMed1993

The peptidergic motoneurone.

Acetylcholine is the classic transmitter in the spinal cord motoneurone. Recent studies have shown that motoneurones also contain calcitonin-gene related peptide (CGRP) and other peptides. In addition to transmitter-like effects, CGRP may also exert trophic actions, as suggested by changes in CGRP expression in motoneurones during development and following experimental perturbations.

Amino Acid Sequence↗

PubMed1992

Thyrotropin-releasing hormone (TRH)-like immunoreactivity in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata with special emphasis on the bulbospinal tract.

The distribution of thyrotropin-releasing hormone (TRH)-like immunoreactivity (LI) has been studied in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata by the use of indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) technique. Furthermore, double-labeling experiments were performed in order to study colocalization of 5-hydroxytryptamine (5-HT)- and substance P-LI. A dense innervation of TRH-immunoreactive (IR) varicose fibers was found in the ventral horn motor nuclei, in the region surrounding the central canal, in the intermediolateral cell column, and in the dorsal horn laminae II and III. In addition, cell bodies harboring TRH-LI were found in the dorsal horn laminae II-IV. In the ventral horn, many of the large cell bodies and their proximal dendrites were totally encapsulated by TRH-IR fibers. From double-labeled sections a high degree of coexistence could be established between TRH-/5-HT-LI, TRH-/substance P-LI, and 5-HT-/substance P-LI in fibers in the motor nuclei; as a consequence, a large proportion of these fibers should harbor TRH-/5-HT-/substance P-LI. A coexistence between TRH-/5-HT-LI could also be demonstrated in the intermediolateral cell column. However, no unequivocal coexistence could be found between TRH-/substance P-LI and 5-HT-/substance P-LI in this region. In the dorsal horn, no clear coexistence could be encountered for any of the above indicated combinations. Electron microscopic analysis of material from the lumbar lateral motor nucleus demonstrated TRH-IR terminals making synapses with large cell bodies and dendrites. In addition, contacts lacking synaptic specializations could also be verified. In the medulla oblongata, with the use of the PAP technique, a large number of cell bodies containing TRH-LI were encountered in the midline raphe nuclei and in nucleus reticularis lateralis. A similar distribution pattern could be found for 5-HT-LI, but no cell bodies containing substance P-LI could be seen in these regions. Chemical analysis of specimens from cervical, thoracic, and lumbar spinal cord revealed higher concentrations of TRH- and 5-HT-LI in the ventral quadrants, whereas substance P-LI dominated in the dorsal quadrants. Thus, the concentrations of TRH-, 5-HT-, and substance P-LI was in accordance with the observed regional variation in density of IR-fibers and varicosities found in the spinal cord. We have shown that TRH-LI has a distribution in the monkey spinal cord and medulla oblongata similar to that previously demonstrated in other species.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗