Improved neuronal tract tracing with stable biocytin-derived neuroimaging agents

Abstract

One of the main characteristics of brains is their profuse connectivity at different spatial scales. Understanding brain function evidently first requires a comprehensive description of neuronal anatomical connections. Not surprisingly a large number of histological markers were developed over the years that can be used for tracing mono- or polysynaptic connections. Biocytin is a classical neuroanatomical tracer commonly used to map brain connectivity. However, the endogenous degradation of the molecule by the action of biotinidase enzymes precludes its applicability in long-term experiments and limits the quality and completeness of the rendered connections. With the aim to improve the stability of this classical tracer, two novel biocytin-derived compounds were designed and synthesized. Here we present their greatly improved stability in biological tissue along with retained capacity to function as neuronal tracers. The experiments, 24 and 96 h postinjection, demonstrated that the newly synthesized molecules yielded more detailed and complete information about brain networks than that obtained with conventional biocytin. Preliminary results suggest that the reported molecular designs can be further diversified for use as multimodal tracers in combined MRI and optical or electron microscopy experiments.

Keywords: Brain mapping; biocytin; biotinidase; histology; neuroanatomical tracers.

Figure 1

Studied biocytin derivatives as neuroanatomical tract tracers.

Scheme 1

Reagents and conditions: (i) acrylonitrile, TEA, MeOH; (ii) d-biotin, PyBroP, DIPEA, dry CH₂Cl₂; (iii) Ra-Ni, H₂, 7 M NH₃/MeOH, 50 psi; (iv) Pd−C (10%), H₂, MeOH, 50 psi; (v) LiOH, THF/MeOH/water (3:2:2).

Scheme 2

Reagents and conditions: (i) TBDMS-Cl/imidazole/DMF; (ii) LiOH−THF/MeOH/H₂O (3:2:2); (iii) MeOH/DCC/CH₂Cl₂; (iv) Pd−C (10%), H₂, MeOH, 50 psi; (v) EDC/NMM/HOBt/DMF; (vi) TBAF/THF; (vii) i-Bu-chloroformate/pyridine/CH₂Cl₂; (viii) HATU/DIPEA/DMF; (ix) TFA/CH₂Cl₂.

Figure 2

Comparative histological studies of L (a), L₁ (b), and L₂ (c) showing the injection site in the cerebral cortex of a rat after a survival time of 24 h. The higher stability of L₁ and L₂ compared with L is visible already after 24 h.

Figure 3

Comparison of L (a), L₁ (b), and L₂ (c) in the cerebral cortex of the rat after a survival time of 96 h. The injection site of L is hardly visible, while the injection sites of L₁ and L₂ do not differ in intensity from those after 24 h.

Figure 4

Comparison of L (a) and L₁ (b, c) after 1 h survival time in the rat brain: (a) axon bundles in the striatum; (b) axons in the white matter emanating from the injection site; (c) same animal, showing axons in the striatum. Both tracers show equally good uptake and transport after 1 h.

Figure 5

Examples of anterograde or retrograde transport of L (a, b), of L₁ (c), and of L₂ (d−f) after injection into the primary motor cortex and a survival time of 24 h. (a, c) axon terminals in the contralateral hemisphere; (b, e) axons running through the striatum; (d) retrogradely stained neurons in a different cortical area; (f) retrogradely stained neurons in the thalamus.

Figure 6

Transport of L₁ (a) and of L₂ (b) after a survival time of 96 h: (a) anterogradely stained ascending axons and some retrogradely stained neurons in the ipsilateral cortex; (b) ascending axons in the contralateral hemisphere.