Overview

Certerra’s Pharmacomap method is based on a proprietary drug-screening technology (Fig.1). An important part of this technology is a high-throughput microscopy, called serial two-photon (STP) tomography, which achieves automated, cellular-resolution imaging of whole mouse brains (Ragan et al., 2012, Nature Methods 9, 255–258). When used to map drug-evoked induction of immediate early genes (IEGs), such as c-fos, STP tomography generates unbiased brainwide maps of drug-evoked neural activation, which we call “pharmacomaps” (Fig. 2).


Because the effects of a drug on the brain are largely determined by which neurons the drug activates, these pharmacomaps provide an unprecedented wealth of high-content, cellular-level information. The main advantages of the Pharmacomap drug-screening services are:

- Automated generation of unbiased maps of drug-evoked activation in the whole mammalian brain at cellular resolution

- Automated computational analysis of the whole brain datasets

- Automated and standardized data registration for visualization and statistical comparisons between different drugs and conditions


In summary, Certerra’s Pharmacomap drug-screening method is the first complete, automated and highly standardized solution to mapping and statistically comparing in vivo drug-evoked effects in the mammalian brain at cellular resolution.

figure1

Figure 1. Generation of pharmacomaps of c-fos activation. (A-D) After drug delivery, the c-fos-GFP brain is imaged as a dataset of 280 coronal sections by STP tomography, a method which integrates two-photon microscopy and tissue sectioning. (E-F) c-fos-GFP-positive cells are automatically detected by machine learning algorithms and the brainwide c-fos-GFP distribution is reconstructed in 3D. (G) The datasets are warped on to a standard “reference” brain volume (top panels) and voxelized for statistical comparisons (bottom panels). (H) c-fos-GFP distribution in voxelized control and experimental brains is compared by series of negative binomial regressions (a false discovery rate of 0.01). All color-coded areas reached statistical significance of P<0.01.

The generation of Pharmacomaps

Drug-evoked pharmacomaps are generated by the following steps (Fig. 1). Transgenic c-fos-GFP mice are treated with a drug or a vehicle for control group (the delivery route may be intraperitoneal, peroral or subcutaneous). After 3 hours to allow peak c-fos-driven GFP expression, the animal is killed and the brains are fixed and imaged by STP tomography. The c-fos-GFP-positive neurons are detected by neural network-based algorithms and the data are registered, voxelized and compared by rigorous statistical methods. This generates unbiased, brainwide, cellular resolution “heat maps” of statistically significant differences in c-Fos-GFP cell counts. Finally, these maps are registered to a mouse brain atlas in order to determine the anatomical brain regions with significant differences in c-fos-GFP expression (Fig. 2).

figure2

Figure 2. Comparison of a typical antipsychotic (A) and two atypical antipsychotic (B, C) pharmacomaps. A subset of structures from the Table 1 (below) is shown as schematic circuits; activated areas are in red, inhibited in blue; arrows denote only major anatomical connections. A) An example of a typical antipsychotic-evoked brain activation, including a major portion of the caudate putamen (CP) and nucleus accumbens (ACB), as well as the olfactory tubercle (OT), prelimbic cortex (PL), lateral septum (LS) and dorsomedial hypothalamus (HYP). The CP and ACB, highlighted in gray, are common structures activated by all three drugs. B) An example of a 1st generation atypical antipsychotic, which activates prelimbic (PL), orbital (ORB), piriform (PIR) and gustatory (GU) cortices, the dorsal and ventral CP, ACB, claustrum (CLA), and superior colliculus (SC). Reciprocal connections between cortex and CLA, unidirectional connections from cortex to CP and ACB, and a multisynaptic pathway between SC and CP are indicated. Note that SC, an important input structure to the striatum via indirect pathways, is activated by both 1st and 2nd generation atypical antipsychotics. Cortical areas (left) and brainstem areas (right) are grouped in dashed ovals. C) An example of a 2nd generation atypical antipsychotic, which activates a partially overlapping pattern, with more cortical areas, including prominent activation of auditory association and entorhinal areas. Parts of the amygdala (AMG), hippocampal formation (HF), and midline thalamus (PVT and RE) also show activation. A subset of cortical areas is repeated at lower left, in association with the hippocampal formation.

table1

Table 1. The list of brain structures representing examples of a typical, 1st and 2nd generation atypical antipsychotic pharmacomaps.

 
 
 
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