We have demonstrated a pathway parallel to generally accepted descriptions of vertebrate brainstem locomotor command circuitry. In addition to projecting directly to the RS system12,19,23-25
as part of the activation system for locomotion, MLR neurons also project to a group of rhombencephalic neurons located laterally in the basal plate. These neurons, identified in this study, are excited by a mixed input that originates in the MLR. Part of this signal comprises fast responses most likely mediated by ionotropic glutamate receptors, but perhaps also by cholinergic nicotinic transmission26
. This early component is followed by a sustained depolarization lasting up to minutes, which is blocked by muscarine receptor antagonist applied to the brainstem in general, or specifically over the neurons. These newly identified neurons project back to RS cells eliciting glutamatergic responses to provide sustained excitatory drive during locomotion (Supplementary Fig 1
). The discovery of this group of neurons explains earlier described excitatory effects of brainstem muscarine receptor activation on vertebrate locomotion15,17
. This discovery also adds a previously unknown component to supraspinal locomotor command circuits with features complementary to known brainstem pathways.
We have anatomically identified bilaterally projecting neurons whose lateral dendritic fields are located precisely within a brainstem location in the rhombencephalon17
, whose response to muscarine excites locomotor command RS neurons. Axons from these muscarinoceptive neurons project into the dendritic field of the RS neurons of the MRRN and paired cell recordings of these neurons and of MRRN RS neurons reveal a monosynaptic response in the RS neurons. The muscarinoceptive cells respond with a sustained Ca2+
signal and oscillating depolarization, both to muscarine, and to microstimulation of the MLR. This excitation in response to muscarine is direct; it is retained after block of action potential-evoked synaptic transmission with TTX in contrast to RS neurons17
. During excitation mediated either by muscarine or electrical stimulation of the MLR, firing activity temporally corresponds to synaptic drive recorded simultaneously in RS neurons. Thus, a group of muscarinoceptive neurons, presumably a subset of brainstem muscarinoceptive neurons, caudal to the MRRN and rostral and lateral to the PRRN, act as a muscarinoceptive relay of sustained excitation from the MLR to the RS system. At low stimulus frequencies of the MLR, these neurons respond with a slower and delayed excitation compared to the response simultaneously recorded in RS neurons. However, during higher frequency MLR stimulation this late excitation corresponds with later and sustained excitation in RS neurons. This sustained component is muscarine receptor-mediated. To complete this parallel circuit, neurons of this muscarinoceptive region project monosynaptic glutamatergic excitation to RS neurons in the ipsilateral and contralateral MRRN.
Thus, transient activation of the MLR provides rapid excitation of the RS system by direct ionotropic glutamatergic and nicotinic receptor activation3,12
, but additionally initiates sustained activation of muscarinoceptive neurons, which provide long-lasting excitation to RS cells. Similar persistent activity following muscarine receptor activation has been reported in thalamus27
, and entorhinal cortex28
. Muscarine directly induced sustained, recurring bursts of activity in a cell population in layer II of rat entorhinal cortex29
. The depolarizing plateaus lasted 2–5s not unlike what we previously described in lamprey RS cells after muscarinic receptor activation17
. Cell populations in entorhinal cortex were also made to oscillate synchronously upon muscarinic receptor activation30,31
. Synchronization may also contribute to increased excitation of RS cells. We have previously shown that a unilateral muscarinic activation of the muscarinoceptive cells produced bilateral excitation in RS cells17
. Therefore, the muscarinoceptive cells may provide a sustained and enhanced excitation to RS cells on both sides, maintaining bilateral coordination for locomotor output. Cellular properties linked directly to activation of muscarine receptors32
may thus play a crucial role in providing additional excitation to RS cells to boost the locomotor output.
Consequently, RS neurons receive two inputs originating from activity in the MLR. A direct and well-characterized component of serial activation of the locomotor system and an indirect, parallel pathway mediated by a very long-lasting muscarinic response in previously unremarked rhombencephalic neurons. This latter, newly identified parallel pathway markedly amplifies the output of the locomotor command system with a long-lasting response of the muscarinoceptive neurons, initiating sustained activity to provide a long-lasting boost to MLR driven motor output. This boost is clearly seen following brief intense stimuli applied to the MLR, in which early activation to the RS system remains but sustained activation of RS neurons is prevented by selective atropine application to the muscarinoceptive region.
Thus, transient activation of the MLR rapidly excites RS neurons by classical ionotropic monosynaptic drive, but can also sustain extended bouts of RS neuron excitation by a newly identified disynaptic pathway utilizing metabotropic muscarine receptors. Additionally, continuous activation of the MLR sustains locomotor activity over long periods3,10
. Activation of command neurons implies an activation of locomotor behaviors. This hitherto unknown parallel pathway plays a substantial role in locomotor control. In the lamprey it is possible to resolve this role because we may access the functional, intact brainstems of semi-intact preparations in which continuous MLR stimulation leads to sustained locomotor activity. Atropine micro-application targeted to the muscarinoceptive region markedly reduced the frequency of the subsequent locomotor activity. This effect was non-linear with respect to stimulus frequency and subsequent locomotor frequencies. Thus, at high stimulus and high locomotor frequency, atropine profoundly reduced locomotor frequencies. At lower frequencies effects of atropine were much less marked.
A muscarinoceptive group of cells amplifying RS cell activity and locomotor output is likely to be common amongst vertebrates. In birds, locomotor behavior is induced by brainstem injections of carbachol, a nonspecific cholinergic agonist. This effect is blocked by the muscarine receptor antagonist, atropine15
. Cholinergic inputs are also believed to activate brainstem neurones in mammals16,33
. A group of muscarinoceptive neurons was recently described in mammals in the ventromedial medulla close to the pontine border34
at a location similar to that of the muscarinoceptive cells in lampreys. These cells receive cholinergic inputs from the pedunculopontine nucleus, known to be part of the mammalian MLR16
. The role of these neurons was not described in relation to locomotion, but the similar properties of these neurons and their homologous location to those described in the present study strongly suggest that they could play a role in amplifying the RS descending signals to boost locomotor output. This would suggest that the muscarinic amplifying mechanism is conserved.
We conclude that brainstem supraspinal locomotor command systems are substantially more complex than previously thought. Anatomically simple serial pathways clearly excite brainstem command neurons. And yet, without a substantial amplification from muscarinoceptive neurons, the resulting locomotion may be short-lived and of much lower frequencies. These newly discovered rhombencephalic muscarinoceptive neurons provide a powerful augmentation of locomotor drive. The boost requires a sustained excitation driven by muscarine receptor activation.