Genetically defined inhibitory neurons in the mouse spinal cord dorsal horn: a possible source of rhythmic inhibition of motoneurons during fictive locomotion

Abstract

To ensure alternation of flexor and extensor muscles during locomotion, the spinal locomotor network provides rhythmic inhibition to motoneurons. The source of this inhibition in mammals is incompletely defined. We have identified a population of GABAergic interneurons located in medial laminae V/VI that express green fluorescent protein (GFP) in glutamic acid decarboxylase-65::GFP transgenic mice. Immunohistochemical studies revealed GFP+ terminals in apposition to motoneuronal somata, neurons in Clarke's column, and in laminae V/VI where they apposed GFP+ interneurons, thus forming putative reciprocal connections. Whole-cell patch-clamp recordings from GFP+ interneurons in spinal cord slices revealed a range of electrophysiological profiles, including sag and postinhibitory rebound potentials. Most neurons fired tonically in response to depolarizing current injection. Calcium transients demonstrated by two-photon excitation microscopy in the hemisected spinal cord were recorded in response to low-threshold dorsal root stimulation, indicating these neurons receive primary afferent input. Following a locomotor task, the number of GFP+ neurons expressing Fos increased, indicating that these neurons are active during locomotion. During fictive locomotion induced in the hemisected spinal cord, two-photon excitation imaging demonstrated rhythmic calcium activity in these interneurons, which correlated with the termination of ventral root bursts. We suggest that these dorsomedial GABAergic interneurons are involved in spinal locomotor networks, and may provide direct rhythmic inhibitory input to motoneurons during locomotion.

Figure 1.

Distribution and morphology of GFP+ neurons. A, Low-power photomicrograph of a cross section of upper lumbar spinal cord demonstrating cluster of GFP+ neurons in medial lamina V/V (B), smaller GFP+ neurons scattered in superficial dorsal horn (C), extensive GFP+ processes in lamina VII (D), and the appearance of GFP+ axonal cross sections in the lateral funiculus (E). F, The medial lamina V/VI GFP+ neurons appear clustered in longitudinal horizontal cross sections. The dashed line approximates the midline. G, Morphology of a GFP+ neuron demonstrated following intracellular fill (in slice) with Neurobiotin demonstrating extensive multipolar dendrites.

Figure 2.

The medial GFP+ neurons are propriospinal. A, Following intracellular fill with Neurobiotin in the hemisected spinal cord followed by tissue sectioning, an axon can be seen extending into the lateral funiculus, where it bifurcates into rostral and caudal branches. B, Labeling with TMRD 2–4 segments rostral or 4–6 segments caudal to study demonstrated few neurons labeled with both GFP (Bi) and TMRD (Bii, overlay in Biii, arrows).

Figure 3.

Spinal targets of medial GFP+ neurons. A, Motoneurons were identified by ChAT immunohistochemistry (white). GFP+ (Ai, Aiii) and GAD+ (Aii, Aiii) boutons (arrows) were identified in apposition to motoneurons following the identification of GFP+ axons coursing into the ventral horn (B). Ci, Axons were also identified coursing into the region of large neurons in Clarke's column, upon which bouton-like structures (arrows) were identified (Cii). D, GFP+ (Di) and GAD+ (Dii) boutons (arrows) were also identified on medial GFP+ neurons (Diii, overlay).

Figure 4.

Electrophysiological properties of medial GFP+ neurons. A, Three different neurons demonstrating a depolarizing sag (arrow) followed by an early rebound potential producing action potentials, and associated with a later, slower rebound depolarization (arrows) (Ai); neither a sag potential nor rebound (Aii); and absence of a sag potential, but an early rebound potential (arrow) producing action potentials (Aiii). Note the large postspike afterhyperpolarizations. B, Another neuron demonstrating an increase in firing with depolarization (Bi–Biv), producing a linear frequency–current relation (Bv).

Figure 5.

Medial GFP+ interneurons (IN) receive primary afferent input. A, Primary afferent boutons were identified by expression of VGLUT1, and were found in apposition to GFP+ (B) neurons (C, overlay). Higher magnification of the areas indicated in C demonstrate bouton-like structures along large (D) and small (E) dendritic branches. F, The threshold for afferent stimulation (stim) in the hemisected spinal cord was determined as the strength of dorsal root stimulation required to elicit a monosynaptic response in the ventral root. G, Stimulation strengths of up to 2 × threshold were used to demonstrate responses in medial GFP+ neurons. The upper trace demonstrates the stimuli, and the lower trace demonstrates calcium transients measured using two-photon excitation microscopy, having loaded the neurons with Fluo-3.

Figure 6.

Medial GFP+ neurons are active during locomotion. A, Few (∼5%) GFP+ neurons express Fos when the animal is quiescent (top), but almost half express Fos following an overground locomotor task (bottom). B, When exposed to the same transmitters that elicit locomotion in the isolated spinal cord, these neurons recorded in slice fire tonically—they are not conditional bursting neurons.

Figure 7.

Medial GFP+ neurons are rhythmically active during fictive locomotion. A, Fictive locomotion was elicited in the hemisected spinal cord (top and middle traces are recordings from L2 ventral roots) while recording calcium transients in medial GFP+ neurons (lower trace). The calcium transients were averaged based either on the start (B) or termination (C) of the L2 ventral root burst, demonstrating that the onset of the calcium transient was slightly delayed in relation to the onset of the ventral root burst. This delay can be seen in the polar plot (D), in which 0° is the onset of the ventral root burst and each point represents the onset of each calcium transient. The vector represents the mean, and the curved arc represents the time to capture a single frame. In the plot in E, 0° represents the termination of the ventral root burst. F–J, Another GFP+ neuron recorded and analyzed as in A–E. In this neuron, the onset of the calcium transient is out of phase with the ventral root burst onset. K, Mean duration (and SD) of the cycle period (onset to onset) of ventral root bursts and calcium transients in seven neurons. L, The mean ratios of cycle duration of the calcium transients compared to the ventral root durations is close to 100% in all neurons, indicating a 1:1 ratio. M, The mean onset of the calcium transients in each of the seven neurons is plotted in a polar plot, with 0° representing the onset of the ventral root burst. N, When these data are plotted in relation to the termination of the ventral root burst, the neuronal activity clusters into two groups, one with onset shortly after ventral root burst termination (first quadrant, as in the neuron depicted in F–J), and the second with onset before burst termination (as in the neuron in A–E). The arcs represent the mean frame capture duration.