Anatomical characterization of a rabbit cerebellar eyeblink premotor pathway using pseudorabies and identification of a local modulatory network in anterior interpositus

Abstract

Rabbit eyeblink conditioning is a well characterized model of associative learning. To identify specific neurons that are part of the eyeblink premotor pathway, a retrograde transsynaptic tracer (pseudorabies virus) was injected into the orbicularis oculi muscle. Four time points (3, 4, 4.5, and 5 d) were selected to identify sequential segments of the pathway and a map of labeled structures was generated. At 3 d, labeled first-order motor neurons were found in dorsolateral facial nucleus ipsilaterally. At 4 d, second-order premotor neurons were found in reticular nuclei, and sensory trigeminal, auditory, vestibular, and motor structures, including contralateral red nucleus. At 4.5 d, labeled third-order premotor neurons were found in the pons, midbrain, and cerebellum, including dorsolateral anterior interpositus nucleus and rostral fastigial nucleus. At 5 d, labeling revealed higher-order premotor structures. Labeled fourth-order Purkinje cells were found in ipsilateral cerebellar cortex in cerebellar lobule HVI and in lobule I. The former has been implicated in eyeblink conditioning and the latter in vestibular control. Labeled neurons in anterior interpositus were studied, using neurotransmitter immunoreactivity to classify individual cell types and delineate their interconnectivity. Labeled third-order premotor neurons were immunoreactive for glutamate and corresponded to large excitatory projection neurons. Labeled fourth-order premotor interneurons were immunoreactive for GABA (30%), glycine (18%), or both GABA and glycine (52%) and form a functional network within anterior interpositus involved in modulation of motor commands. These results identify a complete eyeblink premotor pathway, deep cerebellar interconnectivity, and specific neurons responsible for the generation of eyeblink responses.

Figure 1.

Schematic diagram of the OOM premotor pathway labeled with PRV. A shows the location of 5 PRV injections (green dashed lines) in OOM that include the orbital, preseptal, and pretarsal muscles. B shows the retrograde transsynaptic spread of PRV through the OOM premotor pathway. Directional and sequential spread of PRV through first-order (1°) motor neurons in the facial nucleus (7N), second-order (2°) premotor neurons in the red nucleus (RN) and other areas, and third-order (3°) premotor neurons in the AIN of the cerebellum and other areas.

Figure 2.

Examples of PRV-labeled neurons in the rabbit brain 3 and 4 d after injection. A, PRV-labeled first-order motor neurons are located in the dorsolateral facial nucleus ipsilaterally 3 d after PRV injection into the right OOM. DL, Dorsolateral; L, lateral; Int, intermediate; M, medial; VM, ventromedial subnuclei. B–F, PRV-labeled neurons 4 d after PRV injection. B shows PRV-labeled neurons in the medial vestibular nucleus (MVe) ipsilaterally and absence of PRV labeling in the superior salivatory nucleus (SSN) and abducens nucleus (6N). C shows PRV labeling in the ventral spinal trigeminal nucleus, oral part (Sp5O) ipsilaterally. D, Discrete PRV labeling in the rostral dorsal cochlear nucleus (DC) ipsilaterally. E shows PRV-labeled neurons in the parabrachial nucleus (PB) and Kölliker-fuse nucleus (KF) ipsilaterally. F shows PRV-labeled neurons in the dorsolateral red nucleus (RN) contralaterally. Scale bars: 1 mm.

Figure 3.

Distribution of PRV-labeled neurons in the brain 4 d after PRV injection in the right OOM. A composite of the location of PRV-labeled neurons found across four animals plotted in sequential sections to best represent the pattern of PRV labeling. Sections are arranged caudorostrally (A–K). The arrow indicates the side of PRV injection. Each dot represents one PRV-labeled neuron. 3N, Oculomotor nucleus; 4N, trochlear nucleus; 5N, motor trigeminal nucleus; 7N, motor facial nucleus; 12N, hypoglossal nucleus; AIN, anterior interpositus; CG, central gray; Cn, cuneiform nucleus; DC, dorsal cochlear; Dk, nucleus of Darkschewitsch; LL, lateral lemniscus; dMe, deep mesencephalic nucleus; DN, dentate nucleus; PAG, periaqueductal gray; DpG, deep layer of the superior colliculus; FN, fastigial nucleus; Gi, gigantocellular reticular nucleus; Gr, gracile nucleus; IC, inferior colliculus; InC, interstitial nucleus of Cajal; InG, intermediate gray layer of the superior colliculus; InW, intermediate white layer of the superior colliculus; KF, Kölliker-fuse nucleus; LRt, lateral reticular nucleus; MdD, dorsal medullary reticular nucleus; MdV, ventral medullary reticular nucleus; MG, medial geniculate nucleus; MVe, medial vestibular nucleus; Op, optic nerve layer of the superior colliculus; PaR, pararubral nucleus; PB, parabrachial nucleus; Pn, pontine nucleus; PnO, pontine reticular nucleus, oral; PO, periolivary nucleus; Pr5, principal sensory trigeminal nucleus; RN, red nucleus; RRF, retrorubral field; SN, substantial nigra; Sol, solitary nucleus; Sp5C, spinal trigeminal nucleus, caudal; Sp5I, spinal trigeminal nucleus, interpolar; Sp5O, spinal trigeminal nucleus, oral; SpVe, spinal vestibular nucleus; SuG, superficial gray layer of the superior colliculus; tth, trigeminothalamic tract; Tz, nucleus of the trapezoid body; VC, ventral cochlear nucleus. Scale bar, 5 mm.

Figure 4.

A–D, Examples of PRV-labeled premotor neurons 4.5 d after injection. A shows PRV-labeled neurons in the dorsolateral red nucleus (RN) and pararubral nucleus contralaterally. The oculomotor nucleus (3N), interstitial nucleus of Cajal (InC), and supraoculomotor periaqueductal gray are labeled bilaterally. B shows PRV-labeled neurons in the lateral lemniscus (LL) and periaqueductal gray (PAG) ipsilaterally. C shows a few PRV-labeled neurons in the dorsolateral AIN ipsilaterally. D shows PRV-labeled neurons in the rostral fastigial nucleus (FN) ipsilaterally. Scale bars: 1 mm.

Figure 5.

Distribution of PRV-labeled neurons in the brain 4.5 d after PRV injection in the right OOM. A composite of the location of PRV-labeled neurons found across four animals plotted in sequential sections to best represent the pattern of PRV labeling. Sections are arranged caudorostrally (A–L). The arrow indicates the side of PRV injection. Each dot represents one PRV-labeled neuron. See Figure 3 for abbreviations. Scale bar, 5 mm.

Figure 6.

A–F, Examples of PRV-labeled premotor neurons 5 d after injection. A shows PRV-labeled neurons in the Cu (cuneate nucleus) ipsilaterally. B shows PRV-labeled neurons in the superior colliculus (SC) contralaterally. C shows PRV labeling in the red nucleus (RN) and pararubral nucleus (PaR) mainly contralaterally and deep mesencephalic nucleus (DMe) bilaterally. D shows PRV-labeled neurons in HVI longitudinal zone C-3 ipsilaterally; note AIN PRV-labeled neurons in the same section. E shows a group of PRV-labeled Purkinje cells in HVI zone C-3 ipsilaterally. F shows PRV-labeled cerebellar lobule I (L1) longitudinal zone A ipsilaterally, note PRV-labeling in the rostral fastigial nucleus (FN), lateral and medial vestibular nucleus (MVe) ipsilaterally. Scale bars: A–D, F, 1 mm; E, 200 μm.

Figure 7.

Distribution of PRV-labeled premotor neurons in the DCN 4.5 and 5 d after PRV injection. Sections are arranged rostrocaudally (a–e). Note each dot represents a group of 2–5 PRV-labeled neurons of a typical subject. A shows the distribution of PRV-labeled neurons in the rostral fastigial nucleus (FN) and dorsolateral AIN that are found exclusively on the ipsilateral side 4.5 d after PRV-injection. B shows the distribution of PRV-labeled neurons in the FN, AIN, posterior interpositus nucleus (PIN), and dentate nucleus (DN) that are found bilaterally with ipsilateral predominance 5 d after PRV injection. Scale bar, 5 mm.

Figure 8.

Glutamate, GABA, and glycine immunoreactivity in the cerebellar cortex. Glutamate-immunoreactive granule cells and their numerous axon terminals extending through the molecular layer are seen in the top left panel. GABA-immunoreactive neurons including Purkinje cells, basket, and stellate cells in the molecular layer, and Golgi cells in the granule cell layer are shown in the top right panel. Glycine-immunoreactive neurons in the cerebellar cortex including Lugaro cells in the Purkinje cell layer, and Golgi cells in the granule cell layer are shown in the bottom left panel. A merged image of glutamate-, GABA-, and glycine-immunoreactive neurons is shown in the bottom right panel. Colocalization of GABA and glycine-immunoreactive neurons can be seen in some Golgi and Lugaro cells. Glu, Glutamate; Gly, glycine. Scale bar, 200 μm.

Figure 9.

Glutamate, GABA, and glycine immunoreactivity in the DCN and cerebellar cortex. A section that contains the dorsolateral AIN, dorsal dentate nucleus (DN), and the granule cell layer of the cerebellar cortex is shown. Glutamate-immunoreactive neurons are shown in the top left panel. Note large glutamate-immunoreactive neurons in the DCN and numerous granule cells in the cerebellar cortex. GABA-immunoreactive neurons are shown in the top right panel. Note smaller GABA-immunoreactive neurons and numerous terminals in the DCN and several Golgi cells in the granule cell layer. Glycine-immunoreactive neurons are shown in the bottom left panel. Note small distinctive glycine-immunoreactive neurons evenly distributed in the DCN. A merged image of glutamate, GABA, and glycine-immunoreactive neurons is shown in the bottom right panel. Glu, Glutamate; Gly, glycine. Scale bar, 400 μm.

Figure 10.

Glutamate immunoreactive PRV-labeled neuron in the AIN 4.5 d after injection into the OOM. Glutamate immunoreactivity is shown in the top left panel. Note a large glutamate-immunoreactive neuron at the bottom of the panel indicated by the asterisk. GABA-immunoreactivity is shown in the top middle panel. Four GABA-immunoreactive interneurons are visible and note the presence of glutamate immunoreactivity in their cell bodies. Numerous GABA-immunoreactive terminals are evident apposed to different neuronal processes and glutamate-immunoreactive somas. Glycine-immunoreactivity is shown in the top right panel. Note a small interneuron that is exclusively glycine-immunoreactive. A few glycine-immunoreactive terminals apposing dendrites and somas of glutamate-immunoreactive and GABA-immunoreactive neurons are shown. A PRV-labeled neuron is shown in the bottom left panel. A merged image of glutamate-, GABA-, and glycine immunoreactivity and PRV is shown in the bottom right panel. Note that the PRV-labeled neuron colocalizes with a large glutamate-immunoreactive neuron. A small interneuron immunoreactive for both GABA and glycine is also shown. Glu, Glutamate; Gly, glycine. Scale bar, 40 μm.

Figure 11.

GABA and glycine immunoreactive interneuron labeled with PRV. GABA immunoreactivity is shown in the top left panel. Note two GABA-immunoreactive interneurons and numerous GABA-immunoreactive terminals. Glycine immunoreactivity is shown in the top right panel. Note two Glycine-immunoreactive interneurons and a few and densely packed Glycine-immunoreactive terminals. PRV-labeled fourth-order interneuron and its PRV-labeled terminal are shown in the bottom left panel. A merged image of GABA immunoreactivity, glycine immunoreactivity, and PRV is shown in the bottom right panel. Note that the PRV-labeled interneuron is immunoreactive for both GABA and glycine and its PRV-labeled terminal is immunoreactive solely for glycine. Glu, Glutamate; Gly, glycine. Scale bar 20 μm.

Figure 12.

Organization of the AIN circuit. A schematic representation of different neuronal populations and their connections in the AIN. The premotor pathway of the OOM is shown with black arrows that delineate the direction of PRV labeling. Two representative glutamatergic (GLU) projection neurons are shown. A PRV-labeled third-order GLU neuron projecting to the red nucleus (RN) is depicted with a black arrow. Three types of PRV-labeled fourth-order interneurons are shown to make a direct connection onto the PRV-labeled GLU neuron. Possible connections among different AIN neurons are represented with excitatory (+) and inhibitory (−) terminals (colored triangles). Excitatory input reaches the AIN by mossy fiber (MF) and climbing fiber (CF) collaterals that make connections on GLU neurons and possibly (dashed lines) on different interneurons. Inhibitory input to AIN neurons is provided by Purkinje cells (PC) and local interneurons. 7N, Facial nucleus; GrC, granule cell layer; IO, inferior olive; PF, parallel fibers.