Effects of influenza A virus infections and interferon-gamma on synapse formation and function in hippocampal neurons in culture

The central nervous system (CNS) can be the target for several infections that include those with RNA viruses. The parenchyma of the CNS is considered an immuneprivileged site since it is protected behind the blood-brain barrier, expresses no or only low levels of major histocompatibility complex (MHC) molecules and contains a paucity of antigenpresenting cells that prime an immune response. Nevertheless, viral infections in the brain can be controlled to be cleared or become persistent. This poses the questions concerning by which mechanisms an RNA virus, which must undergo a continuous replication to exist, can be controlled in the brain, and how the function in neurons and neuronal networks is affected by an infection that has either been cleared or become persistent.

This thesis work deals mainly with the second question. Effects on synaptic formation and function were analyzed in primary hippocampal cultures after infections with the neuroadapted WSN/33 strain of influenza A virus, and after exposure to certain molecules involved in host defence against a virus. The latter involved interferon-gamma (IFN-gamma), which has been implied in the control of certain microbe infections in the nervous system in recent studies. In this respect, I have in particular been interested in determining whether viral infections during restricted periods of development, corresponding to those of peaks of synapse formation, can cause persistent changes in synaptic activities.

The viral WSN/33 strain used in this study has previously been shown to selectively infect the hippocampus and the substantia nigra in mice following intracerebral and systemic injections. In my thesis the following observations were made: 1) Infected hippocampal neurons showed reduced voltage-dependent Ca2+ currents in whole-cell patch clamp recordings. 2) By over-expressing the viral nucleoprotein (NP) using the Semliki Forest virus expression system, this protein could be localized to the dendritic spines as visualized by double immunolabeling with anti-NP and anti-alpha-actinin antibodies. The electrophysiological properties of the synapses were not altered in the short-term. After exposure of the hippocampal neurons to the NP coupled to the non-toxic I I amino acid protein transduction domain (PTD) of HIV-TAT, for 5 days during their peak of synapse formation (day 7-12 in culture), a marked reduction in the excitatory postsynaptic currents was seen. 3) The viral infection caused disturbances in synaptic function that persisted even after clearance of the infection in the culture 4) Neuronal networks exposed to INF-gamma during the peak of synapse formation can develop an imbalance between excitatory and inhibitory synaptic activities, which become evident after a latency period.

Altogether, this study shows that synaptic dysfunctions can appear, both as an effect of presence of viral NP that may persist in neurons, and after exposure to IFN-gamma that may be involved in control of the infection. These observations are of interest for an understanding of how viral infections early in life may be associated with functional disturbances in the brain.

List of scientific papers

I. Brask J, Owe-Larsson B, Hill RH, Kristensson K (2001). Changes in calcium currents and GABAergic spontaneous activity in cultured rat hippocampal neurons after a neurotropic influenza A virus infection. Brain Res Bull. 55(3): 421-9.
https://pubmed.ncbi.nlm.nih.gov/11489350

II. Brask J, Chauhan A, Hill RH, Ljunggren HG, Kristensson K (2004). The effects on synaptic activity by influenza A/WSN/33 viral proteins NS1 and NP in hippocampal cultures. [Manuscript]

III. Vikman KS, Owe-Larsson B, Brask J, Kristensson KS, Hill RH (2001). Interferon-gamma-induced changes in synaptic activity and AMPA receptor clustering in hippocampal cultures. Brain Res. 896(1-2): 18-29.
https://pubmed.ncbi.nlm.nih.gov/11277968

IV. Brask J, Kristensson K, Hill RH (2004). Exposure to interferon-gamma during synaptogenesis increases inhibitory activity after a latent period in cultured rat hippocampal neurons. Eur J Neurosci. 19(12): 3193-201.
https://pubmed.ncbi.nlm.nih.gov/15217375

V. Brask J, Kristensson K (2004). Effects of IFN-gamma on influenza A/WSN/33 viral infection in primary cultures of mice hippocampal neurons. [Manuscript]