Strategies for delineating spinal locomotor rhythm-generating networks and the possible role of Hb9 interneurones in rhythmogenesis

Abstract

Despite significant advances in our understanding of pattern generation in invertebrates and lower vertebrates, there have been barriers to the application of the principles learned to the definition of networks underlying mammalian locomotion. Major difficulties have arisen in identifying spinal interneurones in preparations which allow study of neuronal intrinsic properties and the role of identified interneurones in locomotor networks. Recent genetic technologies in which selective expression of fluorescent proteins in specific populations of mouse spinal neurones have provided new avenues of investigation. In this review, we focus on the generation of locomotor rhythm and outline criteria that rhythm-generating neurones might be expected to fulfill. We then examine the extent to which a recently identified population of spinal interneurones, Hb9 interneurones, fulfill these criteria. Finally, we suggest that Hb9 interneurones could be involved in an asymmetric model of locomotor rhythmogenesis through projections of electrotonically coupled rhythm-generating modules to flexor pattern formation half-centres. The principles learned from studying this population of interneurones have led to strategies to systematically evaluate neurones that may be involved in locomotor rhythmogenesis.

Fig. 1

Hb9 interneurones fulfill many of the criteria for rhythm-generating interneurones. A combination of anatomical and electrophysiological studies in Hb9::GFP mice defines the properties of Hb9 interneurones and determines their suitability for rhythm generation. (A) Neurolucida mapping studies of transverse spinal cord sections from mid-thoracic to mid-lumbar levels reveal GFP+ interneurones (green) extending from the thoracic to the mid-lumbar spinal cord. GFP+ somatic and autonomic motoneurones are indicated in brown. The clusters of GFP+ interneurones abutting the ventral commissure are not seen below the mid-lumbar level. By crossing this strain with Hb9^nlslacZ/+ knock-in animals, it was revealed that this population of neurones is Hb9+ (not shown). (B) A representative transverse spinal cord section demonstrates the ventromedial location of Hb9 interneurones (box). (C) An Hb9 interneurone (labeled with anti-GFP, green) is contacted by serotonergic fibres (anti-5-HT, red). (D) VGLUT1 terminals (red) oppose the cell body of an Hb9 interneurone providing evidence that these neurones receive primary afferent input. (E) Fluorescent in situ hybridisation reveals that Hb9 interneurones (green, arrow) contain the mRNA for VGLUT2 (red) thus revealing a glutamatergic transmitter phenotype. (F) Hb9 interneurones (green) are contacted by VLGUT2 positive terminals (red) that are also GFP-positive, raising the possibility that Hb9 interneurones are reciprocally connected. (G) A medium power image indicates the scarcity of VGLUT2+ (red), GFP+ (green) terminals (double positive, yellow, arrow, inset) in lamina IX, making it unlikely that Hb9 interneurones provide significant input to motoneurones. (H) Whole-cell patch clamp recordings from an Hb9 interneurone reveal a prominent post-inhibitory rebound (PIR) potential leading to multiple action potentials when the neurone is released from a hyperpolarising potential (arrow). Hb9 interneurones undergo large oscillations in membrane potential in the presence of NMDA, 5-HT, dopamine and TTX, indicating that they are conditional bursters. Scale bars in inset are 10 mV and 100 ms, and for the oscillations 10 mV and 5 s. (I) Hb9 interneurones (green) are active in locomotor activity as demonstrated by the presence of Fos protein (red) following a locomotor task in the adult. In addition, their activity in the neonatal hemisected spinal cord is in phase with the output of an unidentified ventral root. (Scale bars are 100 μm in panels B, E and I, 10 μm in G, 5 μm in F and D, 2 μm in C, 20 s in panel I. Panels B, C, D, E, F, G and H, and left side of I from Wilson et al. (2005). The right-sided portion of panel I is from Hinckley et al. (2005). With permission.).

Fig. 2

A schematic diagram similar to Brown’s model illustrated, then modified, by Lundberg (1981). This shows a half-centre model for a pattern formation layer in the mammalian locomotor network. The flexor (F) and extensor (E) motoneurones receive input from last-order excitatory interneurones that are reciprocally innervated by inhibitory interneurones (red). Another population of inhibitory interneurones ensures inhibition of the antagonist motor pools. Although this half-centre model may in itself be capable of generating a rhythm, evidence is presented that the rhythm-generating portion of the network is distinct and forms another “layer” (see text). Little is known about the rhythm generator, which is thus represented by a grey box.

Fig. 3

A schematic diagram suggesting a possible flexor-dominated rhythm generator for mammalian locomotor output. The rhythm generator consists of a kernel of Hb9 interneurones (dark green) that are electrotonically coupled to non-Hb9 interneurones (light green). This network produces rhythmic output which excites the corresponding last-order flexor-related interneurones of a half-centre. The rhythmic output also leads to disynaptic inhibition of the extensor-related half-centre interneurones. The rhythm generator is reciprocally innervated by the rhythm generator on the contralateral side to ensure interlimb coordination. In the case of alternating gait, these reciprocal connections will be inhibitory (red). In addition, connections between the bilateral pattern formation layers are also indicated (grey), although the details of these are not known (but see Lundberg, 1981; Jankowska et al., 1967a,b).

Fig. 4

Hypothesis for the regulation of locomotor speed: relationship of V1 and Hb9 interneurones. (A) Locomotor speed slows down (step cycle increases in duration) when the activity of inhibitory V1 interneurones is depressed (Gosgnach et al., 2006). In these transgenic mice, V1 interneurones express the receptor for allatostatin, which is coupled to a G-protein coupled inward rectifying potassium (GIRK) channel. When the ligand is present (AL), V1 interneurones hyperpolarise and thus their output is reduced. This leads to a reversible decrease in locomotor speed. That is, removal of V1 interneurone mediated inhibition leads to a slowing of the rhythm. (B) Depolarisation of Hb9 interneurones increases oscillation period. (C) These findings lead to the hypothesis that inhibitory V1 interneurones innervate Hb9 interneurones. When they are removed, Hb9 interneurones depolarise, leading to an increase in their interburst interval and therefore a reduction in locomotor speed.