Neostigmine Microinjected into Prefrontal Cortex of C57BL/6J (B6) Mice Stimulates Breathing

Abstract

The prefrontal cortex modulates breathing (Neuroscience 123: 17, 2004; J Physiol 591: 6069, 2013) via neurochemical mechanisms that remain to be fully characterized. This study is testing the hypothesis that microinjection of neostigmine bromide, an acetylcholinesterase inhibitor, into the prefrontal cortex of freely behaving mice alters breathing. All procedures involving mice were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Adult male mice (n = 8) were anesthetized and implanted with a 26-gauge guide cannula aimed at the medial prefrontal cortex. One week after recovering from surgery, mice were microinjected (50 nL) with saline (vehicle control) and two concentrations of neostigmine (0.005 and 0.016 nmol/50 nL; 1.5 and 4.8 ng/50 nL). Each mouse received saline and neostigmine in a repeated measures within-subjects design. Respiratory rate (RR) expressed as breaths/min was quantified using whole-body plethysmography. Repeated measures one-way ANOVA showed neostigmine significantly (F(1.9,13.9) = 6.3, p = 0.0116) altered RR. Relative to saline, RR was increased 11.9% by 0.005 nmol and 18.2% by 0.016 nmol neostigmine. Dunnett's post hoc comparisons test revealed that 0.016 nmol significantly (p < 0.05) increased RRwhen compared to saline. Cohen's d = 1.34 for saline vs. 0.016 nmol neostigmine. Histological analyses of microinjection sites confirmed drug delivery into the prelimbic region of the prefrontal cortex. Mean ± S.D. coordinates of the injection sites were 1.8 ± 0.4 mm anterior, 0.3 ± 0.1 mm lateral, and 2.6 ± 0.6 mm ventral relative to bregma (Paxinos and Franklin, 2008). The mechanism by which cholinergic transmission in the prefrontal cortex stimulates breathing remains to be identified. We speculate that the neostigmine-induced increase in RR evoked from the prefrontal cortex may be modulated by projections from the prefrontal cortex to arousal promoting regions of the nucleus pontis oralis (PnO) (J. Neurosci. 29: 871, 2009). Neuroanatomical studies show that the PnO shares reciprocal connections with respiratory neurons in the ponto-medullary brainstem (Lung Cell. Mol. Physiol. 2: L251, 1995). These anatomical data are consistent with functional studies showing that respiratory rate in B6 mice significantly increased in tandem with increases in prefrontal cortex ACh release (Neuroscience 123: 17, 2004). Considered together, the present results support the interpretation that cholinergic neurotransmission in the prefrontal cortex stimulates the drive to breathe. Ongoing studies are further characterizing the site-dependent and concentration-dependent stimulation of breathing by cholinergic transmission in the prefrontal cortex.