Neurosci. Bull. June, 2019, 35(3):447–460 www.neurosci.cn https://doi.org/10.1007/s12264-018-0325-4 www.springer.com/12264 ORIGINAL ARTICLE The Glutamatergic Postrhinal Cortex–Ventrolateral Orbitofrontal Cortex Pathway Regulates Spatial Memory Retrieval 1 2 1 2 1 Xinyang Qi • Zhanhong Jeff Du • Lin Zhu • Xuemei Liu • Hua Xu • 2 2 3 2 1,2 Zheng Zhou • Cheng Zhong • Shijiang Li • Liping Wang • Zhijun Zhang Received: 12 June 2018 / Accepted: 8 September 2018 / Published online: 2 January 2019 Ó Shanghai Institutes for Biological Sciences, CAS 2019 Abstract A deficit in spatial memory has been taken as an memory is relatively unknown. To further illuminate the early predictor of Alzheimer’s disease (AD) or mild function of the UF in spatial memory, we dissected the cognitive impairment (MCI). The uncinate fasciculus POR–vlOFC pathway in mice. We determined that the (UF) is a long-range white-matter tract that connects the POR–vlOFC pathway is a glutamatergic structure, and that anterior temporal lobe with the orbitofrontal cortex (OFC) glutamatergic neurons in the POR regulate spatial memory in primates. Previous studies have shown that the UF retrieval. We also demonstrated that the POR–vlOFC impairment associated with spatial memory deficits may be pathway specifically transmits spatial information to par- an important pathological change in aging and AD, but its ticipate in memory retrieval. These findings provide a exact role in spatial memory is not well understood. The deeper understanding of UF function and dysfunction pathway arising from the postrhinal cortex (POR) and related to disorders of memory, as in MCI and AD. projecting to the ventrolateral orbitofrontal cortex (vlOFC) performs most of the functions of the UF in rodents. Keywords Spatial memory Á Postrhinal cortex Á Ventro- Although the literature suggests an association between lateral orbitofrontal cortex Á Mild cognitive impairment Á spatial memory and the regions connected by the POR– Alzheimer’s disease vlOFC pathway, the function of the pathway in spatial Introduction Xinyang Qi, Zhanhong Jeff Du and Lin Zhu have contributed equally to this work. Spatial memory refers to the part of the memory system & Liping Wang which is in charge of encoding, storing and recalling spatial [email protected] information about the environment and the objects and & Zhijun Zhang agents within it [1]. Spatial memory is required for [email protected] individuals to navigate to goal locations and is vitally 1 important for reaching goals in a spatially extended Department of Neurology, Affiliated ZhongDa Hospital, environment [1, 2]. Patients with a spatial memory deficit Institute of Neuropsychiatry, School of Medicine, Southeast University, Nanjing 210009, China often show disorientation, which is the core symptom in emerging mild cognitive impairment (MCI) and the early 2 Shenzhen Key Lab of Neuropsychiatric Modulation and Collaborative Innovation Center for Brain Science, Chinese stage of Alzheimer’s disease (AD) [3–5]. Illuminating the Academy of Sciences (CAS) Center for Excellence in Brain working mechanism of spatial memory may contribute to Science and Intelligence Technology, the Brain Cognition further understanding of the pathology of these diseases. and Brain Disease Institute for Collaboration Research of the However, the neural mechanism of spatial memory is not Shenzhen Institutes of Advanced Technology at the CAS and the McGovern Institute at Massachusetts Institute of well understood. Functional magnetic resonance imaging Technology, Shenzhen 518055, China (fMRI) has suggested that the temporal [6], frontal [7], and 3 Department of Biophysics, Medical College of Wisconsin, occipital lobes [3] are the critical areas in the neural circuit Milwaukee, WI 53226, USA for spatial memory, but details regarding the function of 123 448 Neurosci. Bull. June, 2019, 35(3):447–460 these areas and fiber tracts in spatial memory remain to be Methods elucidated. The uncinate fasciculus (UF) is a long-range white- Animals matter fiber tract connecting the anterior temporal lobe with the orbitofrontal cortex (OFC), a subregion of the Adult (20 g–30 g) male C57BL/6 mice (Guangdong Med- frontal lobe, through a direct, monosynaptic pathway [8]. ical Laboratory Animal Center, Guangzhou, China), and The temporal segment of the tract originates from the adult male GAD2-Cre mice aged 6 weeks–8 weeks at the anterior part of the parahippocampal cortex and the beginning of experiments, were housed in groups of five temporal pole [9]. The UF belongs to a small group of per cage. The GAD2-Cre mice had Cre recombinase late-myelinating white matter association tracts that expression directed to GAD2-positive neurons without undergo protracted development throughout adolescence disrupting endogenous glutamic acid decarboxylase 2 [10]. So, this slow maturation may open a time window expression. The animals were maintained on a 12/12-h during which these fibers are more vulnerable to risk light/dark cycle (lights on at 08:00, Beijing time) at a factors causing neurological and psychotic disorders [8]. stable temperature (23°C–25°C) and given food and water The exact function of the UF remains unclear, but diffusion ad libitum. All animal experiments were approved by the tensor imaging (DTI) studies have indicated that Institutional Animal Care and Use Committee of Shenzhen microstructural deficits of the UF occur in such conditions Institutes of Advanced Technology, Chinese Academy of as anxiety [11], depression [12], schizophrenia [13], Sciences. psychopathy [14], frontotemporal dementia [15], MCI [16], and AD [17]. Furthermore, some task-fMRI and Anatomy of the POR DTI studies have shown that UF dysfunction is associated with age-related impairment of spatial navigation [18] and The POR is bordered by the following structures: the spatial memory deficits in MCI [19]. Nevertheless, it caudal end of the angular bundle, the caudal part of the remains unclear how the UF participates in spatial memory perirhinal cortex [27], the ventral temporal association area processing; particularly in the encoding, storage, or (TEA), and the dorsal part of the medial entorhinal cortex retrieval stage of spatial memory. (Fig. 1A). The POR is bordered rostrally by the perirhinal The postrhinal cortex (POR) is the rodent homolog of cortex, which was identified by subicular cells in the the primate parahippocampal cortex [20]. Previous studies angular bundle [21, 28]. The POR was differentiated from have confirmed that the POR is rich in GABAergic and the TEA by identification of discrete layers II, III, and IV, glutamatergic neurons [21], and is strongly connected with and the bilaminate appearance of layer V. The POR lies the visual area, laterodorsal thalamic nucleus (LD), hip- above a slight indentation surrounded by the medial pocampal formation, caudate-putamen, and prefrontal entorhinal cortex. In the coronal plane (Fig. 1B–D), the cortex (PFC) [22–24]. As a core region of the PFC, the POR was identified as the region bordered by the rhinal ventrolateral orbitofrontal cortex (vlOFC) mainly receives fissure rostrally and medially, and the TEA at the middle of projections from the POR which may contribute to UF the angular bundle dorsally. At caudal levels, the POR is function in rodents [20]. POR/OFC lesions cause spatial triangular in shape. The dorsal portion is bordered by the memory impairment in rodents [25, 26], suggesting that parasubiculum, which forms one side of a triangle together both the POR and OFC are key regions in the neural circuit with the entorhinal. TEA forms the second side, and the of spatial memory. However, the function of the POR– cortical surface, the third side. vlOFC pathway connecting the two regions in spatial memory has not been investigated in rodents, and its Immunofluorescence Staining structure remains to be elucidated. To further illuminate the function of the UF in spatial Mice were anesthetized with chloral hydrate (10% w/v, memory, we set out to dissect the POR–vlOFC pathway in 300 mg/kg body weight) and perfused transcardially with mice. We aimed to examine the structure of the POR– phosphate-buffered saline (PBS) followed by ice-cold 4% vlOFC pathway and its exact function in spatial memory. paraformaldehyde (PFA, 158127 MSDS, Sigma, St. Louis, MO) (mice for c-fos staining were sacrificed 1.5 h after blue light stimulation). The brain was removed carefully and post-fixed in 4% PFA at 4 °C overnight, then cryoprotected in PBS containing 25% sucrose for 48 h. Coronal sections (40 lm) were cut on a Leica (Wetzlar, Germany) freezing microtome. Free-floating sections were 123 X. Qi et al.: POR–vlOFC Pathway Regulates Spatial Memory Retrieval 449 Fig. 1 Identification of postrhinal cortex (POR) borders and neuronal entorhinal area; PAS, parasubiculum area. E, F POR neurons co- composition in the mouse. A Lateral surface view of the POR borders. labeled for (E) GAD67 (green) and NeuN (red) or (F) CamkII (green) B–D Schematic of the POR shows 3 rostrocaudal levels in the coronal and NeuN (red) are indicated by arrowheads (scale bars, 50 lm). plane. Gray rectangles indicate the cell-counting regions (scale bars, G Proportions of GABAergic (48.4% ± 1.6%) and glutamatergic 250 lm). AP: B, -4.70 mm; C, -4.85 mm; D, -5.00 mm. TEA, (51.4% ± 1.8%) neurons in the POR. Data are presented as temporal association area; PER, perirhinal cortex; Entl, lateral mean ± SEM. rinsed with PBS on a rocking bed (60 rpm, 10 min 9 3 Enhancer, Invitrogen). The sections were then pho- times), and incubated