Introduction to Cortical Neurons
Basic Cell Functions and Applications in Research

Structure

The cerebral cortex is the brain’s outer layer of neural tissue in humans and other mammals. The cerebral cortex plays a vital role in memory, attention, perception, awareness and consciousness (Figure 1). It’s folded and has a much greater surface area in the confined volume of the skull. The fold or ridge is termed as gyrus and the groove or fissure is termed as sulcus. In humans, more than 2/3 of the cerebral cortex is buried in the sulci.¹

The Neocortex

Most of the cerebral cortex is neocortex. However, phylogenetically there are older areas of cortex termed the allocortex. These more primitive areas are located in the medial temporal lobes and are involved with olfaction (smell) and survival functions such as visceral and emotional reactions. In turn, the allocortex has two components: the paleocortex and archicortex. The paleocortex includes the piriform lobe, specialized for olfaction, and the entorhinal cortex. The archicortex consists of the hippocampus, which is a three-layered cortex dealing with encoding declarative memory and spatial functions.2 The neocortex represents the great majority of the cerebral cortex. It has six layers and contains between 10 and 14 billion neurons. The six layers of cortex are numbered with Roman numerals from superficial to deep (Figure 2). Each cortical layer contains different neuronal shapes, sizes and density as well as different organizations of nerve fibers.3-5

Functionally, the layers of the cerebral cortex can be divided into three parts. The supragranular layers consist of layers I to III. The supragranular layers are the primary origin and termination of intracortical connections, which are either associational (i.e., with other areas of the same hemisphere), or commissural (i.e., connections to the opposite hemisphere, primarily through the corpus callosum). The supragranular portion of the cortex is highly developed in humans and permits communication between one portion of the cortex and other regions. The internal granular layer, layer IV, receives thalamocortical connections, especially from the specific thalamic nuclei. This is most prominent in the primary sensory cortices. The infragranular layers, layers V and VI, primarily connect the cerebral cortex with subcortical regions. These layers are most developed in motor cortical areas. The motor areas have extremely small or non-existent granular layers and are often called "agranular cortex". Layer V gives rise to all of the principal cortical efferent projections to basal ganglia, brain stem and spinal cord. Layer VI, the multiform or fusiform layer, projects primarily to the thalamus.⁶ 

Neuropathology

Cortical diseases fall into two major categories, degenerative and developmental. In degenerative conditions such as Alzheimer’s disease, progressive cell loss leads to functional deficits and cognitive decline.⁷ In developmental disorders however, failures in cellular migration, cortical maturation or synaptic pruning, can lead to dysplasias or learning disorders. Cortex neurons have been found to play a major role in these degenerative and developmental disorders. Other neuropathological conditions are caused due to neuronal loss, brain atrophy and gliosis.

In vitro Applications of Cortex Neural Cells in Research

In vitro cortex cell culture systems (primary cell cultures) are considered to be the most valuable tool for studying new techniques in neurology like Neuronal Plasticity and Neurophotonics. In addition, their role in Down’s syndrome, immunocytochemistry, in-silico models, drug screening studies, gene transfer technology, and advanced cell culture models have been extensively studied. Lonza cells and media have been used by different research groups for a better understanding of these applications.

• Neuronal plasticity: Neuronal plasticity refers to the adaptation of neural function and structure. Cortex neurons have been used in understanding the mechanism of neuronal plasticity. When neurons are lost due to stroke, for example, a group of new cortex neurons will begin to perform the functions of the original group. Clinical examples of this phenomenon include taking control over speech and swallowing.⁸
   
• Neurophotonics: Cortex neurons have been used to study the connectivity and function of neuronal circuits. The growing collection of photonics and optical tools offers exciting new possibilities to assess how signals are integrated in cells, how cells are interconnected to form circuits, and how neuronal activity relates to behavior. Recent advances have been made in use of light-based methods for investigating cortical circuits in motor cortex (Figure 3). Two basic approaches are often taken to decipher how motor cortex relates to movements: (1) stimulate or silence motor cortex and measure the resulting effect on skeletal muscle activity and body movements and (2) record motor cortex activity during motor behavior and assess how this correlates with movement parameters. With new and emerging neurophotonics and optogenetic methods, these two basic types of approaches are being pursued at even higher levels of specificity and spatiotemporal precision.⁹

• Down’s syndrome: Cortex neurons in persons with Down’s syndrome (DS) are found to undergo neurodegeneration resulting in Alzheimer’s disease. Studies have been carried out using cortex neurons in order to understand the neuro-degenerative process. It has been seen that in DS, neurons differentiate normally but undergo apoptosis due to increased levels of reactive oxygen species (ROS) resulting in neuronal death. This defect is thought to contribute to the mental degradation in early life and predisposes sufferers to Alzheimer’s in adults.¹⁰
  
  
• Immunocytochemistry: Neuronal nuclear antigen (NeuN) obtained from fetal nervous system is used as a specific marker for neuropathological evaluation and as a prelude to applications in cerebral dysgeneses.¹¹
  
  
• Drug Screening: Microelectrode array (MEA) technology enables advanced drug screening and “disease-in-a-dish” modeling by measuring the electrical activity of cultured networks of neural or cardiac cells. Recent developments in human stem cell technologies, advancements in genetic models, and regulatory initiatives for drug screening have increased the demand for MEA-based assays. A multiwell MEA platform enhanced by optogenic stimulation would enable selective excitation and inhibition of targeted cell types. The system enables finely graded selective control of light delivery during simultaneous recording of network activity in each well. Using human induced pluripotent stem cell (hiPSC) derived cardiomyocytes and rodent primary neuronal cultures, high channel-count light-based excitation and suppression in several proof-of-concept experimental models have been demonstrated. This technique can be used for applications including cardiac safety screening, neural toxicity assessment, and advanced characterization of complex neuronal diseases like Alzheimer’s disease.¹² Lonza cells isolated from neural cortex of rat have been used by certain groups and have been cited in the publications.
   

• Gene Transfer Technology (Transfection): Efficient gene transfer is an important tool for the study of neuronal function and biology. This has proved difficult and inefficient with traditional transfection strategies, which can also be fairly toxic. Even viral mediated gene transfer, although highly efficient, can be time consuming. The recently developed Nucleofector technology, based on optimized electroporation in a cell type–specific solution, enables direct delivery of DNA, small interfering (si) RNA oligonucleotides into the cell nucleus.13 This strategy results in reproducible, rapid, and efficient transfection of a broad range of cells, including primary neurons. In the example below, Nucleofected rat cortical neurons were manipulated to induce neurite sprouting by targeting a protein that repressed neurite outgrowth (Figure 4). Lonza has developed nucleofection kit for mouse and rat neuronal cells which have been used and reported by various groups.

• In-silico models: Neuronal migration, the process by which neurons migrate from their place of origin to their final position in the brain, is a central process for normal brain development and function. Advances in experimental techniques have revealed much about many of the molecular components involved in this process. The molecular machinery to govern the migration process is yet to be fully understood.¹⁴ The computational model integrates isolated subcomponents of the migration process into a dynamic system-level simulation. Extracellular signaling cues, GABA neurotransmitter, Reelin protein, two intracellular regulators, the Lissencephaly1 (LIS1) and Doublecortin (DCX) proteins and the interplay between these four molecules have been found to be crucial in normal migration.

• 3D Models: The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. A simple and reproducible 3D culture approach for generating a laminated cerebral cortex–like structure, named human cortical spheroids (hCSs), from pluripotent stem cells has been developed. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development.¹⁷ Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events.¹⁸ These 3D cultures should allow a detailed study of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.
  
  
  Co-culture Models:
   
• Cortical-Thalamic Neurofluidic: In vitro neuronal network system provides insights into the interactions between the cortex and the thalamus. Until recently, in vivo studies have provided the necessary tools to understand the thalamo-cortical interactions.¹⁹ However, in vivo studies are often limited by the complexities in simultaneous multiple site recordings and by the influence of other regions of the brain in the cortical-thalamic circuitry. Developing an in vitro dissociated co-culture model with cortical and thalamic cells alone may provide tools to circumvent these issues. In one such model, cortical and thalamic cells in Lonza’s neural basal medium, supplemented with 3% FBS and 1% human serum (HS), were cultured in a dual compartment neurofluidic device with micro-channels connecting the two compartments. Micro-channels provided the necessary access paths for neurites to cross-over to the adjacent compartment. The neurofluidic device was then integrated on a planar microelectrode array (MEA) to facilitate electrophysiological measurements from the co-culture system. Neurite growth through the microchannels connecting the compartments was observed and the structural connectivity between the cell types was verified using selective staining and immunofluorescence.²⁰ Co-culture neural models will help in understanding the connectivity pathways involved in the pathological neurodegenerative conditions and their treatment modalities.
   
• Neuron-Microglia: The presence of reactive glia or neuro-inflammation is described in all neurodegenerative diseases and glial activation (especially microglial activation), may contribute to the neuropathology observed. Current research is focused on the study of potential neuroprotective effects of anti-inflammatory agents in experimental models of neurodegeneration occurring in the presence of reactive glia.^21-23 The microglial content in rodent primary mixed neuron-glia cultures is usually low. As microglial cells are believed to play a critical role in the neurotoxicity induced by reactive glia, the low amount of microglia present makes it difficult to study cell activation and the resulting neurotoxicity. An experimental strategy to overcome the low amount of microglial cells could be the use of co-culture approaches, where variable amounts of BV-2 microglial cells (maintained in DMEM + 5% FBS from Lonza) are added on top of mixed neuron-glia cultures.²⁴ The inhibition of neuro-inflammation has been postulated as a putative target in the treatment of neurodegenerative diseases.
   
   

Therapeutic Applications of Cortex Neural Cells in Research

• Regenerative medicine: Various studies have been carried out in order to screen compounds that can help overcome inhibition of regeneration based on their ability to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. E18 cortical neurons from Lonza were used in one of these studies. The cells were rapidly thawed and resuspended in neural medium with 5% FBS. The study produced four “hit compounds,” which act with nanomolar potency on several different neuronal types and on several distinct substrates relevant to glial inhibition. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions and potentially allows for regeneration of optic nerve axons in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.¹⁵
  
  
• Restorative medicine: Repair of peripheral nerve injuries remains a major challenge in restorative medicine. Effective therapies in conjunction with surgical nerve repair to improve nerve regeneration and functional recovery are being investigated. It has been demonstrated by many studies that Photobiomodulation (PBM) supports this therapy.¹⁶ Rat cortical neurons from Lonza have been widely used to optimize parameters of PBM. The findings indicate that infra-red light with optimized parameters promote accelerated nerve regeneration and improved functional recovery in surgical repaired peripheral nerve injury.

References

1. Kandel, Eric R.; Schwartz, James H.; Jessell, Thomas M. “Principles of Neural Science” (Fourth Edition.) (2000); 324

2. http://www.dartmouth.edu/~rswenson/NeuroSci/chapter11.html

3. Shipp, Stewart. "Structure and function of the cerebral cortex". Current Biology. (2007); 17(12)
 

4. Jones EG. "Viewpoint: the core and matrix of thalamic organization". Neuroscience. (1998); 85 (2): 331–45
 

5. Creutzfeldt, O. 1995. Cortex Cerebri.
 

6. Natasha Warren et.al; Oxford Journals National Institutes of Health. (1999); 627–635
 

7. Dimitri P Agamanolis; Neuropathology- An interactive course for medical students and residents (2011).
 

8. Benjamin A. Suter, Naoki Yamawaki,a Katharine Borges, Xiaojian Li, Taro Kiritani, Bryan M. Hooks, and Gordon M. G. Shepherd; Neurophotonics (2014)
 

9. Busciglio, Jorge; Yankner, Bruce A. Nature; London (1995): 776-779
 

10. Harvey B Sarnat, David Nochlin, Donald E Born; Brain and Development (1998); 20(2): 88-94
 

11. Isaac P. Clements, Daniel C. Millarda, Anthony M. Nicolinia, Amanda J. Preyera , Robert Griera , Andrew Heckerlinga , Richard A. Bluma , Phillip Tylera , K. Melodi McSweeneyb , Yi-Fan Lub , Diana Hallb, James D. Rossa; “Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications” ; Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation (2016).
 

12. Annette Gärtner, Ludovic Collin, Giovanna Lalli ; “Nucleofection of Primary Neurons”; Methods in Enzymology (2006); 406: 374-388
 

13. Yaki Setty, Chih-Chun Chen, Maria Secrier, Nikita Skoblov, Dimitrios Kalamatianos and Stephen Emmott; “How neurons migrate: a dynamic in-silico model for neuronal migration in developing cortex”, BMC Systems Biology(2011); 5: 154
 

14. Jennifer Gordon, Shohreh Amini and Martyn K white , “General Overview of neuronal cell culture”; Methods Mol Biology (2014)
 

15. Lynn C. Usher,Andrea Johnstone,Ali Ertu¨rk, Ying Hu, Dinara Strikis, Ina B. Wanner, Sanne Moorman, Jae-Wook Lee, Jaeki Min, Hyung-Ho Ha, Yuanli Duan, Stanley Hoffman, Jeffrey L. Goldberg, Frank Bradke, Young-Tae Chang, Vance P. Lemmon, and John L. Bixby; “A Chemical Screen Identifies Novel Compounds That Overcome Glial-Mediated Inhibition of Neuronal Regeneration” ; The Journal of Neuroscience (2010); 30(13): 4693–4706
 

16. Juanita J. Anders, Helina Moges, Xingjia Wu, Issac D. Erbele, Stephanie L. Alberico, Edward K. Saidu, Jason T. Smith and Brian A. Pryor; “Invitro and Invivo Optimization of Infrared Laser Treatment for Injured Peripheral Nerves”; Lasers in Surgery and Medicine (2014); 46: 34-45.
 

17. Anca M Paşca, Steven A Sloan, Laura E Clarke,Yuan Tian,Christopher D Makinson, Nina Huber, Chul Hoon Kim, Jin-Young Park,Nancy A O'Rourke, Khoa D Nguyen, Stephen J Smith, John R Huguenard,Daniel H Geschwind, Ben A Barres& Sergiu P Paşca; “Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture”; Nature Methods(2015); 12,671-678.
 

18. Dolmetsch, R. & Geschwind, D.H. “The human brain in a dish: the promise of iPSC-derived neurons”; Cell (2011)145, 831–834.
 

19. Nicolelis MAL. “Computing with thalamocortical ensembles during different behavioural states”; Journal of Physiology-London (2005), 566 (1):37-47.
 

20. Kanagasabapathi TT, Massobrio P, Tedesco M, Martinoia S, Wadman WJ, Decré MMJ. “An experimental approach towards the development of an in vitro cortical-thalamic co-culture model”; Proc. of IEEE Engineering in Medicine and Biology Society (EMBC) (2011), pp 648 – 651.
 

21. Lee H, Kim YO, Kim H, Kim SY, Noh HS, “Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia”; FASEB Journal (2003) 17: 1943-1944.
 

22. Qian L, Xu Z, Zhang W, Wilson B, Hong J-S, Simomenine, “A natural dextrorotatory morphinan analog, is anti-inflammatory and neuroprotective through inhibition of microglial NADPH oxidase”; Journal of Neuroinflamm (2007) 4: 23-36.
 

23. Pan XD, Chen XC, Zhu YG, Zhang J, Huang TW, “Neuroprotective role of tripchlorolide on inflammatory neurotoxicity induced by lipopolysaccharide-activated microglia”; Biochem Pharmacology (2008),76: 362-372.
 

24. Núria Gresa-Arribas , Cristina Viéitez , Guido Dentesano , Joan Serratosa , Joseph Saura and Carme Solà; “Modelling Neuroinflammation In Vitro: a Tool to Test the Potential Neuroprotective Effect of Anti-Inflammatory Agents”; PLoS, (2012) Volume 7, Issue 9.