Characterization of PF-6142, a Novel, Non-Catecholamine Dopamine Receptor D1 Agonist, in Murine and Nonhuman Primate Models of Dopaminergic Activation.

Selective activation of dopamine D1 receptors remains a promising pro-cognitive therapeutic strategy awaiting robust clinical investigation. PF-6142 is a key example from a recently disclosed novel series of non-catechol agonists and partial agonists of the dopamine D1/5 receptors (D1R) that exhibit pharmacokinetic (PK) properties suitable for oral delivery. Given their reported potential for functionally biased signaling compared to known catechol-based selective agonists, and the promising rodent PK profile of PF-6142, we utilized relevant in vivo assays in male rodents and male and female non-human primates (NHP) to evaluate the pharmacology of this new series. Studies in rodents showed that PF-6142 increased locomotor activity and prefrontal cortex acetylcholine release, increased time spent in wakefulness, and desynchronized the EEG, like known D1R agonists. D1R selectivity of PF-6142 was supported by lack of effect in D1R knock-out mice and blocked response in the presence of the D1R antagonist SCH-23390. Further, PF-6142 improved performance in rodent models of NMDA receptor antagonist-induced cognitive dysfunction, such as MK-801-disrupted paired-pulse facilitation, and ketamine-disrupted working memory performance in the radial arm maze. Similarly, PF-6142 reversed ketamine-induced deficits in NHP performing the spatial delayed recognition task. Of importance, PF-6142 did not alter the efficacy of risperidone in assays predictive of antipsychotic-like effect in rodents including pre-pulse inhibition and conditioned avoidance responding. These data support the continued development of non-catechol based D1R agonists for the treatment of cognitive impairment associated with brain disorders including schizophrenia.

Evoked potentials as a translatable biomarker to track functional remyelination.

Enhancing remyelination is a key therapeutic strategy for demyelinating diseases such as multiple sclerosis. To achieve this goal, a central challenge is being able to quantitatively and longitudinally track functional remyelination, especially with translatable biomarkers that can be performed in both preclinical models and in the clinic. We developed the methodology to stably measure multi-modal sensory evoked potentials from the skull surface over the course of months in individual mice and applied it to a genetic mouse model of oligodendrocyte ablation and demyelination. We found that auditory and somatosensory evoked potential latencies reliably increased over time during the early phase of the model and recovered spontaneously and almost completely during a later phase. Histological examination supported the interpretation that the evoked potential latency changes dynamically reflect changes in CNS myelination. Specifically, we found reduction of myelination in corresponding brain regions at the time that sensory evoked potentials were maximally impacted. Importantly, we also found that myelination levels recovered when evoked potential latencies recovered. Other changes known to associate with demyelination were also observed at the time of delayed evoked potentials, including the emergence of white matter vacuoles and increased markers for activated microglia and macrophages; these changes also fully reversed by the time that evoked potentials recovered. Our results support the hypothesis that skull-surface recorded evoked potential latencies can dynamically track CNS myelination changes. The methods developed here allow for longitudinally tracking functional myelination changes in vivo in preclinical rodent models with a quantitative biomarker that can also be applied clinically and will facilitate translational development of CNS remyelinating therapies.

Kainic acid-induced recurrent mossy fiber innervation of dentate gyrus inhibitory interneurons: possible anatomical substrate of granule cell hyper-inhibition in chronically epileptic rats.

Kainic acid-induced neuron loss in the hippocampal dentate gyrus may cause epileptogenic hyperexcitability by triggering the formation of recurrent excitatory connections among normally unconnected granule cells. We tested this hypothesis by assessing granule cell excitability repeatedly within the same awake rats at different stages of the synaptic reorganization process initiated by kainate-induced status epilepticus (SE). Granule cells were maximally hyperexcitable to afferent stimulation immediately after SE and became gradually less excitable during the first month post-SE. The chronic epileptic state was characterized by granule cell hyper-inhibition, i.e., abnormally increased paired-pulse suppression and an abnormally high resistance to generating epileptiform discharges in response to afferent stimulation. Focal application of the gamma-aminobutyric acid type A (GABA(A)) receptor antagonist bicuculline methiodide within the dentate gyrus abolished the abnormally increased paired-pulse suppression recorded in chronically hyper-inhibited rats. Combined Timm staining and parvalbumin immunocytochemistry revealed dense innervation of dentate inhibitory interneurons by newly formed, Timm-positive, mossy fiber terminals. Ultrastructural analysis by conventional and postembedding GABA immunocytochemical electron microscopy confirmed that abnormal mossy fiber terminals of the dentate inner molecular layer formed frequent asymmetrical synapses with inhibitory interneurons and with GABA-immunopositive dendrites as well as with GABA-immunonegative dendrites of presumed granule cells. These results in chronically epileptic rats demonstrate that dentate granule cells are maximally hyperexcitable immediately after SE, prior to mossy fiber sprouting, and that synaptic reorganization following kainate-induced injury is temporally associated with GABA(A) receptor-dependent granule cell hyper-inhibition rather than a hypothesized progressive hyperexcitability. The anatomical data provide evidence of a possible anatomical substrate for the chronically hyper-inhibited state.

Hippocampal granule cell activity and c-Fos expression during spontaneous seizures in awake, chronically epileptic, pilocarpine-treated rats: implications for hippocampal epileptogenesis.

The process of postinjury hippocampal epileptogenesis may involve gradually developing dentate granule cell hyperexcitability caused by neuron loss and synaptic reorganization. We tested this hypothesis by repeatedly assessing granule cell excitability after pilocarpine-induced status epilepticus (SE) and monitoring granule cell behavior during 235 spontaneous seizures in awake, chronically implanted rats. During the first week post-SE, granule cells exhibited diminished paired-pulse suppression and decreased seizure discharge thresholds in response to afferent stimulation. Spontaneous seizures often began during the first week after SE, recruited granule cell discharges that followed behavioral seizure onsets, and evoked c-Fos expression in all hippocampal neurons. Paired-pulse suppression and epileptiform discharge thresholds increased gradually after SE, eventually becoming abnormally elevated. In the chronic epileptic state, interictal granule cell hyperinhibition extended to the ictal state; granule cells did not discharge synchronously before any of 191 chronic seizures. Instead, granule cells generated only low-frequency voltage fluctuations (presumed "field excitatory postsynaptic potentials") during 89% of chronic seizures. Granule cell epileptiform discharges were recruited during 11% of spontaneous seizures, but these occurred only at the end of each behavioral seizure. Hippocampal c-Fos after chronic seizures was expressed primarily by inhibitory interneurons. Thus, granule cells became progressively less excitable, rather than hyperexcitable, as mossy fiber sprouting progressed and did not initiate the spontaneous behavioral seizures. These findings raise doubts about dentate granule cells as a source of spontaneous seizures in rats subjected to prolonged SE and suggest that dentate gyrus neuron loss and mossy fiber sprouting are not primary epileptogenic mechanisms in this animal model.

"Dormant basket cell" hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat.

The "dormant basket cell" hypothesis suggests that postinjury hippocampal network hyperexcitability results from the loss of vulnerable neurons that normally excite insult-resistant inhibitory basket cells. We have reexamined the experimental basis of this hypothesis in light of reports that excitatory hilar mossy cells are not consistently vulnerable and inhibitory basket cells are not consistently seizure resistant. Prolonged afferent stimulation that reliably evoked granule cell discharges always produced extensive hilar neuron degeneration and immediate granule cell disinhibition. Conversely, kainic acid-induced status epilepticus in chronically implanted animals produced similarly extensive hilar cell loss and immediate granule cell disinhibition, but only when granule cells discharged continuously during status epilepticus. In both preparations, electron microscopy revealed degeneration of presynaptic terminals forming asymmetrical synapses in the mossy cell target zone, including some terminating on gamma-aminobutyric acid-immunoreactive elements, but no evidence of axosomatic or axoaxonic degeneration in the adjacent granule cell layer. Although parvalbumin immunocytochemistry and in situ hybridization revealed decreased staining, this apparently was due to altered parvalbumin expression rather than basket cell death, because substance P receptor-positive interneurons, some of which contained residual parvalbumin immunoreactivity, survived. These results confirm the inherent vulnerability of dendritically projecting hilar mossy cells and interneurons and the relative resistance of dentate inhibitory basket and chandelier cells that target granule cell somata. The variability of hippocampal cell loss after status epilepticus suggests that altered hippocampal structure and function cannot be assumed to cause the spontaneous seizures that develop in these animals and highlights the importance of confirming hippocampal pathology and pathophysiology in vivo in each case.