Unveiling a Novel Memory Center in Humans: Neurochemical Identification of the Nucleus Incertus, a Key Pontine Locus Implicated in Stress and Neuropathology

Background The nucleus incertus (NI) was originally described by Streeter in 1903, as a midline region in the floor of the fourth ventricle (4V) of the human brain with an ‘unknown’ function. More than a century later, the neuroanatomy of the NI including its forebrain target regions has been described in lower vertebrates, but not in humans. Therefore, we examined the neurochemical anatomy of the human NI using several markers, including the neuropeptide, relaxin-3 (RLN3), and began to explore the distribution of the NI-related RLN3 innervation of the hippocampus. Methods Histochemical staining of serial, coronal sections (30 µm) of control human postmortem pons was conducted to reveal the presence of the NI by detection of immunoreactivity (IR) for the neuronal marker, microtubule-associated protein-2 (MAP2), two markers present in rat NI, glutamic acid dehydrogenase (GAD)-65/67 and corticotrophin releasing hormone receptor 1 (CRHR1), and RLN3, which is highly expressed in a major population of NI neurons in diverse species. RLN3 and vesicular GABA transporter 1 ( vGAT1 ) mRNA was detected by multiplex, fluorescence in situ hybridization. Postmortem pons sections containing the NI from an Alzheimer’s disease (AD) case were immunostained for phosphorylated-tau (AT8 antibody), to explore potential relevance to neurodegenerative diseases. Lastly, sections of human hippocampus were stained to detect RLN3-IR and somatostatin (SST)-IR, as SST is expressed in interneurons targeted by RLN3 projections in rodents. Results In the dorsal, anterior-medial region of the human pons, neurons containing RLN3– and MAP2-IR, and RLN3/vGAT1 mRNA-positive neurons were observed in an anatomical pattern consistent with that of the NI in other species. GAD65/67– and CRHR1-immunopositive neurons were also detected within this area. Furthermore, RLN3– and AT8-IR were co-localized within NI neurons of an AD subject. Lastly, RLN3-IR was detected in neurons within the CA1, CA2, CA3, and DG areas of the hippocampus, in the absence of RLN3 mRNA. In the DG, RLN3– and SST-IR were co-localized in a small population of neurons. Conclusions Aspects of the anatomy of the human NI are shared across species, including a population of RLN3-expressing neurons and a RLN3 innervation of the hippocampus. Accumulation of phosphorylated-tau in the NI suggests its possible involvement in AD pathology. Further characterization of the neurochemistry of the human NI will increase our understanding of its functional role in health and disease. ![Figure][1] Graphical Abstract Created with BioRender.com ### Competing Interest Statement The authors have declared no competing interest. * ACD : Advanced Cell Diagnostics AD : Alzheimer’s disease ADRC : Alzheimer’s Disease Research Center ADRD : Alzheimer’s disease and related dementias BBDP : Brain Bank Donation Program BSA : Bovine serum-albumin CCK : Cholecystokinin CGM : Central gray of the medulla CRH : Corticotrophin releasing hormone CRHR1 : Corticotrophin releasing hormone receptor 1 DAB : 3,3′-Diaminobenzidine D2R : D2-like dopamine receptor EP : Ependymal GABA : γ-Aminobutyric acid GAD : Glutamic acid decarboxylase H&E : Hematoxylin and Eosin HG : Hypocellular gap IHC : Immunohistochemistry IR : Immunoreactivity ISH : In situ hybridization LC : Locus coeruleus MAP2 : Microtubule-associated protein-2 MCH : Melanin-concentrating hormone MCH-1 : Melanin-concentrating hormone receptor-1 mlf : Medial longitudinal fasciculus NMB : Neuromedin-B NI : Nucleus incertus ON : Overnight OX1 : Orexin receptor-1 OX2 : Orexin receptor-2 PAG : Periaqueductal gray PBS : Phosphate buffered saline PBS-T : Phosphate-buffered saline with Tween® 20 detergent PRn : Pontine raphe nucleus RLN3 : Relaxin-3 RT : Room temperature RXFP3 : Relaxin-family peptide-3 receptor SHS : Septohippocampal system SN : Substantia nigra TBS-T : Tris-buffered saline with 0.1% Tween® 20 detergent vGAT1 : Vesicular GABA transporter-1 4V : Fourth ventricle 5-HT1A : Serotonin-1A receptor [1]: pending:yes