Shakya Shrestha, Sony
Input properties of four populations of spinocerebellar tract neurons in the cat and the rat thoraco-lumbar spinal cord.
PhD thesis, Institute of Neuroscience and Psychology.
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The cerebellum receives information from the hindlimbs through several populations of spinocerebellar tract neurons. Although the role of these neurons has been established in electrophysiological experiments, the relative contribution of afferent fibres and central neurons to their input, their organization and mechanisms of control of transmission has only been estimated approximately so far. The present study aimed to investigate the input properties of four populations of spinocerebellar tract neurons: dorsal spinocerebellar tract neurons located in Clarke´s column (ccDSCT) and in the dorsal horn (dhDSCT) and ventral spinocerebellar tract (VSCT) neurons including spinal border (SB) neurons. There were three major aims: (1) to investigate the excitatory inputs to four types of spinocerebellar tract neurons in the cat and rat thoraco-lumbar spinal cord; (2) to analyze the inhibitory inputs to four types of spinocerebellar tract neurons in the cat and rat thoraco-lumbar spinal cord; (3) to determine the origin of excitatory and inhibitory inputs to four types of spinocerebellar tract neurons in the cat and rat thoraco-lumbar spinal cord.
Two series of experiments were carried out. In the first series of experiments in cats, spinocerebellar tract neurons were identified electrophysiologically and labelled intracellularly with rhodamine-dextran and Neurobiotin. In the second series of experiments in rats, cells were labelled by retrograde transport of b-subunit of Cholera toxin (CTb) from the cerebellum. In addition, to address the third aim, reticulospinal (RetS) and corticospinal (CS) terminals were identified by anterograde transport of CTb from the caudal medulla and hindlimb sensory motor cortex respectively in rats along with labelling of spinocerebellar tract neurons by retrograde injection of Fluorogold in the cerebellum. Following this, immunohistochemistry was carried out.
The first aim was achieved by utilizing the difference in the immunohistochemistry of glutamatergic terminals of peripheral afferents and of central neurons with vesicular glutamate transporters, VGLUT1 or VGLUT2, respectively. All SB neurons with dominating inhibitory input from the periphery possessed very few VGLUT1 contacts and remarkably higher numbers of VGLUT2 contacts. In VSCT neurons with excitatory primary afferent input, the number of VGLUT1 contacts was relatively high although VGLUT2 contacts likewise dominated. In contrast, DSCT neurons were associated with numerous VGLUT1 contacts; ccDSCT neurons with strong input from group I afferents had higher density of VGLUT1 contacts than dhDSCT neurons with major input from group II and cutaneous afferents.
In order to fulfill the second aim, quantification of contacts formed by inhibitory axon terminals on spinocerebellar tract neurons along with excitatory terminals was carried out. Inhibitory axon terminals were characterised as either GABAergic, glycinergic or both GABAergic/glycinergic by using antibodies against vesicular GABA transporter (VGAT), glutamic acid decarboxylase (GAD) and gephyrin. Similarly, excitatory terminals were characterised by using combination of VGLUT1 and 2. The comparison revealed the presence of much higher proportions of inhibitory than excitatory contacts on SB neurons but similar proportions were found on VSCT, ccDSCT and dhDSCT neurons. In all types of cell, the majority of inhibitory terminals were glycinergic. The density of contacts was higher on somata and proximal in comparison with distal dendrites of SB and VSCT neurons but more evenly distributed in ccDSCT and dhDSCT neurons.
To achieve the third aim, a series of immunohistochemical reactions was performed to characterize contacts that originate from proprioceptors, different types of interneurons and descending RetS and CS pathways. Among the four populations of spinocerebellar tract neurons, ccDSCT neurons had the highest proportion of contacts formed by VGLUT1 terminals double labeled with parvalbumin (PV) which indicated that majority of direct excitatory sensory inputs to ccDSCT neurons are derived from proprioceptors. A small proportion of excitatory and inhibitory contacts on these neurons originated from Calbindin/ Calretinin/ PV expressing neurons. Quantitative analysis revealed that SB and VSCT neurons have significantly higher numbers of appositions from VGLUT2 expressing RetS axon terminals than DSCT neurons. A small proportion of the RetS contacts on these neurons were VGAT positive. In contrast, DSCT neurons had higher numbers of appositions made by CS axon terminals in comparison to SB and VSCT neurons.
The present findings provide a new basis for understanding the organization and functional connectivity of four populations of spinocerebellar tract neurons and strengthen previous indications of their functional differentiation. SB and VSCT neurons principally receive inputs from spinal and supraspinal neurons although direct input from primary afferents is also stronger in VSCT neurons. DSCT neurons have major direct input from primary afferents and also to some extent from the CS pathway but monosynaptic inputs from proprioceptors dominated in ccDSCT neurons and dhDSCT neurons have mixed proprioceptive and low threshold cutaneous afferent input.
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