Hans Holmberg

A survey of spinal dorsal horn neurones encoding the spatial organization of withdrawal reflexes in the rat

The withdrawal reflex pathways to hindlimb muscles have an elaborate spatial organization in the rat. In short, the distribution of sensitivity within the cutaneous receptive field of a single muscle has a spatial pattern that is a mirror image of the spatial pattern of the withdrawal of the skin surface ensuing on contraction in the respective muscle. In the present study, a search for neurones encoding the specific spatial input-output relationship of withdrawal reflexes to single muscles was made in the lumbosacral spinal cord in halothane/nitrous oxide-anaesthetized rats. The cutaneous receptive fields of 147 dorsal horn neurones in the L4-5 segments receiving a nociceptive input and a convergent input from A and C fibres from the hindpaw were studied. The spatial pattern of the response amplitude within the receptive fields of 118 neurones was quantitatively compared with those of withdrawal reflexes to single muscles. Response patterns exhibiting a high similarity to those of withdrawal reflexes to single muscles were found in 27 neurones located in the deep dorsal horn. Twenty-six of these belonged to class 2 (responding to tactile and nociceptive input) and one belonged to class 3 (responding only to nociceptive input). None of the neurones tested (n=20) with reflex-like response patterns could be antidromically driven from the upper cervical cord, suggesting that they were spinal interneurones. With some overlap, putative interneurones of the withdrawal reflexes to the plantar flexors of the digits, the plantar flexors of the ankle, the pronators, the dorsiflexors of the ankle, and a flexor of the knee, were found in succession in a mediolateral direction. It is concluded that neurones that are able to encode the specific spatial input-output organization of the withdrawal reflexes to single muscles do exist in the deep dorsal horn. Such reflex encoders appear to have a “musculotopic” organization. A hypothesis of the organization of the withdrawal reflex system is presented.

Postnatal development of the nociceptive withdrawal reflexes in the rat: a behavioural and electromyographic study.

1. The postnatal development of nociceptive withdrawal reflexes was studied. In awake intact rats, forelimb, hindlimb and tail reflexes were recorded on videotape. In decerebrate spinal rats, electromyography (EMG) was used to record nociceptive withdrawal reflexes in musculi extensor digitorum longus (EDL), peronei, gastrocnemius‐soleus (G‐S) and biceps posterior‐semitendinosus (BP‐ST). Thermal (short‐lasting CO2 laser pulses) and mechanical stimulation were used. 2. In adults, nociceptive withdrawal reflexes were typically well directed and reflex pathways to single hindlimb muscles had functionally adapted receptive fields. By contrast, at postnatal day (P) 1‐7, the nociceptive withdrawal reflexes were often inappropriate, sometimes producing movements towards the stimulation, and EMG recordings revealed unadapted variable receptive fields. With increasing age, the nociceptive withdrawal reflexes progressively became well directed, thus producing localized withdrawal. Both withdrawal movements and spatial organization of the receptive fields were adult‐like at P20‐25. 3. Up to P25, reflex thresholds were more or less constant in both intact awake rats and spinal decerebrate rats, except in G‐S in which no nociceptive withdrawal reflexes were evoked from P20 on. After P25, mechanical, but not thermal, thresholds increased dramatically. 4. EMG recordings revealed that during the first three postnatal weeks, the latency of the CO2 laser‐evoked nociceptive withdrawal reflexes decreased significantly in peronei and BP‐ST, but not in EDL, and thereafter increased significantly in peronei, BP‐ST and EDL. The magnitude of the nociceptive withdrawal reflexes in these muscles increased markedly between P7 and P20 and showed little change thereafter. 5. Possible mechanisms underlying the postnatal tuning of the nociceptive withdrawal reflexes are discussed.

Spinal Sensorimotor Transformation: Relation between Cutaneous Somatotopy and a Reflex Network

The projection of primary afferents onto spinal interneurons constitutes the first step in sensorimotor transformations performed by spinal reflex systems. Despite extensive studies on spinal somatotopy, uncertainties remain concerning the extent and significance of representational overlap and relation to spinal reflex circuits. To address these issues, the cutaneous projection from the hindpaw and its relation to the topography of lamina V neurons encoding withdrawal reflex strength (“reflex encoders”) was studied in rats. Thin and coarse primary afferent terminations in laminas II and III–IV, respectively, were mapped by wheat germ agglutinin-horseradish peroxidase and choleragenoid tracing. The functional weights of these projections were characterized by mapping nociceptive and tactile field potentials and compared with the topography of reflex encoders. Both anatomical and physiological data indicate that thin and coarse skin afferent input is spatially congruent in the horizontal plane. The representation of the hindpaw in the spinal cord was found to be intricate, with a high degree of convergence between the projections from different skin sites. “Somatotopic disruptions” such as the representation of central pads medial to that of the digits were common. The weight distribution of the cutaneous convergence patterns in laminas III–IV was similar to that of lamina V reflex encoders. This suggests that the cutaneous convergence and features such as somatotopic disruptions have specific relations to the sensorimotor transformations performed by reflex interneurons in the deep dorsal horn. Hence, the spinal somatotopic map may be better understood in light of the topography of such reflex systems.

Developmental Tuning in a Spinal Nociceptive System: Effects of Neonatal Spinalization

Recent studies indicate a modular organization of the nociceptive withdrawal reflex system. Each module has a characteristic receptive field, closely matching the withdrawal movement caused by its effector muscle. In the rat, the strength of the sensory input to each module is tuned during the first postnatal weeks, i.e., erroneous spinal connections are depressed, and adequate connections are strengthened. To clarify if this tuning is dependent on supraspinal structures, the effect of a complete neonatal spinal cord transection on the postnatal tuning of withdrawal reflexes was studied. The nociceptive receptive fields of single hindlimb muscles and compound withdrawal reflexes were examined in decerebrate unanesthetized and awake rats, respectively. Noxious thermal CO2 laser stimulation was used to evoke reflex responses. Neonatal spinal cord transection resulted in a disrupted reflex organization in the adult rat, resembling that previously found in neonatal rats. The receptive fields of single hindlimb muscles exhibited abnormal distribution of sensitivity not matching the withdrawal action of the effector muscles. Likewise, the composite nocifensive movements, as documented in the awake rat, often resulted in erroneous movements toward the stimulus. It is concluded that withdrawal reflexes do not become functionally adapted in rats spinalized at birth. These findings suggest a critical role for supraspinal systems in the postnatal tuning of spinal nociceptive systems.

Developmental Adaptation of Rat Nociceptive Withdrawal Reflexes after Neonatal Tendon Transfer

Nociceptive withdrawal reflexes (NWRs) were studied in adult rats in which the movement patterns produced by single muscles had been altered by neonatal tendon transfer. NWRs evoked by cutaneous noxious mechanical and thermal (CO2-laser) stimulation were recorded using electromyography in a decerebrate spinal preparation. The sensitivity distribution within the receptive fields of the NWRs of the extensor digitorum longus and the peronei muscles exhibited changes corresponding to the altered movement patterns. No detectable change of NWRs was found in normal muscles whose receptive fields overlapped that of the modified muscle. Furthermore, NWRs of muscles that regained an essentially normal function after neonatal tendon transfer did not differ from normal. It is proposed that a developmental experience-dependent mechanism, which takes into account the hindlimb movement pattern caused by contraction of single muscles, underlies the functionally adapted organization of adult NWRs.

Perforated silicon nerve chips with doped registration electrodes: in vitro performance and in vivo operation

An in vitro model was developed for the study of signal transduction between a Cu-wire, mimicking a neural signal source, and recording electrodes on perforated silicon chips. Phosphorous doped electrodes were used to achieve an all-silicon device. The model was used to study signal amplitude as a function of the spatial position, and distance to the signal source. Recordings of the signal crosstalk to neighboring electrodes on the chips were made. It was found that the amplitude decreased by a factor of two at a distance of 50 /spl mu/m between the electrode surface and the signal source. The chip electrode signal crosstalk was found to be 6 dB using an external reference electrode. Improvements were accomplished with an on chip reference electrode giving a crosstalk suppression of 20 dB. Impedance analysis showed that doped silicon electrodes displayed similar characteristics as Cu-electrodes at frequencies above 3 kHz. Sieve electrodes were implanted in the rat sciatic nerve and following a 10-week nerve regeneration period the dorsal and ventral (L5) roots in the spinal cord were stimulated. Compound action potentials were recorded via the chip. Stimulating the regenerated sciatic nerve via the sieve electrode also induced lower leg muscle contraction activity.

Functional connections are established in the deafferented rat spinal cord by peripherally transplanted human embryonic sensory neurons

Functionally useful repair of the mature spinal cord following injury requires axon growth and the re‐establishment of specific synaptic connections. We have shown previously that axons from peripherally grafted human embryonic dorsal root ganglion cells grow for long distances in adult host rat dorsal roots, traverse the interface between the peripheral and central nervous system, and enter the spinal cord to arborize in the dorsal horn. Here we show that these transplants mediate synaptic activity in the host spinal cord. Dorsal root ganglia from human embryonic donors were transplanted in place of native adult rat ganglia. Two to three months after transplantation the recipient rats were examined anatomically and physiologically. Human fibres labelled with a human‐specific axon marker were distributed in superficial as well as deep laminae of the recipient rat spinal cord. About 36% of the grafted neurons were double labelled following injections of the fluorescent tracers MiniRuby into the sciatic and Fluoro‐Gold into the lower lumbar spinal cord, indicating that some of the grafted neurons had grown processes into the spinal cord as well as towards the denervated peripheral targets. Electrophysiological recordings demonstrated that the transplanted human dorsal roots conducted impulses that evoked postsynaptic activity in dorsal horn neurons and polysynaptic reflexes in ipsilateral ventral roots. The time course of the synaptic activation indicated that the human fibres were non‐myelinated or thinly myelinated. Our findings show that growing human sensory nerve fibres which enter the adult deafferentated rat spinal cord become anatomically and physiologically integrated into functional spinal circuits.

Developmental adaptation of withdrawal reflexes to early alteration of peripheral innervation in the rat.

1. In adult decerebrate spinal rats whose plantar nerves (PLN) had been transected at either postnatal day 1 (P1) or P21 the nociceptive withdrawal reflexes (NWR) of musculi extensor digitorum longus (EDL), peroneus longus (PER) and semitendinosus (ST) were characterized with respect to receptive field (RF) organization, magnitude and time course, using electromyography. Thermal (short CO2 laser pulses) and mechanical (calibrated pinch) stimulation were used. The innervation patterns in normal and lesioned adult rats were assessed by acute nerve lesions. 2. The spatial organization of the mean mechano‐ and thermonociceptive RFs of all the muscles studied was similar to normal in both P1‐ and P21‐lesioned rats, although in some P21‐lesioned rats atypical EDL RFs were encountered. 3. In P1‐lesioned rats thermo‐NWR of PER and EDL had normal magnitudes, while mechano‐NWR were reduced. In P21‐lesioned rats both thermo‐ and mechano‐NWR of these muscles had reduced magnitudes. Except for thermo‐NWR of ST in P1‐lesioned rats, which were increased, NWR of ST had normal magnitudes in both P1‐ and P21‐lesioned rats. The time course of thermonociceptive NWR of the muscles studied were near normal in both P1‐ and P21‐lesioned rats. 4. Acute nerve lesions in adult P1‐lesioned rats revealed an essentially abolished contribution to NWR from the PLN. Instead, the contribution to NWR from other hindpaw nerves, such as the superficial and deep peroneal nerves, was dramatically increased. By contrast, in P21‐lesioned rats, the regenerated PLN contributed significantly to the NWR. 5. It is concluded that despite profound alterations of plantar hindpaw innervation induced by early PLN transection the cutaneous nociceptive input to NWR attained an essentially normal spatial organization. An experience‐dependent mechanism is suggested to be instrumental in adapting the reflex connectivity to the peripheral innervation.

Clinical and electrophysiologic outcome in patients with neovascular glaucoma treated with and without bevacizumab.

Purpose To investigate the clinical and electrophysiologic effect of a single intravitreal injection of bevacizumab for neovascular glaucoma (NVG) after ischemic central retinal vein occlusion (iCRVO). Methods Nineteen eyes from 19 patients with NVG secondary to iCRVO were randomly allocated to either an intravitreal bevacizumab injection and panretinal photocoagulation (PRP) (10 eyes) or PRP alone (9 eyes). The primary outcome measure was the change in the total retinal function 6 months after treatment, demonstrated by full-field electroretinography (ERG). Secondary outcomes included visual acuity, intraocular pressure (IOP), glaucoma medication, additional IOP-lowering treatment, and the presence of ocular neovascularization before treatment, and 1 week, 2 months, and 6 months after treatment. Results The regression of ocular neovascularization in the bevacizumab/PRP group was confirmed 1 week after injection. Patients in both study groups had very poor visual acuity at baseline. This remained unchanged. There was no significant difference in the mean IOP between the groups at any point in time. The a-wave amplitudes of combined rod-cone response were significantly decreased after 6 months in the bevacizumab/PRP group (p=0.028), compared with the baseline values. The a- and b-wave amplitudes of combined rod-cone response and the b-wave amplitudes of the 30-Hz flicker response were also markedly reduced compared with the PRP group (–60%, −43%, −47% vs +23%, −36%, −16%, respectively). Conclusions This study suggests that intravitreal injection of bevacizumab is valuable in the treatment of NVG by hastening the resolution of neovascularization, while the full-field ERG results indicate that bevacizumab may reduce the photoreceptor function in NVG patients.

Pattern recognition of nerve signals using an artificial neural network

By using a microfabricated nerve chip with integrated electrodes through which peripheral nerves can regenerate and make electrical connects, it should be possible to control a remote prosthesis by processing the detected nerve signals. In this study different artificial neural networks have been employed for classification of such complex patterns of signals. The signals were obtained from four electrodes detecting muscle activity in a rat hindlimb as a consequence of applied stimulus to the rat right hindpaw. These signals recorded at four different sites resembles a situation of a nerve chip with four electrodes, which implies that one might be able to use the same strategy when analyzing data from a four-electrode chip to obtain information from the nervous system. Here, the authors address the usefulness of different network topologies for analyzing measured in vivo data from an implanted perforated nerve chip.