Structure


Hippocampal neurons (Figure 1) play a major role in the functioning of the human brain. Humans and other mammals have two hippocampi, one in each side of the brain. The hippocampus belongs to the limbic system and plays an important role in the consolidation of information from short to long-term memory, and enables navigation via spatial memory. The hippocampus can be seen as a ridge of gray matter tissue, elevating from the floor of each lateral ventricle in the region of the inferior or temporal horn.  The cortex thins from six layers to the three or four layers that make up the hippocampus. The term hippocampal formation is used to refer to the hippocampus proper and its related parts. The neural layout and pathways within the hippocampal formation are very similar in all mammals.1

 

Image of Lonza Rat Hippocampal Cells  

Figure 1. Lonza's Primary Rat Hippocampal Neurons

 

The hippocampus (Figure 2), including the dentate gyrus, has the shape of a curved tube, which has been compared to a seahorse, and a ram's horn (Cornu Ammonis). Its abbreviation CA is used in naming the hippocampal subfields: CA1, CA2, CA3, and CA4.

 

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It can be distinguished as an area where the cortex narrows into a single layer of densely packed pyramidal neurons, which curl into a tight U shape. One edge of the "U," – CA4, is embedded into the backward-facing, flexed dentate gyrus.2 The hippocampus is described as having an anterior and posterior part (in primates) or a ventral and dorsal part in other animals. Both parts are of similar composition but belong to different neural circuits.3 In rat, the two hippocampi resemble a pair of bananas, joined at the stems by the commissure of fornix (also called the hippocampal commissure). In primates, the part of the hippocampus at the bottom, near the base of the temporal lobe, is much broader than the part at the top. This means that in cross-section the hippocampus can show a number of different shapes, depending on the angle and location of the cut.

Image depicting the regions of the Hippocampus
Figure 2. The Regions of the Hippocampus.  Source: J. A. Kiernan modified from Edinger, 1899 (L. Edinger, The Anatomy of the Central Nervous System of Man and of Vertebrates in General, F.A. Davis, Philadelphia, Pa, USA, 5th edition, 1899, Edited by W. S. Hall assisted by P. L. Holland and E. P. Carlton.) Epilepsy Research and Treatment Vol 2012, Article ID 176157, 12 pages 

 

Function


Memory: Hippocampal neurons play a critical role in the formation of new memories and in the detection of new surroundings, occurrences and stimuli. It is involved in declarative memory; that is memories that can be stated verbally such as facts and figures. However, studies have shown that damage to the hippocampus does not affect a person's ability to learn a new skill such as playing a musical instrument or solving certain types of puzzles.4 

 

Spatial navigation and spatial memory: There are several navigational cell types within the brain that are either present in the hippocampus itself or strongly connected to it.  One such example are speed cells present in the medial entorhinal cortex.5 Together these cells form a network that serves as spatial memory. When the hippocampus is dysfunctional, orientation is affected; people may have difficulty in remembering how they arrived at a location and how to proceed further.6 Getting lost is a common symptom associated with  amnesia. Studies with animals have shown that an intact hippocampus is required for initial learning and long-term retention of some spatial memory tasks, in particular ones that require finding the way to a hidden goal.     

 

Behavioral inhibition: Damage to the hippocampus causes hyperactivity and affects the ability to inhibit previously learned responses.   

 

Anxiety and Depression: The involvement of the hippocampus in mood disorders is suggested by magnetic resonance imaging (MRI) studies demonstrating a small reduction in hippocampal volume in depressed patients.

 

Stress Regulation: A less well known function of the hippocampus is its role as a negative feedback regulator of the Hypothalamic Pituitary Axis (HPA). The high concentration of adrenal steroid receptors in the hippocampus and the hippocampal projections to the hypothalamus provide an indirect link between the hippocampus and regulation of the stress response.8,9

 

 

In vitro Neural Research Applications


In vitro neural cell culture systems (primary cell cultures and cell lines) are one of the most valuable research tools in areas like neuropathology, drug screening studies, gene-transfer technology, and three dimensional (3D) cell culture models. Lonza primary cells and media have been used by different research groups for a better understanding of these applications.

 

Neuropathology


Schizophrenia: The hippocampus is crucial for normal brain function, especially for the encoding and retrieval of multimodal sensory information. Neuropsychiatric disorders such as temporal lobe epilepsy, amnesia, and the dementias are associated with structural and functional abnormalities of specific hippocampal neurons. Various studies have used neuronal cells and media from Lonza in order to understand the role of hippocampus in the pathophysiology of schizophrenia.10 In contrast to neurodegenerative disorders, total hippocampal cell number is not markedly decreased in schizophrenia. However, the expression of several genes, including those related to the GABAergic system, neurodevelopment, and synaptic function, is decreased in schizophrenia. Taken together, recent studies of hippocampal cell number, protein expression, and gene regulation point towards an abnormality of hippocampal architecture in schizophrenia.11 

 

Epilepsy: The hippocampus is the most widely studied brain region in both human and experimental epilepsy. Sclerosis of the hippocampus in epilepsy was first noted in 1825.12,13 In the modern era of epilepsy surgical programs for the management of drug-resistant epilepsy, hippocampal sclerosis (HS) is one of the common pathologies.14 

 

Alzheimer’s Disease: It is believed that in Alzheimer's disease (AD) some areas of the brain are particularly vulnerable to specific degenerative processes and that they could exhibit neuronal dysfunction in the earliest stage of the disease. The implications of the hippocampus in memory processes are very well known and it is likely that the hippocampus would be among the first areas of the brain affected by the pathogenic mechanisms occurring in AD. (Figure 3) A study demonstrated and confirmed a significant neuronal loss of hippocampus in AD, as compared to an age-matched control group.15 Additionally, it seems that this decrease of hippocampal neuronal density was more prominent at the CA1 and CA3 hippocampal areas. This could have important implications in the design of therapeutic and investigative strategies of AD. Rat Hippocampal Neurons and Primary Neuron Growth Medium (PNGM™) from Lonza have been used in order to study the significant degeneration of hippocampal neurons in AD.16

 

Image of MRI scans showing hippocampal atrophy of both sides in an-85-year-old female with advanced AD 
 Figure 3.  MRI scans showing hippocampal atrophy of both sides in an-85-year-old female with advanced AD.  Source: Ann Indian Acad Neurol. 2012 Oct-Dec; 15(4): 239–246.doi:  10.4103/0972-2327.104323

 

Three Dimensional (3D) Models


Two-dimensional substrates have a limitation in the replication of 3D cellular environments and the absence of 3D factors in cellular developments could limit the biological outcome in the culture. Moreover, cells cultured on the 2D substrate have to adapt to rigid and flat surfaces while natural in vivo environments have large influences from microenvironments derived from extracellular matrix (ECM) and cell-cell interactions.17 

 

Biofidelic 3D culture:  The purpose of this model is to provide an in vivo-like environment to investigate the behavior of brain cellular systems outside of the body while maintaining the highest possible relevance to the system’s natural environment.18 The model consists of cells that are freshly isolated from rat brain tissue. A few studies have used E18 cells from Lonza cultured in PNGM™  to study the 3D models. The model not only kept the cells alive and functional but it also demonstrated the fundamental biological/electrical/anatomical features of a developing brain at a multicellular systems level over a long period of time.19,20 Silk biomaterial was chosen for its compatibility with  versatile uses in neurobiological studies.21 

 

RAFT™ 3D cultures: The RAFT™ System creates 3D cell cultures inside a high-density collagen scaffold that more closely mimics the natural environment of cells. For example, it is possible to develop and study blood-brain barrier models using the RAFT system. The blood-brain barrier prevents the entry of most large hydrophilic molecules and many potentially harmful toxins from the blood into the brain. On the other hand it also prevents the entry of many therapeutic agents into the brain. Considerable efforts are made to develop therapeutics that can cross the blood-brain-barrier and these efforts can be supported by high-value 3D in vitro blood-brain-barrier models.22 Lonza continues to develop and support the RAFT™ System with additional optimized protocols that allow for applying standard histological, biochemical and imaging techniques to 3D cultures (Figure 4).

 

 

blood brain barrier model in raft

Figure 4. Blood-Brain Barrier Model in RAFT™Cultures. Figure A shows primary human astrocytes in RAFT™ Cultures.  Figure B shows co-culture of astrocytes with brain endothelial cells hCMEC/D3 and transport of glucose-coated gold nanoparticles in primary human astrocytes and/or brain endothelial cells (hCMEC/D3) . Data courtesy of Gromnicova et al (2013) PlosOne 8(12)

 

Gene Transfer Technology

Efficient gene transfer is an important tool for studying the biology of neuronal function. The recently developed Nucleofector technology, based on electroporation in a celltype–specific solution, enables direct delivery of DNA and small interfering (si) RNA oligonucleotides into the cell nucleus.23 This strategy results in reproducible, rapid, and efficient transfection of a broad range of cells, including primary neurons. Lonza has developed protocols for successful transfection of post-mitotic neurons. Primary hippocampal neurons were transfected with pmaxGFP® Vector (Lonza), or double transfected with pSyn-GFP (eGFP under the control of the neuron-specifi c synapsin promoter, and pDsRed monomer-C1 RFP (CMV promoter, BD Pharmingen) with 30-50% efficiency and normal neural development. Lonza Nucleofection Kits for Mouse and Rat Hippocampal cells have been used and reported by various other groups.24     

 

Cancer Therapy Neurotoxicity 

The overall aim of current treatments for cancer (such as radiotherapy and chemotherapy) is to prevent aberrant cell division of cell populations associated with malignancy.25 There is accumulating clinical evidence that chemotherapeutic agents induce neurological side effects, including memory deficits and mood disorders, in cancer patients who have undergone chemotherapeutic treatments. Chemotherapy-induced neurodegeneration & hippocampal dysfunctions are measured by in vivo and in vitro approaches. These investigations are helpful in determining how best to further explore the causal mechanisms of chemotherapy-induced neurological side effects and in providing direction for the future development of novel optimized chemotherapeutic agents.26

 

Drug Screening

In recent years the need of efficient in vitro  neuropharmacological and neurotoxicological testing  is increasing, as there are new directives to restrict animal use for laboratory tests.27 New experimental strategies based on alternative methods, in which the use of time, materials, and animals is reduced and refined or animal use is completely replaced, are required. Arrays of releasable polystyrene micro-rafts to generate thousands of uniform, mobile neuron mini-cultures have been fabricated. These mini-cultures sustain synaptically-active neurons which can be easily transferred, thus increasing screening throughput by >30-fold. Compared to conventional methods, micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. A study validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics.28 This affordable high-throughput approach has the potential to transform drug discovery in neuroscience.

 

 

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References


1.      Martin, JH. Lymbic system and cerebral circuits for emotions, learning, and memory. Neuroanatomy: text and atlas (2003); McGraw-Hill Companies. p. 382

2.      Amaral D and Lavenex P. Hippocampal Neuroanatomy. The Hippocampus Book. (2006); Oxford University Press.

3.      Moser MB, Moser EI. "Functional differentiation in the hippocampus". Hippocampus (1998); 8 (6): 608–619

4.      Diana RA, Yonelinas AP and Ranganath C. Imaging recollection and familiarity in the medial temporal lobe: a three-component model. Trends in Cognitive Sciences (2007); 11 (9): 379–386

5.      Chiu YC, Algase D, Whall A, Liang J, Liu HC, Lin KN and Wang PN. Getting lost: directed attention and executive functions in early Alzheimer's disease patients. Dementia and Geriatric Cognitive Disorders (2004); 17 (3): 174–180

6.      Solstad T, Boccara CN, Kropff E, Moser MB and Moser EI. Representation of geometric borders in the entorhinal cortex. Science (2008); 322 (5909): 1865–1868

7.      Campbell S and MacQueen G. The role of the hippocampus in the pathophysiology of major depression. Journal of Psychiatry Neuroscience (2004); 29: 417–426

8.       deKloet ER, Vreugdenhil E, Oitzl MS and Joëls M. Brain corticosteroid receptor balance in health and disease. Endocrinology Reviews (1998); 19: 269–301 

9.      Ulrich-Lai YM and Herman JP. Neural regulation of endocrine and autonomic stress responses. Nature Reviews Neuroscience (2009); 10: 397–409

10.  Monya B. Neurons from reprogrammed cells. Nature Methods (2011); 8: 905–909

11.  Heckers S and C. Konradi C. Hippocampal neurons in Schizophrenia. Journal of Neural Transmission (2002); 109(0): 891–905

12.  Sommer W. Erkrankungdes Ammonshornesalsaetiologisches Moment der Epilepsie. ArchPsychiatrNervenkr (1880); 361–375

13.  Thom M. Hippocampal sclerosis: progress since Sommer. Brain Pathology (2009); 19: 565–572 

14.  Maria T, Review: Hippocampal Sclerosis in epilepsy: a neuropathology review. Journal of Neuropathology Applied Neurobiology. (2014);  40(5): 520–543

15.  Padurariu M, Ciobica A, Mavroudis I, Fotiou D, Baloyannis S. Hippocampal neuronal loss in CA1 and CA3 areas of Alzheimer’s disease patients.  Psychiatria Danubina (2012); 24(2):  152-158

16.  Jenny K. Method development for siRNA silencing in primary hippocampus culture by means of Cellaxess electroporation. Molecular Biotechnology Programme, Uppsala University School of Engineering (2009).

17.  Sang JY, Jongmin K, Chang-Soo L, and Yoonkey N. Simple and novel three dimensional neuronal cell culture using a mesh scaffold. Experimental  Neurobiology (2011); 20(2): 110–115

18.  Ren M, CDE, Tang-Schomer MD and  Özkucur N. A biofidelic 3D culture model to study the development of brain cellular systems. Scientific Reports (2016); 6: 2495

19.  Narayanan, NS, Cavanagh, JF, Frank, MJ. & Laubach M. Common medial frontal mechanisms of adaptive control in humans and rodents. Nature Neuroscience (2013); 16: 1888–1895

20.  Sauvageot CM. & Stiles CD. Molecular mechanisms controlling cortical gliogenesis. Current Opinion in Neurobiology (2002); 12: 244–249

21.  Tang-Schomer MD. Et al. Bioengineered functional brain-like cortical tissue. Proceedings of National Academy of Sciences (2014); 111; 13811–13816

22.  http://www.lonza.com/products-services/bio-research/primary-cells/raft-3d-cell-culture-system/raft-3d-kits-applications.aspx

23.  Galina D, Martin H, Corinna T, Mika OR, Markus D, Gregor S and Michael Nix  AD. Rapid and efficient electroporation-based gene transfer into primary dissociated neurons. Journal of Neuroscience Methods (2003); 130: 65-73

24.  Manuel Z, Sabine T, Markus Z and Fabian T. High Throughput Nucleofection® of Primary Rat Hippocampal Neurons. (2009); Lonza Whitepaper.

25.  Pereira DG, Hollywood R, Bevilaqua MC, da Luz AC, Hindges R, Nardi AE and Thuret S. Consequences of cancer treatments on adult hippocampal neurogenesis: implications for cognitive function and depressive symptoms. Neuro Oncology (2014); 16(4): 476-92

26.  Miyoung Y and Changjong Moon. Neurotoxicity of cancer chemotherapy. Neural Regenerative Research (2013); 8(17): 1606–1614

27.  Johnstone AF, Gross GW, Weiss DG, Schroeder OH, Gramowski A and Shafer TJ. Microelectrode arrays: a physiologically based neurotoxicity testing platform for the 21st century. Neurotoxicology (2010); 31(4): 331-350

28.  Mark N, Raluca D, Angela MM, Yuli W, Benjamin D. Philpot Nancy LA, and Anne MT. Transferable neuronal mini-cultures to accelerate screening in primary and induced pluripotent stem cell-derived neurons. Science Reports (2015); 5: 8353