Protocol
Elucidation of neuronal circuitry: protocol(s) combining intracellular labeling, neuroanatomical tracing and immunocytochemical methodologies

https://doi.org/10.1016/S1385-299X(01)00065-4Get rights and content

Abstract

We describe a protocol combining either intracellular biotinamide staining or anterograde biotinylated dextran amine (BDA) tracing with retrograde horseradish peroxidase (HRP) labeling and immunocytochemistry in order to map physiologically identified neuronal pathways. Presynaptic neurons including their boutons are labeled by either intracellular injection of biotinamide or extracellular injection of BDA while postsynaptic neurons are labeled with HRP via retrograde transport. Tissues are first processed to detect HRP using a tetramethylbenzidine and sodium-tungstate method. Biotinamide or BDA staining is then visualized using an ABC-diaminobenzidine-Ni method and finally the tissue is immunocytochemically stained using choline acetyltransferase (ChAT) or parvalbumin antibodies and a peroxidase-anti-peroxidase method. After processing, biotinamide, BDA, HRP and immunocytochemical staining can readily be distinguished by differences in the size, color and texture of their reaction products. We have utilized this methodology to explore synaptic relationships between trigeminal primary afferent neurons and brainstem projection and motoneurons at both the light and electron microscopic levels. This multiple labeling methodology could be readily adapted to characterize the physiological, morphological and neurochemical properties of other neuronal pathways.

Section snippets

Type of research

Neural pathways of the central nervous system (CNS) provide a fundamental substrate for understanding brain function and behavior. Currently no powerful method is available for mapping neuronal pathways consisting of more than two neurons. Classically, physiologists have utilized the latency between electrical stimulation of a remotely located neuronal element and an electrophysiologically recorded response to distinguish between monosynaptic and polysynaptic neuronal circuits. However, it is

Time required

To complete the entire protocol in which horseradish peroxidase tracing is combined with intracellular biotinamide labeling and immunocytochemistry requires 5–5.5 days. If biotinylated dextran amine is incorporated into the methodology, 12–14 days are needed to complete the protocol, due to the additional time necessary for BDA transport. The specific breakdown of time required is as follows:

  • •

    Anterograde axonal transport using biotinylated dextran amine (BDA): 7–9 days

  • •

    Retrograde horseradish

Animals

  • •

    Adult, male Sprague–Dawley rats, weighing 300–350 g (Harlan, Indianapolis, IN) were housed in an animal facility approved by the Association for the Accreditation of Laboratory Animal Care International (AALAC). Animals were housed at 22–25°C with a 12:12 h light–dark cycle and were allowed free access to food and water. All procedures involving animals were carried out in accordance with the NIH guide for the care of animals in research and were approved by the Institutional Animal Care and

Retrograde neuronal labeling via horseradish peroxidase (HRP)

(1) Anesthetize rats with sodium pentobarbital (40 mg/kg, i.p.) and administer atropine sulfate (0.16 mg/kg, i.p.).

(2) To retrogradely label motoneurons, inject a solution consisting of 20% HRP and 1% WGA-HRP into the innervated muscle with a microsyringe or directly into the muscle nerve via a micropipette. Our experience with masticatory and upper esophageal muscles indicates ∼5–15 μl are optimal for muscle injections while 2–3 μl are sufficient for nerve injections. We have also found that a

Identification and differentiation of HRP, biotinamide, BDA and immunocytochemical labeling

Neurons retrogradely labeled with HRP were readily identified by the presence of HRP reaction product in the cytoplasm of their somata and dendrites. By using the modified TMB-ST method described here, HRP reaction product appeared as a black, coarse granular, crystalline material. Horseradish peroxidase reaction product was heterogeneously distributed throughout the cell body as well as the large and medium diameter dendrites of neurons retrogradely labeled with HRP (Fig. 1, Fig. 2). In

Discussion

The combined methodologies described in this protocol provide a reliable means to characterize the morphological, physiological and neurochemical properties of neuronal pathways. While we have recently utilized these methodologies to examine neuronal pathways from trigeminal primary afferent neurons to the thalamus, trigeminal motor nucleus and spinal cord [14], [29], [31], this strategy readily could be employed to explore other neuronal systems.

Combined intracellular biotinamide staining and retrograde HRP labeling

  • 1.

    Inject horseradish peroxidase (HRP) and allow a 24–48-h survival time.

  • 2.

    Physiologically identify neurons and intracellularly inject them with biotinamide.

  • 3.

    Perfuse and fix animal.

  • 4.

    Cut serial sections on a vibratome.

  • 5.

    Histochemically react tissue to visualize retrograde HRP labeling.

  • 6.

    Histochemically react tissue to visualize intracellular biotinamide staining.

  • 7.

    Embed tissue, cut ultrathin sections and observe on electron microscope.

Intracellular biotinamide staining combined with immunocytochemistry

  • 1.

    Physiologically identify and inject neurons with biotinamide.

  • 2.

    Fix and

Essential references

The following are essential references: Refs. [13], [14], [18], [27], [28], [29], [35].

Acknowledgements

This work was supported by NIH DE10132 and DC04096. We thank E. Wade for excellent technical assistance.

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