Immunocytochemistry and Neurobiology

Abstract

Immunocytochemistry enables the localization of transmitter‐related antigens in tissue sections at either the light microscopic or the electron microscopic level. In the case of neuropeptides and certain transmitters (e.g. serotonin) it has been possible to produce antibodies directed against the putative transmitter itself. In other cases it has not been possible to produce useful antibodies against transmitters but antibodies have been raised against enzymes involved in transmitter metabolism (e.g. tyrosine hydroxylase, glutamic acid decarboxylase) and these antibodies are suitable markers for transmitter systems. Successful immunostaining with an antibody depends on a number of factors, two of the most important being the fixation of the antigen in the tissue and the visualization of the primary antibody once it has bound to the antigen. Techniques available for the visualization of bound primary antibody include the indirect‐labelled immunofluorescence procedure and the unlabelled peroxidase‐antiperoxidase (PAP) procedure. Direct‐labelled immunocytochemistry is not now widely used but is likely to become increasingly important with the introduction of monoclonal antibodies and the development of techniques for the simultaneous localization of multiple antigens. Monoclonal antibody procedures also allow the production of antibodies against antigens which are difficult to purify such as certain transmitter markers (e.g. choline acetyltransferase) and constituents of neuronal membranes. Immunocytochemistry allows the production of detailed maps of the distribution of putative transmitters in the nervous system and in combination with tract tracing procedures is being used increasingly to identify transmitters in neuronal circuits. It has also been important in establishing the transmitter status of various neuroactive compounds in single neurones. Immunocytochemistry can be carried out on post‐mortem samples and is providing information on transmitter distribution in normal and abnormal human brain.

Adrenalectomy decreases nerve growth factor in young adult rat hippocampus.

“…Serial coronal sections (40 ,um thick) were cut on a Bright cryostat. The sections were incubated with monoclonal ChAT antibodies (Boehringer Mannheim), at a dilution of 1:25 in phosphatebuffered saline, for 24 hr at 40C and then processed with the peroxidase-antiperoxidase method (20,21). A quantitative analysis of lightly and darkly stained immunoreactive nerve cells in the SLB of Adx and sham-operated rats was carried out on seven consecutive sections following a rostrocaudal direction.…”

Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes.

“…Furthermore, no antibodies with this unique staining pattern were detected when mice were "sham-immunized" with complete Freund's adjuvant or when hybridomas were generated after immunization with native oocyte membranes and other antigens following the use of similar drastic immunotolerization. In addition, other researchers (8)(9)(10)(11)(12)(16)(17)(18) used a large variety of neural antigens and immunization procedures and found interesting cerebellum-specific antibodies, but none were like the one described here.…”

“…Production of monoclonal antibodies (mAbs) to the diverse molecular constituents of the nervous system is contributing greatly to our knowledge of the molecular basis of brain function (8)(9)(10)(11)(12). We now report an approach used to produce a set of mAbs recognizing an epitope present almost exclusively in the cerebellar pinceau terminals and not detected in any of the other four types of synaptic contacts made by the cerebellar basket cell axons.…”

“…No other example of this voluminous and complex synapse is known in the mammalian nervous system. The exact function of the pinceau is not yet known, although it has been proposed that it mediates chemical and electrical inhibition of the Purkinje cell (3-6); and loss of basket cells has been shown to cause severe locomotor deficits (7).Production of monoclonal antibodies (mAbs) to the diverse molecular constituents of the nervous system is contributing greatly to our knowledge of the molecular basis of brain function (8)(9)(10)(11)(12). We now report an approach used to produce a set of mAbs recognizing an epitope present almost exclusively in the cerebellar pinceau terminals and not detected in any of the other four types of synaptic contacts made by the cerebellar basket cell axons.…”

Neuroanatomy of the spinal cord: current research and future prospects

“…This technique allows specific antigens to be localised in tissue sections at the light and electron micro scopic level and has many applications in the field of neuroscience (Cuello, Priestley and Sofroniew, 1983). In the spinal cord one of the most fruitful applications has been for mapping the distribution of putative neurotransmitters (Hunt, 1983) and, unlike some of the techniques described earlier, this approach can be applied to human post mortem tissue (Pearson, Brandeis and Cuello, 1982; see also Fig.…”