Adeno-associated virus (AAV) has many advantages such as small size, high safety, and low immunogenicity, making it widely used in the study of nervous system diseases. What key points should be considered for AAV applications in neural tissues?

 

Selection of Tissue-Specific Promoters in the Nervous System

AAV is an important vector for gene therapy and a common tool for labeling neural circuits. Various AAV serotypes combined with tissue-specific promoters exhibit strong tropism for neural cells, making the choice of promoter a crucial step in vector design. Here are a few examples of nervous system-specific promoters:

  • hSyn Neuron-Specific Promoter

The hSyn promoter is derived from the human SYN1 gene, which produces the Synapsin I protein, a neuron-specific protein primarily expressed at the axon terminal, particularly on the membrane of synaptic vesicles, regulating axon genesis and synapse formation.

  • GAD67-Specific Promoter

GAD67 is a specific promoter for GABAergic neurons. GAD1 and GAD2 genes encode two isoforms, GAD65 and GAD67, which produce GABA, an inhibitory neurotransmitter. Therefore, GAD1 and GAD2 promoters can serve as specific promoters for inhibitory neurons.

  • c-fos-Specific Promoter

In neuroscience, the c-fos protein is commonly used as a marker of neuronal activity. c-fos is a transcription factor that regulates the expression of genes in the cell nucleus in response to external stimuli.

  • CaMKIIa Promoter

The CaMKIIa promoter is specific to glutamatergic neurons in the forebrain and is a specific promoter for excitatory neurons in the neocortex and hippocampus. It is regulated by the Ca2+/calmodulin-dependent serine/threonine-specific protein kinase complex.

 

Common Injection Methods for AAV in Neural Tissue

The choice of injection method varies depending on the target site. For systemic injection, tail vein injection is generally used; for cerebrospinal fluid system injection, intraventricular and intrathecal injections are preferred; for specific brain regions or spinal cord locations, stereotactic injection is used. Systemic and cerebrospinal fluid injections are more commonly used in clinical translation, as they are better accepted by patients and facilitate the progression of experiments. After intravenous injection, the virus spreads throughout the body via the bloodstream. Due to the use of specific promoters, the gene carried by the intravenously injected virus is expressed only in specific neural tissues. Intraventricular injection allows cerebrospinal fluid to carry the virus throughout the brain.

and spinal cord. Intrathecal injection involves injecting drugs into the spinal canal through lumbar puncture, allowing the cerebrospinal fluid to distribute the drug. Stereotactic brain or spinal cord injection is precise, requiring a small injection dose, and allows for accurate control of the injection site using a stereotactic apparatus.

Promoter Characteristics
hSyn Derived from SYN1 gene; promotes Synapsin I; expressed in axon terminals.
GAD67 Targets inhibitory neurons; involved in GABA production.
c-fos Marker of neuron activation; regulates gene expression in response to stimuli.
CaMKIIα Specific to forebrain excitatory neurons; regulated by Ca2+/calmodulin kinase.
GFAP Expressed in glial cells; maintains structural integrity of astrocytes.
MBP Important for myelin sheath formation; targets myelinating cells.
NSE Involved in glycolysis; expressed in neurons and neuroendocrine cells.
CNP Expressed in oligodendrocytes; important for myelin sheath maintenance.
MECP2 Methyl-CpG binding protein 2; involved in gene regulation and neurological function, specific to neurons.
TUBA1A Tubulin Alpha 1a promoter; specific to neurons, important for cytoskeleton structure.
hVGAT Human Vesicular GABA Transporter; specific to inhibitory neurons involved in GABA transport.
mDLx Mouse Distal-less Homeobox; used to target specific subsets of GABAergic neurons.
fPV Specific to fast-spiking parvalbumin-expressing interneurons, a subset of GABAergic neurons.
fNPV Targets GABAergic neurons excluding parvalbumin-expressing subsets.
fSST Specific to somatostatin-expressing interneurons, another subset of GABAergic neurons.
fEV Fast excitatory viral promoter; targets excitatory neurons broadly.

 

Visualization of Neurons

In visualizing neurons, AAV plays an important role. The nervous system is unique, with neurons having cell bodies and very long projections that form a complex and extensive information network. To analyze neural network functions, neuronal tracing experiments are needed to explore the connections between neurons. rAAV2-retro, which allows for retrograde axonal transport, is commonly used. When injected into the nervous system, the gene carried by the virus is retrogradely transported to the cell body and expressed in the neurons. The level of transgene expression achieved by rAAV2-retro through retrograde transport is sufficient for neural circuit function tracing and targeted manipulation of the neuronal genome.

 

AAV can also undergo anterograde transport. In 2017, Professor Li Zhang’s team published an article in *Neuron* demonstrating for the first time that AAV can anterogradely transsynaptically transmit across single synapses. High-titer AAV2/1-hSyn-Cre can effectively and specifically mark neurons and their outputs across single synapses. Through neuronal tracing, researchers gain a more intuitive understanding of the direction and destination of AAV, providing a visual tool for studying the functional and structural applications of AAV in the brain.

Figure 1 Directed Evolution of rAAV2-retro
Figure 1: Directed Evolution of rAAV2-retro

Studying Neural Circuits

As research on single, dual, and triple axonal projections and retrograde tracing progresses, neural circuit tracing studies are increasing. For example, labeling ChAT, NE, and dopamine neurons. Visualizing brain connectivity maps reveals highly complex networks. As neuroscience progresses, researchers aim to study not only static images but also dynamic real-time images. This has led to the development of AAV calcium imaging, which involves delivering calcium imaging components to specific brain regions of mice. This allows researchers to observe specific neurons in response to stimuli over time, providing real-time monitoring of neuronal activity.

The basic principle of neuronal calcium imaging is to use special fluorescent dyes or calcium ion indicators to show changes in calcium ion concentration within neurons through fluorescence intensity. Rapid imaging of calcium ion changes reflects neuronal activity.

GCaMPs are the result of the fusion of a single green fluorescent protein (GFP), calmodulin (CaM), and a myosin light chain kinase fragment (M13). Calmodulin is inactive without Ca2+, but in the presence of Ca2+, the M13 from actin binds to CaM, causing EGFP to change conformation and become fluorescent. This process is reversible, allowing real-time monitoring of neuronal activity. Due to its high sensitivity and signal-to-noise ratio, GCaMPs are increasingly used in in vivo calcium imaging studies. Researchers inject AAV carrying CaMP components into different brain regions of mice, providing stimuli such as pain, smell, or itch to study neuronal firing activity. Compared to electrophysiology, which can only study a small number of neurons, calcium imaging allows researchers to study the electrical activity of large numbers of neurons.

Figure 2 Neuronal Calcium Imaging
Figure 2: Neuronal Calcium Imaging (Jihae Oh et al., Korean J Physiol Pharmacol, 2019)

Intervening in Neuronal Activity

Current brain science research not only aims to observe neuronal activity but also to intervene in it to restore certain neuronal functions. This led to the birth of optogenetics. Initially, lentiviral vectors were used for optogenetics, but AAVs have higher transfection efficiency and strength, and are easier to obtain and store. Therefore, AAVs have gradually become the preferred method for delivering optogenetic materials. The experiment is relatively simple: an electrode is implanted in the mouse brain, AAV delivers the optogenetic protein to neurons, and then light is used to trigger neuronal electrical activity, thereby manipulating mouse behavior.

Optogenetics is a technique that combines optics and genetics. Light-sensitive proteins respond selectively to the passage of cations or anions (such as Cl-, Na+, H+, K+) when stimulated by light of specific wavelengths, causing changes in membrane potential and selectively exciting or inhibiting cells. The application of optogenetic technology consists of three main components: light-sensitive proteins, delivery of light-sensitive protein expression, and delivery of light.

Figure 3 Subcellular Targets of Optogenetics
Figure 3: Subcellular Targets of Optogenetics (Benjamin R. Rost et al., Neuron, 2017)

Chemical Genetics

Chemical genetics, similar to optogenetics, emerged earlier. Since 1991, when Stader designed a mutated β2-adrenergic receptor, and in 2007, when Armbruster and Roth developed DREADDs (designer receptors exclusively activated by designer drugs), chemical genetics has been widely used in neural research. The basic principle is to use small chemical molecules to control the excitation or inhibition of target cells. The experimental process involves selecting the appropriate chemical genetic receptor, choosing the right viral vector (such as AAV), administering the small chemical molecule, and then recording or observing changes in animal behavior. Compared to optogenetic receptors, chemical genetic receptors include the excitatory hM3Dq and the inhibitory hM4Di. After constructing the virus and injecting it into the brain, and waiting for peak AAV expression (about 3 weeks), a small molecule substance is used to activate the virus for behavioral observation.

In summary, the three techniques for visualizing neurons each have their advantages and disadvantages. Calcium imaging detects cell activation and is used to identify functionally related brain regions and neurons. Optogenetics and chemical genetics can manipulate neuronal and neural circuit physiological activities for functional validation. Optogenetics offers high temporal precision and bidirectional regulation but is invasive. Chemical genetics is non-invasive and low toxicity but has low temporal precision and can only regulate in one direction. Researchers can design experiments based on their needs.

 

References:

  • Gowanlock et al., “A designer AAV variant permits efficient retrograde access to projection neurons,” Neuron, 2016.
  • Zingg et al., “AAV-mediated Anterograde Transsynaptic Tagging,” Neuron, 2017.
  • Rost et al., “Optogenetic Tools for Subcellular Applications in Neuroscience,” Neuron, 2017.
  • Oh et al., “Imaging and analysis of genetically encoded calcium indicators,” Korean J Physiol Pharmacol, 2019.
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