Maternal Inflammation Increases the Number of Basal Forebrain Cholinergic Neurons and Alters Neuregulin-1 Expression in the Hippocampus

Background: Alterations within the cholinergic basal forebrain are implicated in autistic spectrum disorders and schizophrenia. In the rodent basal forebrain the differentiation of cholinergic neurons from bipotential precursors occurs during midgestation. These neurons then project axons to the hippocampus where they form synapses perinatally. Several genetic susceptibility factors for both autism and schizophrenia are associated with synaptogenesis and synapse signaling. An alteration in cholinergic development could affect the transcription and/or protein expression of key synaptic molecules in the hippocampus. Maternal inflammation is recognized as contributing to neurodevelopmental disorders. Moreover, our lab has found that a cocktail of factors from inflamed brain microglia produces excess cholinergic neuron development in vitro.

Objectives: Experiments were designed to determine whether prenatal maternal infection alters cholinergic differentiation in the fetal basal forebrain and whether there is an accompanying change in expression of specific synapse-related molecules in the perinatal hippocampus. Molecules examined were Shank3, Neuregulin-1 (NRG1), α7 nicotinic acetylcholine receptor (α7nAChR), NMDA receptor (NMDAR), the sodium-potassium chloride transporter (NKCC1) and the potassium chloride co-transporter (KCC2).

Methods: The viral mimic polyinosinic-polycytidylic acid was administered to pregnant mice at embryonic day 12.5 (E12.5) to produce maternal inflammation. The activity of choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine biosynthesis was measured in basal forebrain. Transgenic mice expressing enhanced green fluorescent protein (EGFP) along with ChAT and under control of the ChAT promoter were used to assess the number of cholinergic neurons in the basal forebrain.  GFP+ cells were randomly and systematically sampled with 3D counting frames using Stereo Investigator Stereology software (MBF Bioscience). Real-time polymerase chain reaction (RT-PCR) quantified the relative mRNA transcript levels of synaptic molecules and western blot with antibody to NRG1 detected protein and revealed molecular weights of isoforms.

Results: Following maternal poly (I:C) administration, a modest but significant increase in ChAT activity is seen at embryonic day 16.5 (E16.5), E18.5 and postnatal day 1 (P1) in the basal forebrain. Meanwhile the number of ChAT+ cells in the basal forebrain increases at E16.5 and more than doubles at P1. In the P1 hippocampus of offspring of dams injected with poly (I:C) message levels of Shank-3, α7nAChR, NMDAR, NKCC1 and KCC2 do not differ from controls  While there was no difference in total message level of NRG1 between pups from poly (I:C)-treated and control dams, RT-PCR revealed two distinct transcripts. One was predominant in control samples and the other predominant in offspring of treated moms. Western blot with αNRG1 revealed the existence of several neuregulin-1 isoforms. All control samples contained a strong band between 75 and 100 kDa that was greatly reduced in all poly (I:C) samples. This band corresponds to reported molecular weights for type III neuregulin-1.

Conclusions: Maternal immune activation results in increased ChAT activity and greater numbers of cholinergic neurons in the fetal basal forebrain. In addition there is an associated reduction in protein expression of type III neuregulin-1 in the hippocampus of P1 offspring of dams injected with poly (I:C).