Stephen M. Ho

Transplantation of nasal olfactory tissue promotes partial recovery in paraplegic adult rats

Recent reports have highlighted the potential therapeutic role of olfactory ensheathing cells for repair of spinal cord injuries. Previously ensheathing cells collected from the olfactory bulbs within the skull were used. In humans a source of these cells for autologous therapy lies in the nasal mucosa where they accompany the axons of the olfactory neurons. The aim of the present study was to test the therapeutic potential of nasal olfactory ensheathing cells for spinal cord repair. Olfactory ensheathing cells cultured from the olfactory lamina propria or pieces of lamina propria from the olfactory mucosa were transplanted into the transected spinal cord. Three to ten weeks later these animals partially recovered movement of their hind limbs and joints which was abolished by a second spinal cord transection. Control rats, receiving collagen matrix, respiratory lamina propria or culture medium, did not recover hind limb movement. Recovery of movement was associated with recovery of spinal reflex circuitry, assessed using the rate-sensitive depression of the H-reflex from an interosseous muscle. Histological analysis of spinal cords grafted with olfactory tissue demonstrated nerve fibres passing through the transection site, serotonin-positive fibres in the spinal cord distal to the transection site, and retrograde labelling of brainstem raphe and gigantocellularis neurons from injections into the distal cord, indicating regeneration of descending pathways. Thus, olfactory lamina propria transplantation promoted partial restoration of function after relatively short recovery periods. This study is particularly significance because it suggests an accessible source of tissue for autologous grafting in human paraplegia.

Effects of Different Anesthetics on the Paired-Pulse Depression of the H Reflex in Adult Rat

Hyperreflexia is a common feature of spinal cord injury (SCI), and changes in reflex excitability have been reported to be useful in assessing treatments in animal models of cord damage. However, spinal reflexes are known to be dependent on anesthetic level. As a preliminary to its use in SCI, the excitability of the Hoffman reflex (H reflex) has been assessed under several commonly used anesthetics. The H reflex was recorded in the distal foot muscles (dorsal interossei) of adult rats, while the lateral plantar nerve was stimulated. Five different anesthetics were used: ketamine, halothane, Nembutal, Etomidate, and Saffan. Recording and stimulating electrodes were inserted directly through the skin to minimize the surgical procedure for each experiment, allowing repeated recording to be made in the same animal on a weekly basis. Suppression of the H reflex was tested using twin-pulse stimulation. Halothane and ketamine produced suppression of the H response when interpulse intervals were shortened to less than 1 s. The H-reflex suppression profiles recorded under Etomidate, Saffan, and Nembutal anesthesia were less sensitive to the stimulation rate, with little reduction until intervals were 200 ms or less. The suppression profiles of halothane and ketamine resemble that seen in unanesthetized humans, whereas that under the other anesthetics tried here resembles that observed in spinal-cord-injured animals. The results suggest a preferential action of some anesthetics on descending pathways involved in reflex modulation and the importance of assessing reflex excitability under anesthetics such as ketamine or halothane.

Rhythmic motor activity and interlimb co‐ordination in the developing pouch young of a wallaby (Macropus eugenii)

1 The forelimb motor behaviour of developing wallaby was studied. A clock‐like alternating movement was reactivated whenever the animal was removed from the pouch. 2 Forelimb stepping frequency increased during the first 3 weeks of development, while the phase relationship remained constant. Forelimb activity could be affected by altering the afferent feedback from the contralateral limb, or an increase in ambient temperature. 3 In vitro experiments were performed using an isolated brainstem‐spinal cord preparation from animals up to 6 weeks postnatal. Fictive locomotor activity could be evoked by electrical stimulation or bath‐applied NMDA (< 10 μm). 4 Bath‐applied strychnine (10‐25 μm) and bicuculline (10‐50 μm) disrupted the phase relationship between motor pools, while rhythmic motor discharge remained in the absence of these inhibitory pathways. 5 The present findings indicate that the pattern generator that underlies the robust forelimb movement during the first journey to the pouch is retained for different motor functions during in‐pouch development. The neural network that underlies such behaviour can be divided into two major components, a rhythm generator within each hemicord, and a pattern co‐ordinating pathway which involve both glycinergic and GABAergic interneurones.

Afferent arrival and onset of functional activity in the trigeminothalamic pathway of the rat

In this study, a novel in vitro slice preparation has been used to study the anatomical and physiological development of the trigeminothalamic pathway in the prenatal and neonatal rat. Anterograde tracing studies showed that the most rostral trigeminal fibres had reached the cephalic flexure by embryonic day (E)15, and entered the diencephalon by E16. By E17 the first few fibres had reached the ventroposteromedial thalamic nucleus (VPM) where they terminated in growth cones. The projection was more substantial and fibres had begun branching by E18, and arbors were more elaborate by E19. The fibres densely filled the nucleus by the day of birth (PO). The physiological studies showed that postsynaptic responses to stimulation of the trigeminal nerve or principal sensory nucleus (Pr5) could first be recorded at E17. Reliable responses to stimulation of either the nerve or Pr5 were recorded from E18 on. Stimulation of Pr5 enabled both axonal and synaptic signals to recorded in VPM. A GABAergic influence was acting to decrease the overall level of excitability in the thalamus from E18. In prenatal animals, the excitatory response was primarily mediated by NMDA receptors, and by P1 a non-NMDA mediated component was beginning to appear. These results demonstrate that the capacity for axonal conduction in the trigeminothalamic fibres and synaptic transmission in the thalamus are present from the time that anatomical connections are first established.

Afferent ingrowth and onset of activity in the rat trigeminal nucleus

A novel in vitro preparation, consisting of the rat brainstem with the trigeminal ganglion attached, has been used to study the anatomical and functional development of the trigeminal nucleus from embryonic day (E)13 to postnatal day (P)6. Neurobiotin injections into the trigeminal ganglion showed that primary afferents had reached the trigeminal tract by E13 and had grown simple, mainly unbranched, collaterals into all levels of the nucleus by E15. By E17, these collaterals were extensively branched, with occasional boutons present. Patches of intense neurobiotin‐labelled terminals, corresponding to whisker‐related patterns, were first seen at E20 and became clearer over the next few days. Terminal arbours at this stage were relatively localized and densely branched, with many boutons. Responses from the trigeminal nucleus were recorded with suction electrodes, following stimulation of the trigeminal ganglion. Recordings from the main sensory nucleus showed a postsynaptic response was first present at E15. At E16, bath application of AP5 and DNQX showed that the response contained both NMDA and AMPA components, with NMDA predominating (75%). The NMDA : AMPA ratio remained high until P1, then gradually declined to 50% by P6. The postsynaptic response was also reduced by bath application of bicuculline, indicating the presence of a GABAA‐mediated excitatory component. GABAergic excitation was present at all ages but was maximal from E20 to P1, the age at which whisker‐related patterns are developing. It is hypothesized that both GABAergic excitation and NMDA receptor activation play a role in the consolidation of trigeminal connections, and are thus important in the development of whisker‐related patterns.

Spontaneous activity in the perinatal trigeminal nucleus of the rat

The role of electrical activity in establishing appropriate connections in the trigeminal pathway remains controversial. We report here a novel observation of spontaneous activity in the perinatal trigeminal sensory nucleus between embryonic day (E) 16 to postnatal day (P) 8. Most of these spontaneous bursts had the same polarity and were of comparable amplitude to the trigeminal nerve-evoked response. This synchronized activity was abolished by bicuculline or kynurenic acid. Recording from the teased trigeminal root during the spontaneous bursts also showed a corresponding afferent depolarization. We speculate that the GABAergic depolarization of pre- and postsynaptic cells may contribute to the generation of the spontaneous activity and its propagation throughout the trigeminal sensory pathway, even in the absence of activity initiated from peripheral sensory receptors.

Morphological development of afferent segregation and onset of synaptic transmission in the trigeminothalamic pathway of the wallaby (Macropus eugenii).

A light and electron microscopic study has been made of the time of formation of whisker‐related patterns in trigeminothalamic afferents and the onset of synapse formation between afferents and cells in the ventroposteromedial nucleus (VPM) of the marsupial mammal, the wallaby, by labelling afferents with a carbocyanine dye. A parallel in vitro study was made of the functional development of the trigeminothalamic pathway to the VPM. Evoked synaptic responses could be recorded in the VPM from the time that afferents first reached the VPM at postnatal day 15 (P15). At all stages, the excitatory response comprised both N‐methyl‐D‐aspartate‐ and non‐N‐methyl‐D‐aspartate‐mediated components. At P40, the response decreased markedly in duration, coinciding with the onset of synaptogenesis. This implies that transmission is occurring prior to synapse formation, probably through transmitter release from growth cones. At P50, synaptic responses became dominated by a fast, non‐N‐methyl‐D‐aspartate potential, and this coincided with the first appearance of whisker‐related patterns in the VPM. A gamma‐aminobutyric acid (subtype A)‐mediated, inhibitory component was also present from the time of afferent arrival. These findings support the idea that functional interactions between afferents and their targets may play a role in pattern formation in the somatosensory thalamus. J. Comp. Neurol. 399:47–60, 1998.

Retinocollicular synaptogenesis and synaptic transmission during formation of the visual map in the superior colliculus of the wallaby (Macropus eugenii)

Spontaneous retinal activity has been implicated in the development of the topographic map in the superior colliculus (SC) but a direct demonstration that it reaches the colliculus is lacking. Here we investigate when the retinocollicular projection is capable of transmitting information from the retina in a marsupial mammal, the wallaby (Macropus eugenii). The projection develops postnatally, allowing in vivo analysis throughout development. Quantification of retinocollicular synaptogenesis has been combined with electrophysiology of the development and characteristics of retinocollicular transmission, including in vivo and in vitro recording in the same animals. Prior to postnatal day (P) 12–14 in vitro recording detected only presynaptic activity in retinal axons in the colliculus, in response to stimulation of the optic nerve. Postsynaptic responses, comprising both N‐methyl‐d‐aspartate (NMDA) and non‐NMDA responses, were first detected in vitro at P12–14 and retinal synapses were identified. In contrast, postsynaptic responses to optic nerve stimulation could not be detected in vivo until P39, around the time that retinal axons begin arborizing. Around this age density and numbers of total synapses began increasing in the retinorecipient layers of the colliculus. By P55–64, the numbers of retinal synapses had increased significantly and density and numbers of retinal and total synapses continued to increase up to P94–99. During this time the map is undergoing refinement and degenerating axons and synapses were present. The discrepancy between in vitro and in vivo onset of functional connections raises the question of when retinal activity reaches collicular cells in the intact, unanaesthetized animal and this will require investigation.

GABAergic modulation of axonal conduction in the developing rat retinotectal pathway.

The effect of gamma-aminobutyric acid (GABA) on nerve conduction was investigated in the developing rat optic nerves in vitro. Antidromic compound action potentials (CAP) were suppressed by GABA, with increasing attenuation in older preparations. Enhancement of CAP amplitude using bicuculline was observed in postnatal days 4-5 suggesting the presence of endogenous GABA activity. Our findings suggest that the role of electrical activity in the refinement of the retinotectal projection could be limited by the GABAergic action on axonal conduction.

Early detection of optic nerve-evoked response in the superior colliculus of the neonatal rat

Optic nerve-evoked responses were measured in the superior colliculus (SC) of neonatal rats in vivo from postnatal day (P) 0 to P11. At P1, a biphasic response was recorded in superficial layers and the amplitude diminished as the electrode penetrated into the deeper layers of the SC. By P2, a similar response, with a fast positive-going potential followed by a more prolonged negative potential was observed at the surface. The polarity of the response reversed as the electrode was moved into the deeper laminae of the SC. Such a reversal in the polarity of optic nerve-evoked responses resembled those observed in more mature preparations. Using current source density analysis, a single pair of source-sink could be identified following optic nerve stimulation at P2, and this changed to a more complex pattern by P11. Our results suggest that synaptic transmission in the retinocollicular pathway of the rat is functional as early as P2.