Highlights d Glutamatergic neurons of the PPN and CnF show distinct biophysical differences d CnF neurons produce bilateral muscle activation and involuntary locomotor activity d PPN neurons elicit long-lasting muscle activity and decreased overall motor output

Number of words: Abstract: 147; Main Text: 4669. 22 23 Acknowledgments: 24 This research was supported by an NIH grant NS100824 (J.M.S.), a NJ-DOH grant 25 CSCR20IRG008 (J.M.S.), a NARSAD Young Investigator Award (J.M.S.), the Hungarian 26 National Brain Research Program (B.P.), the OTKA Bridging Fund of the University of 27 Debrecen (B.P.) and Rutgers University. The authors are grateful to Dr. Péter Szücs 28 Abstract 42The mesencephalic locomotor region (MLR) serves as an interface between higher-order 43 motor systems and lower motor neurons. The excitatory module of the MLR is composed 44 of the pedunculopontine nucleus (PPN) and the cuneiform nucleus (CnF), and their 45 activation has been proposed to elicit different modalities of movement, but how the 46 differences in connectivity and physiological properties explain their contributions to motor 47 activity is not known. Here we report that CnF glutamatergic neurons are 48 electrophysiologically homogeneous and have short-range axonal projections, whereas 49 PPN glutamatergic neurons are heterogeneous and maintain long-range connections, 50 most notably with the basal ganglia. Optogenetic activation of CnF neurons produced 51 fast-onset, involuntary motor activity mediated by short-lasting muscle activation. In 52 contrast, activation of PPN neurons produced long-lasting increases in muscle tone that 53 reduced motor activity and disrupted gait. Our results thus reveal a differential contribution 54 to motor behavior by the structures that compose the MLR. 55 56 58 composed of the pedunculopontine nucleus (PPN) and the cuneiform nucleus (CnF) 59 which has been typically described as an output station of forebrain systems reaching 60 lower motor circuits 1-3 . Early experiments defined the MLR by demonstrating that 61 electrical stimulation of this region induced a locomotor response in decorticated cats 4,5 . 62More recently, optogenetic experiments revealed that the motor function of the MLR is 63 dependent on excitatory transmission from glutamatergic neurons 6,7 , which is the most 64 3 prominent cell type in the MLR 8,9 . In the last two decades, a role for these circuits in gait 65 and posture has been proposed 10-13 . Moreover, degeneration of neurons in the MLR may 66 underlie some of the motor impairments in Parkinson's disease [14][15][16][17][18][19] . Deep brain 67 stimulation into the PPN has been shown to produce some improvements in abnormal 68 gait based on the idea that the output from the MLR is excitatory 20-23 . However, it is not 69 fully understood how excitatory MLR neurons contribute to motor behavior and how motor 70 functions are associated with different neuronal types in the MLR. 71 72 The PPN, the largest component of the MLR, is highly heterogeneous. It is composed of 73 three neurotransmitter-defined cell types: cholinergic, GABAergic and glutamatergic 74 neurons. Among PPN glutamatergic neurons, a high degree of variability has been 75 reported in their neurochemical composition 8 , connectivity 24 and firing properties 2...

The pedunculopontine nucleus (PPN) is a part of the reticular activating system which is composed of cholinergic, glutamatergic and GABAergic neurons. Early electrophysiological studies characterized and grouped PPN neurons based on certain functional properties (i.e., the presence or absence of the A-current, spike latency, and low threshold spikes). Although other electrophysiological characteristics of these neurons were also described (as high threshold membrane potential oscillations, great differences in spontaneous firing rate and the presence or absence of the M-current), systematic assessment of these properties and correlation of them with morphological markers are still missing. In this work, we conducted electrophysiological experiments on brain slices of genetically identified cholinergic neurons in the PPN. Electrophysiological properties were compared with rostrocaudal location of the neuronal soma and selected morphometric features obtained with post hoc reconstruction. We found that functional subgroups had different proportions in the rostral and caudal subregions of the nucleus. Neurons with A-current can be divided to early-firing and late-firing neurons, where the latter type was found exclusively in the caudal subregion. Similar to this, different parameters of high threshold membrane potential oscillations also showed characteristic rostrocaudal distribution. Furthermore, based on our data, we propose that high threshold oscillations rather emerge from neuronal somata and not from the proximal dendrites. In summary, we demonstrated the existence and spatial distribution of functional subgroups of genetically identified PPN cholinergic neurons, which are in accordance with differences found in projection and in vivo functional findings of the subregions. Being aware of functional differences of PPN subregions will help the design and analysis of experiments using genetically encoded opto- and chemogenetic markers for in vivo experiments. Electronic supplementary material The online version of this article (10.1007/s00018-019-03025-4) contains supplementary material, which is available to authorized users.

The pedunculopontine nucleus (PPN), a structure known as a cholinergic member of the reticular activating system (RAS), is source and target of cholinergic neuromodulation and contributes to the regulation of the sleep–wakefulness cycle. The M-current is a voltage-gated potassium current modulated mainly by cholinergic signaling. KCNQ subunits ensemble into ion channels responsible for the M-current. In the central nervous system, KCNQ4 expression is restricted to certain brainstem structures such as the RAS nuclei. Here, we investigated the presence and functional significance of KCNQ4 in the PPN by behavioral studies and the gene and protein expressions and slice electrophysiology using a mouse model lacking KCNQ4 expression. We found that this mouse has alterations in the adaptation to changes in light–darkness cycles, representing the potential role of KCNQ4 in the regulation of the sleep–wakefulness cycle. As cholinergic neurons from the PPN participate in the regulation of this cycle, we investigated whether the cholinergic PPN might also possess functional KCNQ4 subunits. Although the M-current is an electrophysiological hallmark of cholinergic neurons, only a subpopulation of them had KCNQ4-dependent M-current. Interestingly, the absence of the KCNQ4 subunit altered the expression patterns of the other KCNQ subunits in the PPN. We also determined that, in wild-type animals, the cholinergic inputs of the PPN modulated the M-current, and these in turn can modulate the level of synchronization between neighboring PPN neurons. Taken together, the KCNQ4 subunit is present in a subpopulation of PPN cholinergic neurons, and it may contribute to the regulation of the sleep–wakefulness cycle.

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