Electron microscopical identification of 3,3′,5,5′-tetramethylbenzidine-reacted horseradish peroxidase after retrograde axoplasmic transport

Stürmer, Claudia; Bielenberg, Katrin; Spatz, W.B.

doi:10.1016/0304-3940(81)90181-6

Cited by 35 publications

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“…Secondly it appeared that most of the TMB reaction product (the oxidized TMB) dissolves when the tissue is routinely processed for electron microscopy, especially during the dehydration and osmification steps. Several solutions to this problem have been proposed: (1) speeding up the dehydration step (Sturmer et al, 1981), ( 2 ) using chemical dehydration with dimethoxypropane (Holstege, 1987), (3) changing the osmification procedure by using a lower pH, a higher temperature and a shorter time (Sakumoto et al, 1980;Carson & Mesulam, 1982), and (4) stabilizing the TMB reaction product with DAB (Lemann et al, 1985). These precautions result in a better preservation of the TMB reaction product, but some loss has to be accepted.…”

Section: T H E Horseradish Peroxidase T E C H N I Q U Ementioning

confidence: 99%

Anterograde tracing in the brain using autoradiography and HRP‐histochemistry. A comparison at the ultrastructural level

Holstege

Vrensen

1988

Journal of Microscopy

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K E Y WORDS. Analysis, anterograde tracing, autoradiography, comparison, electron microscopy, horseradish peroxidase histochemistry , neuro-anatomy , 3,3 ', 5,5 ' -tet ramethylbenzidine. S U M M A R YThe present paper decribes the use of 'H-leucine and WGA-HRP as anterograde tracers in the brain at the ultrastructural level. In addition the different aspects of the two tracing techniques are compared, including the analysis of the results, the reliability and relative efficiency. I N T R O D U C T I O NThe nervous system is a most complex organ, involved in such diverse functions as monitoring the outside world, organizing and directing movement, learning and memory, maintaining the internal environment of the body, etc. For these purposes internal and external sensory information are integrated within the central nervous system and may eventually result in the initiation and execution of a particular movement by the appropriate motor areas. In order to fulfil these complex tasks it is a prerequisite that the various parts of the brain are functionally interconnected. Functional interconnection within the brain necessarily implies structural connections between distinct neuronal cell groups or areas. It is the aim of anterograde tracing studies to unravel the intricate afferent connections (input) of the various neuronal cell groups. However, for the understanding of the integrative function of neurons it is not sufficient to determine from which area(s) of the brain afferent input is conveyed to a particular neuronal cell group. It is of equal importance to determine which transmitters are used in a particular system and whether their action is excitatory or inhibitory. For the latter purpose electro-physiologica1 techniques are most appropriate. However, in general they measure the overall effect of a specific input to a neuron without detailed knowledge of the local circuitry. Anterograde tracing techniques at the ultrastructural level are meant to fill this gap by studying the local characteristics of the afferent systems, i.e. the strategic location of their terminals on the neuronal membrane. It is determined whether synaptic contacts are established and whether they are located on a cell soma (axo-somatic), on a dendrite (axodendritic) or on another terminal (axo-axonic). The size and shape of the terminals and their synaptic vesicles as well as the proximity of other synapses may be important parameters all of which determine the functional properties of a particular afferent connection. At present the *To whom all correspondence should be addressed. 0 1988 The Royal Microscopical Society 233 234 J . C . Holsrege and G . F . J . M . Vrensentwo main techniques for anterograde tracing at the ultrastructural level are the autoradiographic technique and the horseradish peroxidase (HRP) technique. In this paper a critical comparison of these two methods at the electron microscopical (EM) level is presented, including a discussion on the analysis of the EM autoradiographs of brain tissue. Fig. 1. Electron micro...

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Section: T H E Horseradish Peroxidase T E C H N I Q U Ementioning

confidence: 99%

Anterograde tracing in the brain using autoradiography and HRP‐histochemistry. A comparison at the ultrastructural level

Holstege

Vrensen

1988

Journal of Microscopy

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Ultrastructural identification of labeled neurons in the dorsal motor nucleus of the vagus nerve following injections of horseradish peroxidase into the vagus nerve and brainstem

McLean

Hopkins

1982

J of Comparative Neurology

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The efferent connections of two types neurons in the dorsal motor nucleus of the vagus nerve (DMV) were studied in the cat by light and electron microscopy following horseradish peroxidase (HRP) injections into the cervical vagus nerve or brainstem. After injections of HRP into the vagus nerve, up to 80% of medium-sized neurons averaging 26 x 20 micrometers in 1-micrometer-thick sections were retrogradely labeled while no small neurons were labeled in the DMV. Incubation with either diaminobenzidene (DAB) or p-phenylenediamine-pyrocatechol (PPD-PC) chromogens yielded electron-dense reaction products localized mainly in lysosomes. Identification of label at the ultrastructural level was facilitated by omitting lead citrate staining and by counting numbers of lysosomes, which were higher in labeled neurons. Quantitative comparisons of the dimensions of labeled and unlabeled somata demonstrated that retrograde transport and incorporation of HRP had no effect on cell size within the 2-3-day survival times used in this study. In order to determine whether neurons in the DMV project to higher levels of the brain stem, large injections of HRP (1-3 microliters) were made into the pons, mesencephalon, hypothalamus, and amygdala. After injections of HRP into the brainstem, only small neurons, measuring 17 x 10 micrometers, were retrogradely labeled. Approximately 90% of the small neurons remained unlabeled following the HRP injections. The ultrastructural features of the labeled small neurons included an invaginated nucleus, low cytoplasmic/nuclear ratio, and relatively fewer organelles than the medium-sized neurons. A quantitative analysis of labeled and unlabeled small neurons demonstrated that the labeled neurons were significantly larger than the unlabeled small neurons. Thus, two populations of small neurons may exist in the DMV. One population appears to have ascending projections to higher levels of the brainstem while the other more numerous population may be interneurons or project for only short distances.

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Corticotectal terminals in the superior colliculus of the rabbit: A light‐ and electron microscopic analysis using horseradish peroxidase (HRP)‐tetramethylbenzidine (TMB)

Holländer

Schönitzer

1983

J of Comparative Neurology

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The projection from the striate cortex to the superior colliculus was studied light- and electron microscopically by means of anterogradely transported horseradish peroxidase and tetramethylbenzidine histochemistry. Labeled boutons were found in the stratum zonale (SZ) and in the stratum griseum superficiale (SGS), not in stratum opticum (SO). There are two maxima of frequency of labeled boutons, one in middle SGS at about 500 microns depth, and a smaller one in upper SGS at about 200 microns depth. Such a bimodal distribution of corticotectal terminals has not been described in any species before. Labeled myelinated axons were found in SGS and SO with a maximal frequency in middle SGS at about 400 microns depth. The myelinated axons in SZ, which are commonly considered to be of cortical origin, were not labeled. The labeled cortical terminals contained numerous round synaptic vesicles and predominantly dark mitochondria. They formed usually asymmetrical synapses and contacted dendrites, some of which contained synaptic vesicles. Occasionally, labeled boutons were observed which definitely did not belong to the type that is generally considered to be of cortical origin.

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Electron microscopical identification of 3,3′,5,5′-tetramethylbenzidine-reacted horseradish peroxidase after retrograde axoplasmic transport

Cited by 35 publications

References 8 publications

Anterograde tracing in the brain using autoradiography and HRP‐histochemistry. A comparison at the ultrastructural level

Anterograde tracing in the brain using autoradiography and HRP‐histochemistry. A comparison at the ultrastructural level

Ultrastructural identification of labeled neurons in the dorsal motor nucleus of the vagus nerve following injections of horseradish peroxidase into the vagus nerve and brainstem

Corticotectal terminals in the superior colliculus of the rabbit: A light‐ and electron microscopic analysis using horseradish peroxidase (HRP)‐tetramethylbenzidine (TMB)

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