However, a previous study in the hamster concluded that there is a large degree of convergence in the rodent tecto-LPN pathway (Mooney et al., 1984). found that all tracer-labeled cortical terminals, as well as vGLUT1 antibody-labeled terminals, are small profiles with round vesicles (RS profiles) that innervate small caliber dendrites. Tracer-labeled tecto-LPN terminals, as well as vGLUT2 antibody-labeled terminals, were medium-sized profiles with round vesicles (RM profiles). Tecto-LPN terminals were significantly larger than cortico-LPN terminals, and contacted significantly larger dendrites. These results indicate that within the tectorecipient zone of the rat LPN, cortical terminals are located distal to tectal terminals, and that vGLUT1 and vGLUT2 antibodies may be used as markers for cortical and tectal terminals respectively. Finally, comparisons of the synaptic patterns formed by tracer-labeled terminals with those of vGLUT antibody-labeled terminals suggest that individual LPN neurons receive input from multiple cortical and tectal axons. We suggest that the tectorecipient LPN constitutes a third category of thalamic nucleus (second order) that integrates convergent tectal and cortical inputs. This organization may function to signal the movement of novel or threatening TNFSF10 objects moving across the visual field. studies of the rat LPN, we have characterized the tectorecipient zone of the rat LPN using histochemical markers, tract tracing and electron microscopy. Methods A total of 19 hooded rats were used for these experiments. Five rats received injections of Fluorogold (FG, Fluorochrome LLC, Denver, CO) in the LPN to label corticothalamic and tectothalamic cells by retrograde transport. Four rats received injections of biotinylated dextran amine (BDA, 3,000 MW; Molecular Probes, Eugene, OR) in the visual cortex to label corticothalamic terminals by anterograde transport. Three rats received injections of phaseolus vulgaris leucoagglutinin (PHAL, Vector Laboratories, Burlingame, CA) in the SC to label tectothalamic terminals by anterograde transport. Brains from an additional 4 rats were used for the immunocytochemical localization of the type 1 and type 2 vesicular glutamate transporters (vGLUT1 and Valemetostat tosylate vGLUT2). Finally, 3 rats received injections of ibotenic acid (MP Biomedicals, Aurora, OH) in the SC to determine whether vGLUT1 or vGLUT2 staining of the LPN was diminished after the destruction of tectothalamic cells. All procedures conformed to the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the University of Louisville Animal Care and Use Committee. Tract tracing The rats were anesthetized with intraperitoneal injections of sodium pentobarbital (initially 50mg/kg, with supplements injected as needed to maintain anesthesia) or intramuscular Valemetostat tosylate injections of ketamine and xylazine (initially 100 mg/kg and 6.7 mg/kg respectively, with supplements to maintain anesthesia). They were placed in a stereotaxic apparatus and prepared for aseptic surgery. BDA (5% in saline) PHAL (2.5% in water), or FG (10% in saline) were injected through a glass micropipette (20C30 m Valemetostat tosylate tip diameter) using 5 A of continuous positive current for 10C20 minutes. 2 L of ibotentic acid (2% in saline) was ejected from a glass micropipette (10C20 m tip diameter) using a PV830 pneumatic PicoPump (WPI, Sarasota, FL). After a survival time of one week, the rats were perfused transcardially with artificial cerebrospinal fluid (ACSF) or Tyrode solution followed by a fixative solution of 4 % paraformaldehyde, or 2.5C3% paraformaldehyde and 1C1.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 (PB). The brain was removed from the skull, and the thalamus was sectioned to a thickness of 50 m using a vibratome and placed in PB. The transported FG was revealed by incubating sections overnight at 4C in the goat anti-FG antibody diluted 1:10,000. The next day the sections were incubated one hour in a biotinylated rabbit anti-goat antibody (Vector) diluted 1:100, followed by 2 hours in a solution containing a complex of avidin and biotinylated horseradish peroxidase (ABC). The transported PHAL was revealed by incubating sections overnight at 4C in the biotinylated goat anti-PHAL antibody diluted 1:200, followed by 2 hours in ABC. The transported BDA was revealed by incubating sections overnight at 4C in ABC solution. Sections containing FG, PHAL or BDA were subsequently reacted with nickel intensified diaminobenzidine (DAB) for five to 10 minutes, and washed in PB. Sections were then mounted on slides for light microscopic examination, or prepared for electron microscopy. Light level photographs were taken using a digitizing camera (Spot RT, Diagnostic Instruments Incorporated, Sterling Heights, MI), and terminal distributions were plotted using a Neurolucida system. Antibody characterization The antibodies used in this study are listed in Table 1. Preabsorption of the type 1 and type 2 vesicular glutamate transporter (vGLUT1 and vGLUT2) antiserums with their corresponding immunogen peptides (Chemicon catalogue #AG208 and AG209; 1 g/ml immunogen peptide added to the diluted antibody) eliminated all terminal staining in tissue sections containing the thalamus and cortex (mouse, rat, tree shrew and cat tissue perfusion fixed with 4 % paraformaldehyde, or 2% paraformaldehyde and 2% glutaraldehyde for previous studies). Western blot.