Molecular annotation of integrative feeding neural circuits

Abstract

The identity of higher-order neurons and circuits playing an associative role to control feeding is unknown. We injected pseudorabies virus, a retrograde tracer, into masseter muscle, salivary gland, and tongue of BAC-transgenic mice expressing GFP in specific neural populations and identified several CNS regions that project multisynaptically to the periphery. MCH and orexin neurons were identified in the lateral hypothalamus, and Nurr1 and Cnr1 in the amygdala and insular/rhinal cortices. Cholera toxin β tracing showed that insular Nurr1(+) and Cnr1(+) neurons project to the amygdala or lateral hypothalamus, respectively. Finally, we show that cortical Cnr1(+) neurons show increased Cnr1 mRNA and c-Fos expression after fasting, consistent with a possible role for Cnr1(+) neurons in feeding. Overall, these studies define a general approach for identifying specific molecular markers for neurons in complex neural circuits. These markers now provide a means for functional studies of specific neuronal populations in feeding or other complex behaviors.

Figure 1. Brainstem Cranial Nerve Motor Nuclei are the First CNS Infected Targets Following PRV Injection Into Peripheral Tissues

A) PRV-152, a Bartha PRV strain genetically modified to express EGFP constitutively (Smith et al., 2000) was used in the present studies. B) PRV-152 was injected bilaterally into the submandibular salivary gland (SAL), the masseter muscle (MAS), or unilaterally into the posterior part of the tongue (T_P). 36, 48 or 60 hrs after the injections, EGFP immunofluorescence was analyzed in the brains of infected animals. The motor nucleus for the indicated cranial nerves was the first region where PRV infection was detected, following PRV injection into the indicated tissues. The motor nucleus of the cranial nerve V (Mo5) was the first brainstem area infected after PRV-152 injection into the masseter muscle (MAS), the motor nucleus of the cranial nerve VII (7N) and the superior salivatory nucleus (SuS) were identified after PRV injection into the salivary gland (SAL). Finally, the motor nucleus of the cranial nerve XII (12N) was the only place where PRV was readily observed after PRV injection into the posterior region of the tongue (T_P).

Figure 2. Brain Areas Infected by PRV After Injection Into Multiple Peripheral Tissues

A) Distribution of forebrain areas infected by PRV-152 96hr after five distinct PRV injections (SAL, MAS, T_P, T_L, T_A) were made into peripheral tissues. This analysis revealed that several brain areas were infected by PRV in each circuit studied (color- coded columns). Very few brain regions were common to the descending neural circuits projecting to all peripheral tissues injected. These regions included the lateral hypothalamus, basolateral and central amygdala and insular, ectorhinal and perirhinal cortices (see multiple color-coded rows in the lower-right corner). B) Symmetrical, non-proportional, 5-way Venn diagram illustrating the number of PRV-infected brain regions after injection in 1 to 5 peripheral sites. It can be appreciated the vast number of subsets (32) to be considered when analyzing multiple circuits and how our proposed approach can identify brain regions belonging to only one such subset. n=3 per injection site.

Figure 3. Triple PRV Labeling in the LH Identifies a Neuronal Population Projecting to Masseter Muscle, Salivary Gland and Tongue

A triple PRV injection paradigm (PRV-152 in T_P, PRV-614 in SAL and PRV-BaBLU in MAS) was used to identify triple labeled neurons. PRV-BaBLU, is isogenic to PRV-152 and PRV-614, and encodes LacZ (see scheme at the bottom). Triple labeled neurons were observed (arrows) in the LH, as well as neurons displaying single (arrowheads) or double (double arrowheads) PRV infection.

Figure 4. Colocalization of PRV-Encoded mRFP and Transgenic EGFP Reveals Markers for Neurons in Feeding Neuronal Circuits

Elavl3⁺/PRV⁺ neurons (A) were detected in the rhinal cortex following a SAL PRV injection. Lhx6⁺/PRV⁺ neurons (B) were detected in the insular cortex after a MAS PRV injection. Cnr1⁺/PRV⁺ neurons (C) were detected in the insular cortex following a PRV injection into SAL. ; Gabrb2⁺/PRV⁺ neurons (D) were detected in the insular cortex following a PRV injection into SAL. Lrig2⁺/PRV⁺ neurons were detected in the insular cortex following PRV injections into SAL (E) and MAS (F). Nurr1⁺/PRV⁺ neurons were detected in the insular cortex following SAL (G), MAS (H) and Brown Adipose Tissue (I) PRV injections. (J) Diagram showing our approach to identify markers for neuronal populations from a specific neural circuit (as indicated by their susceptibility to PRV infection). Mice in which EGFP is known to be expressed in a specific brain region are infected in the periphery with PRV-614 (encodes mRFP) and in those cases where both mRFP (from PRV) and EGFP (from the transgenic mouse) are detected in the same neuron, it can be asserted that the gene whose promoter directs EGFP expression is a marker for neurons integrated into the circuit projecting to the tissue infected by PRV-614.

Figure 5. Nurr1 is a Marker of Neurons in Neural Circuits Projecting to Both Salivary Gland and Masseter Muscle

PRV-BaBLU and PRV-614 were injected in MAS and SAL of Nurr1::EGFP transgenic mice (see scheme at the bottom). Nurr1⁺ neurons contributing to both neural circuits (positive for both viral reporters) were identified in the insular cortex (A) and the amygdala (B).

Figure 6. Cholera Toxin β Tracing Confirms Connectivity of Insular Nurr1⁺ And Cnr1⁺ Neurons Predicted by PRV Tracing

Cholera toxin β conjugated to Alexa Fluor 594 was injected into the LH or amygdala of Cnr1::EGFP and Nurr1::EGFP mice (n=3). One week after the stereotaxic injection of the tracer, it was detected in Nurr1⁺ neurons after injections in the amygdala (A) and also in Cnr1⁺ neurons following injections into the LH (B).

Figure 7. Insular Cnr1⁺ Neurons are Sensitive to Fasting

(A) Insular cortex Cnr1 mRNA levels were measured in wild type mice fed ad libitum vs. mice that had fasted for 36 hrs. Using a Taqman assay it was determined that there was a significant increase in Cnr1 mRNA following a 36h fasting period (p=0.01, n=5). (B) We also quantified the number of c-Fos⁺/Cnr1⁺ neurons in the insular cortex of Cnr1::EGFP mice in mice with unrestricted access to food vs. mice that had fasted for 36hrs (n=3 per group). It was observed that the number of c-Fos⁺/Cnr1⁺ neurons in the posterior insular cortex (p-INS) doubled following fasting (p=0.040); this was also valid for its agranular (p-Aip; p=0.036) and visceral (p-VISC; p=0.023) subdivisions. All data are represented as mean +/- SEM; Student's t test was used for statistical analysis. Significance was assumed for p values < 0.05.