Dendritic regression of striatal spiny projection neurons (SPNs) is a pathological hallmark of Parkinson’s disease (PD). Here we investigate how chronic dopamine denervation and dopamine replacement with L-DOPA affect the morphology and physiology of direct pathway SPNs (dSPNS) in the rat striatum. We used a lentiviral vector optimized for retrograde labeling (FuG-B-GFP) to identify dSPNs in rats with 6-hydroxydopamine (6-OHDA) lesions. Changes in morphology and physiology of dSPNs were assessed through a combination of patch-clamp recordings and two photon microscopy. The 6-OHDA lesion caused a significant reduction in dSPN dendritic complexity. Following chronic L-DOPA treatment, dSPNs segregated into two equal-sized clusters. One group (here called “cluster-1”), showed sustained dendritic atrophy and a partially normalized electrophysiological phenotype. The other one (“cluster-2”) exhibited dendritic regrowth and a strong reduction of intrinsic excitability. Interestingly, FosB/∆FosB induction by L-DOPA treatment occurred preferentially in cluster-2 dSPNs. Our study demonstrates the feasibility of retrograde FuG-B-GFP labeling to study dSPNs in the rat and reveals, for the first time, that a subgroup of dSPNs shows dendritic sprouting in response to chronic L-DOPA treatment. Investigating the mechanisms and significance of this response will greatly improve our understanding of the adaptations induced by dopamine replacement therapy in PD.
Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber–forming (PF-forming) cerebellar granule cells (Cgcs). Postnatal Pkn1–/– animals showed a defective PF–Purkinje cell (PF-PC) synapse formation. In vitro, Pkn1–/– Cgcs exhibited deregulated axonal outgrowth, elevated AKT phosphorylation, and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly, Pkn1–/– Cgcs had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels, and reduced density of presynaptic specifications in Pkn1–/– Cgcs. In line with our in vitro data, Pkn1–/– mice showed AKT hyperactivation, elevated NeuroD2 levels, and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgcs and subsequently the correct PF-PC synapse formation.
Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.
CaV1.4 L-type calcium channels are predominantly expressed in photoreceptor terminals playing a crucial role for synaptic transmission and, consequently, for vision. Human mutations in the encoding gene are associated with congenital stationary night blindness type-2. Besides rod-driven scotopic vision also cone-driven photopic responses are severely affected in patients. The present study therefore examined functional and morphological changes in cones and cone-related pathways in mice carrying the CaV1.4 gain-of function mutation I756T (CaV1.4-IT) using multielectrode array, patch-clamp and immunohistochemical analyses. CaV1.4-IT ganglion cell responses to photopic stimuli were seen only in a small fraction of cells indicative of a major impairment in the cone pathway. Though cone photoreceptors underwent morphological rearrangements, they retained their ability to release glutamate. Our functional data suggested a postsynaptic cone bipolar cell defect, supported by the fact that the majority of cone bipolar cells showed sprouting, while horizontal cells maintained contacts with cones and cone-to-horizontal cell input was preserved. Furthermore a reduction of basal Ca2+ influx by a calcium channel blocker was not sufficient to rescue synaptic transmission deficits caused by the CaV1.4-IT mutation. Long term treatments with low-dose Ca2+ channel blockers might however be beneficial reducing Ca2+ toxicity without major effects on ganglion cells responses.
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