Preterm Birth Impedes Structural and Functional Development of Cerebellar Purkinje Cells in the Developing Baboon Cerebellum

Abstract

Human cerebellar development occurs late in gestation and is hindered by preterm birth. The fetal development of Purkinje cells, the primary output cells of the cerebellar cortex, is crucial for the structure and function of the cerebellum. However, morphological and electrophysiological features in Purkinje cells at different gestational ages, and the effects of neonatal intensive care unit (NICU) experience on cerebellar development are unexplored. Utilizing the non-human primate baboon cerebellum, we investigated Purkinje cell development during the last trimester of pregnancy and the effect of NICU experience following premature birth on developmental features of Purkinje cells. Immunostaining and whole-cell patch clamp recordings of Purkinje cells in the baboon cerebellum at different gestational ages revealed that molecular layer width, driven by Purkinje dendrite extension, drastically increased and refinement of action potential waveform properties occurred throughout the last trimester of pregnancy. Preterm birth followed by NICU experience for 2 weeks impeded development of Purkinje cells, including action potential waveform properties, synaptic input, and dendrite extension compared with age-matched controls. In addition, these alterations impact Purkinje cell output, reducing the spontaneous firing frequency in deep cerebellar nucleus (DCN) neurons. Taken together, the primate cerebellum undergoes developmental refinements during late gestation, and NICU experience following extreme preterm birth influences morphological and physiological features in the cerebellum that can lead to functional deficits.

Keywords: NICU; Purkinje cell; baboon; cerebellum; electrophysiology; fetal development; non-human primate; preterm birth.

Figure 1

Morphological development of baboon cerebellum during the last trimester of the pregnancy. (A) Experimental paradigm comparing baboon gestational age (GA) compared to human GA. (B) Cerebellar sections from E-Pre, M-Pre, Term, and NICU animals immunostained to show Purkinje cell bodies, dendrites, and axons expressing calbindin (CB, green). (C) Granule cell nuclei stained with DAPI (red) in the external granule layer (EGL) and internal granule layer (IGL), with CB labeling of Purkinje dendrites in the molecular layer (ML) and Purkinje cell bodies in the Purkinje layer (PL) in E-Pre, M-Pre, Term, and NICU cerebellar sections. (D–G) Summary of EGL width (D), ML width (E), Purkinje cell density (F), and Purkinje cell soma diameter (G) at each gestational age. Data are represented as ± SEM. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 2

Purkinje cell intrinsic properties are altered in the NICU condition. (A) Top, confocal images of Purkinje cells Alexa dye-filled during whole-cell recording. Scale = 20 m. Bottom, representative traces of action potentials (APs) induced by 200 pA current injections from Purkinje cells from E-Pre, M-Pre, Term, and NICU baboon neonates. Inset, expanded view of action potentials in box. (B–D) Summary of AP number (B) in response to current injection from 50 to 200 pA, inter-spike interval (ISI; C), and rheobase (D) from each group. (E) Representative phase plot of an AP (dV/dt), demonstrating the threshold (red arrow), peak (grey line), and amplitude (voltage difference from the threshold to the peak). (F–H) Summary of threshold (F), amplitude (G), and halfwidth (H) of a single AP from each gestational age. Data are represented as ± SEM. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 3

Spontaneous postsynaptic currents (sPSCs) increase in frequency throughout gestation and are reduced after NICU experience. (A) Representative traces of sPSCs recorded from Purkinje cells of E-Pre, M-Pre, Term, and NICU neonates. (B, C) Summary of sPSC amplitude (B) and frequency (C) at each group. Data are represented as ± SEM. * and *** represent p < 0.05, and p < 0.001, respectively.

Figure 4

DCN cell APs are impacted by NICU experience. (A) Spontaneous AP recordings from DCN neurons from E-Pre, M-Pre, and NICU baboon neonates. (B) Summary of spontaneous AP frequency in DCN neurons from each group. (C) Representative traces of action potentials induced by 200 pA current injections from DCN neurons from E-Pre, M-Pre, and NICU animals. (D–I) Summary of DCN neuron AP number (D), inter-spike interval (ISI; E), rheobase (F), threshold (G), amplitude (H), and half-width (I) from each group. Data are represented as ± SEM. *, and ** represent p < 0.05, and p < 0.01, respectively.