Modified climbing fiber/Purkinje cell synaptic connectivity in the cerebellum of the neonatal phencyclidine model of schizophrenia

Environmental perturbations during the first years of life are a major factor in psychiatric diseases. Phencyclidine (PCP), a drug of abuse, has psychomimetic effects, and neonatal subchronic administration of PCP in rodents leads to long-term behavioral changes relevant for schizophrenia. The cerebellum is increasingly recognized for its role in diverse cognitive functions. However, little is known about potential cerebellar changes in models of schizophrenia. Here, we analyzed the characteristics of the cerebellum in the neonatal subchronic PCP model. We found that, while the global cerebellar cytoarchitecture and Purkinje cell spontaneous spiking properties are unchanged, climbing fiber/Purkinje cell synaptic connectivity is increased in juvenile mice. Neonatal subchronic administration of PCP is accompanied by increased cFos expression, a marker of neuronal activity, and transient modification of the neuronal surfaceome in the cerebellum. The largest change observed is the overexpression of Ctgf, a gene previously suggested as a biomarker for schizophrenia. This neonatal increase in Ctgf can be reproduced by increasing neuronal activity in the cerebellum during the second postnatal week using chemogenetics. However, it does not lead to increased climbing fiber/Purkinje cell connectivity in juvenile mice, showing the complexity of PCP action. Overall, our study shows that administration of the drug of abuse PCP during the developmental period of intense cerebellar synaptogenesis and circuit remodeling has long-term and specific effects on Purkinje cell connectivity and warrants the search for this type of synaptic changes in psychiatric diseases.

Phencyclidine (PCP), a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist initially developed for its properties as an anesthetic, became a popular drug of abuse in the 1960s (1, 2). Nowadays, PCP is often mixed with other drugs, in particular marijuana, and a US 2013 report estimated that PCP-related emergency department visits increased more than 400% between 2005 and 2011 (https://www.samhsa.gov/data/sites/default/files/DAWN143/DAWN143/sr143-emergency-phencyclidine-2013.htm). PCP has important psychotomimetic effects, such as alterations of body image, feelings of estrangement and loneliness, and disorganization of thought. Repeated use of PCP induces persistent symptoms found in schizophrenia, including both positive (hallucinations, psychosis…), cognitive and negative (social withdrawal) effects. PCP also produces regressive symptoms in schizophrenic patients. These observations led to the NMDA hypothesis of schizophrenia and the development of animal models using both acute and chronic PCP administration to study the pathophysiology of this disease (3). Because schizophrenia is now considered a developmental disorder, neonatal administration of PCP in rodents has been tested and shown to produce a wide range of behavioral alterations in the adult, including spatial memory deficits (4, 5) and a deficit in social novelty discrimination (6–8). Some studies found defects in prepulse inhibition (PPI) of the startle response, a sensorimotor gating task used both in animal models and in humans as a behavioral marker of psychiatric disorders (4, 9, 10). Interestingly, these PPI deficits last even after withdrawal, which is not the case when PCP is administered in adulthood, suggesting that this aspect of the disease is better modeled by neonatal administration of PCP. Finally, PCP abuse during pregnancy has been associated with neurobehavioral defects (11) and with long-term consequences on social behavior and motor control in children (12), further highlighting the need to understand the consequences of PCP exposure on the development of neuronal networks.In the subchronic neonatal PCP model, the drug is administered three times during the second postnatal week in rodents (Fig. 1A), a period of intense neuronal growth and synaptogenesis. This developmental stage is particularly sensitive to early environmental stressors associated with increased risk of developing schizophrenia in humans (13). Histopathological and genetic studies have revealed that schizophrenia is a synaptopathy. Mutations and expression changes have been found in genes coding for synaptic proteins (14–19) (a meta-analysis is in ref. 20). Postmortem studies of the cortex of patients with schizophrenia have revealed deficits in dendritic arborization, spine densities, and the number of parvalbumin interneurons (21–25). Similar spine and cellular deficits have been reported in the PCP neonatal model (5, 26, 27), as well as impaired function of both excitatory and inhibitory synapses (28–30). It is, however, still unclear whether these common alterations in schizophrenia and the neonatal PCP model are due to the direct inhibition of NMDA function or a complex interaction between perturbations of neuronal activity and genetic factors.Open in a separate windowFig. 1.Neonatal PCP administration does not modify the cytoarchitecture of the cerebellum and the spontaneous activity of PCs. (A) Experimental design. PCP (10 mg/kg) or vehicle is injected subcutaneously in mouse pups at P7, P9, and P11. Morphological and electrophysiological analyses are performed at P30. (B) Parasagittal cerebellar sections from the vermis of P30 mice were immunolabeled with an anti-calbindin antibody to stain PCs in their entirety and reveal cerebellar cytoarchitecture. Quantification of the mean area of cerebellar slices showed no significant differences between the two conditions (mean ± SEM; vehicle: n = 12 animals; PCP: n = 12; P = 0.0962, Student’s t test). Representative images from PCs and their dendritic tree in lobule VI reveal similar morphology in sections from P30 vehicle- and PCP-treated animals. Quantification of the thickness of the molecular layer (ML) in the lobule VI, measured as the length from the beginning of the primary dendrite of PCs in the Purkinje cell layer (PCL) to the upper extremity of the ML, reveals no significant difference between vehicle- and PCP-treated animals (lobule VI is shown here; mean ± SEM; vehicle: n = 14 animals; PCP: n = 16 animals; P = 0.964, unpaired Student’s t test). (Scale bars: Upper, 1,000 µm; Lower, 50 µm.) (C) High-density MEAs were used to record PC spontaneous spiking in acute cerebellar slices from P30 mice. An example of the recorded electrical activity in a cerebellar slice from a vehicle-treated mouse is shown with each pixel representing one channel and units showing high activity in red and low activity in blue. A representative trace of recordings from one channel is shown for each condition. Spike sorting using the SpyKING CIRCUS software allowed for estimation of the mean firing rate and mean interspike interval (ISI) CV and CV2. No significant difference was detected in any of these parameters (mean ± SEM; vehicle: n = 7 animals; PCP, n = 9 animals; unpaired Student’s t test).While initially thought to be a motor-related structure, it is now well established that the cerebellum also plays a role in cognitive processes (31, 32), such as spatial navigation (33), language (34), reward (35), and social cognition (36). In addition, while schizophrenia has been primarily thought of as a disease of the prefrontal cortex or hippocampus, the cerebellum has emerged as a potential actor in this pathology. Schizophrenic patients often present a decreased cerebellar volume (37–39) as well as neurological soft signs, a type of sensorimotor impairment that implicates the cerebellum (40). Interestingly, neurological soft signs have been correlated with a poor outcome and greater negative and cognitive symptoms (41–43). A significant correlation between negative symptoms in schizophrenia and diminished connectivity between the dorsolateral prefrontal cortex and vermal posterior cerebellum was found in a study of whole-brain connectivity using resting-state functional magnetic resonance imaging in schizophrenic patients (44). It is, however, still unknown whether synaptic deficits are present in the cerebellum of schizophrenic patients.NMDA receptors are present in many neurons of the olivocerebellar circuit, such as molecular layer interneurons (45), granule cells (GCs) (46, 47), inferior olivary neurons (IONs) (48), and Purkinje cells (PCs) themselves (49). In the cerebellum, NMDA receptors participate in the control of neuronal survival (50), circuit maturation, and function (51–53), suggesting that neonatal administration of PCP, an NMDA antagonist, could directly impact the development of the olivocerebellar circuit. In this study, we combined morphological, electrophysiological, and molecular approaches to study the olivocerebellar network in the PCP neonatal model. Lasting synaptic changes were detected in juvenile mice, in particular at the climbing fiber (CF)/PC excitatory synapses, which are key for cerebellar computation. PCP was also found to induce a transient misregulation of the expression pattern of genes coding for membrane and secreted proteins in the cerebellum. The largest misregulation was found for connective tissue growth factor (Ctgf), a gene previously documented as a biomarker in schizophrenic patients. Reproducing the transient misregulation of Ctgf during the second postnatal week using chemogenetics was not sufficient to recapitulate the long-term synaptic changes induced by PCP in the olivocerebellar network. Altogether, neonatal subchronic administration of PCP leads to acute changes in gene expression and long-term synaptic connectivity modifications in the olivocerebellar circuit.