Isaura Tavares

GABA decreases in the spinal cord dorsal horn after peripheral neurectomy

A significant fall in the number of GABA-immunoreactive cells in laminae I-III of the rat spinal cord occurred in the somatotopic area of projection of the sciatic nerve after nerve transection. The decrease started at 2 weeks post-neurectomy, and at 4 weeks ipsilateral mean cell numbers were approximately 72% of contralateral control values. Similarly, the concentration of GABA in spinal homogenates was significantly reduced 4 weeks post-neurectomy. These data, together with our recent finding of an increase in spinal GABA during chronic inflammation of the hindlimb, suggest that the level of GABA in the dorsal horn is regulated by the amount of primary afferent input.

Increase in GABAergic Cells and GABA Levels in the Spinal Cord in Unilateral Inflammation of the Hindlimb in the Rat

The effects of chronic peripheral inflammation on spinal cord γ‐aminobutyric acid (GABA) were examined in the rat. Following the injection of complete Freunds adjuvant in the left hindlimb footpad an increased number of immunoreactive cells occurred in ipsilateral laminae I‐III of the dorsal horn from L3 to L5. GABA‐immunoreactive cells were more numerous than contralaterally 1 week after the onset of the inflammation, reached maximal numbers after 3–4 weeks, and declined thereafter. Differences from control sides were statistically significant except at week 6. GABA levels in homogenates of the ipsilateral lumbar enlargement were increased significantly at 4 weeks. Since increases in GABA occurred in the spinal cord zone of projection of the nerves supplying the inflamed foot, the central response is surmised to result from the increased nociceptive input arriving from the periphery. However, the transmission from primary axons to GABA interneurons is not likely to be monosynaptic since profiles containing glutamate decarboxylase or GABA immunoreactivity are known to be predominantly presynaptic, and rarely postsynaptic, to primary afferent endings in electron micrographs in the rat. The findings support the function attributed to spinal GABA in modulating nociceptive input at segmental level.

Neurons in the superficial dorsal horn of the rat spinal cord projecting to the medullary ventrolateral reticular formation express c-fos after noxious stimulation of the skin

The nociceptive nature of the neurons of the superficial dorsal horn (laminae I-III) which project to the medullary ventrolateral reticular formation is studied in the rat. Medullary injections of Fluoro-Gold showed exclusive retrograde labeling of laminae I-III cells when the tracer filled a zone intermediate between the lateral tip of the lateral reticular nucleus and the spinal trigeminal nucleus, pars caudalis. This zone is here called VLMlat. Following noxious mechanical or thermal stimulation of the skin, double-labeled neurons, which stained retrogradely and were Fos-immunoreactive, prevailed in laminae I and IIo. Double-labeled neurons were few in lamina IIi after thermal stimulation and entirely lacking in lamina III after the two kinds of stimulation. Findings in lamina I confirm previous electrophysiological data (see Menétrey et al., J. Neurophysiol., 52 (1984) 595-611) showing that lamina I cells projecting to the ventrolateral reticular medulla convey noxious messages. The occurrence of numerous double-labeled cells in lamina IIo suggests that this lamina is also involved in nociceptive transmission to the VLMlat.

Carrageenan-induced inflammation of the hind foot provokes a rise of GABA-immunoreactive cells in the rat spinal cord that is prevented by peripheral neurectomy or neonatal capsaicin treatment

&NA; An increase in the number of &ggr;‐aminobutyric acid (GABA)‐immunorcactive cells is reported in the superficial dorsal horn of the rat spinal cord upon unilateral inflammation of the hind foot caused by subcutaneous carrageenan injection. The rise of GABAergic cells was restricted to the ipsilateral dorsal horn, reaching a peak value of 23.4% over the contralateral side 4 days after carrageenan injection. Sciatic neurectomy or neonatal capsaicin treatment prevented this effect. These findings suggest that dorsal horn GABA is up‐regulated by the increase of noxious inflow conveyed by unmyelinated C fibers from the inflamed tissues.

Descending projections from the caudal medulla oblongata to the superficial or deep dorsal horn of the rat spinal cord

The location of neurons in the caudal medulla oblongata that project to the superficial or deep dorsal horn was studied in the rat, by means of retrograde labelling from confined spinal injection sites. The tracer cholera toxin subunit B was injected into laminae I–III (fuve rats) or I–V (three rats) at C4–7 spinal segments. Neurons projecting to the superficial dorsal horn were located in the dorsomedial part of the dorsal reticular nucleus ipsilaterally, the subnucleus commissuralis of the nucleus tractus solitarius bilaterally, and a region occupying the lateralmost part of the ventrolateral reticular formation between the lateral reticular nucleus and the caudal pole of the spinal trigeminal nucleus, pars caudalis, bilaterally. Neurons projecting to the deep dorsal horn, which were only labelled when laminae I–V were filled by the tracer, occurred in the dorsomedial and ventrolateral parts of the dorsal reticular nucleus and in the ventral reticular nucleus bilaterally. A few cells were located in the above described lateralmost portion of the ventrolateral reticular formation bilaterally and in the ventral portion of the ipsilateral cuneate nucleus. In the light of previous data demonstrating that dorsal horn neurons project to the dorsal reticular nucleus, the ventrolateral reticular formation, and the nucleus tractus solitarius, and that neurons in these three medullary regions are involved in pain inhibition at the spinal level, the descending projections demonstrated here suggest the occurrence of spino-medullary-spinal loops mediating the analgesic actions elicited in each nucleus upon the arrival of nociceptive input from the dorsal horn.

Descending projections from the medullary dorsal reticular nucleus make synaptic contacts with spinal cord lamina I cells projecting to that nucleus: An electron microscopic tracer study in the rat

An ultrastructural study is made of the synaptic contacts occurring between structures labelled anterogradely and retrogradely in the superficial dorsal horn following injections of cholera toxin subunit B or horseradish peroxidase in the dorsal reticular nucleus of the medulla oblongata of the rat. Both tracers revealed labelled axonal boutons in lamina I with round synaptic vesicles and a few large granular vesicles making asymmetrical synaptic contacts upon labelled somata and dendrites. After injections of Phaseolus vulgaris leucoagglutinin in the dorsal reticular nucleus, labelled boutons identical to those revealed by the two other tracers were presynaptic to unlabelled somata and dendrites. In addition, dorsoreticular neurons were labelled retrogradely following injections of cholera toxin subunit B into the superficial dorsal horn of the cervical enlargement. These observations show the occurrence of a reciprocal connection between dorsal reticular and lamina I neurons. Considering the putative excitatory nature of the axodendritic contacts in lamina I, a positive feedback circuit is suggested, whereby the nociceptive signals transmitted to the dorsal medullary reticular formation by marginal neurons are intensified.

Minocycline completely reverses mechanical hyperalgesia in diabetic rats through microglia-induced changes in the expression of the potassium chloride co-transporter 2 (KCC2) at the spinal cord.

Aim: Neuronal hyperactivity at the spinal cord during mechanical hyperalgesia induced by diabetes may result from a decrease in the local expression of the potassium chloride co‐transporter 2 (KCC2), which shifts the action of the neurotransmitter γ‐amminobutiric acid (GABA) from inhibitory to excitatory. In this study, we evaluated the effects of spinal microglia inhibition or brain‐derived neurotrophic factor (BDNF) blockade on KCC2 expression, spinal neuronal activity and mechanically induced pain responses of streptozotocin (STZ)‐diabetic rats.

Brain afferents to the medullary dorsal reticular nucleus: a retrograde and anterograde tracing study in the rat

The medullary dorsal reticular nucleus (DRt) was recently shown to belong to the supraspinal pain control system; neurons within this nucleus give origin to a descending projection that increases spinal nociceptive transmission and facilitates pain perception [Almeida et al. (1999), Eur. J. Neurosci., 11, 110–122]. In the present study, the areas of the brain that may modulate the activity of DRt neurons were investigated by using of tract‐tracing techniques. Injection of a retrograde tracer into the DRt resulted in labelling in multiple areas of the brain. In the contralateral orbital, prelimbic, infralimbic, insular, motor and somatosensory cortices labelling was prominent, but a smaller ipsilateral projection from these same areas was also detected. Strong labelling was also noted in the central amygdaloid nucleus, bed nucleus of stria terminalis and substantia innominata. Labelled diencephalic areas were mainly confined to the hypothalamus, namely its lateral and posterior areas as well as the paraventricular nucleus. In the mesencephalon, the periaqueductal grey, red nucleus and deep mesencephalic nucleus were strongly labelled, whereas, in the brainstem, the parabrachial nuclei, rostroventromedial medulla, nucleus tractus solitarius, spinal trigeminal nucleus, and the parvocellular, dorsal, lateral and ventral reticular nuclei were the most densely labelled regions. All deep cerebellar nuclei were labelled bilaterally. These data suggest that the DRt integrates information from the somatosensory, antinociceptive, autonomic, limbic, pyramidal and extrapyramidal systems while triggering its descending facilitating action upon the spinal nociceptive transmission.

Noxious-evoked c-fos expression in brainstem neurons immunoreactive for GABAB, µ-opioid and NK-1 receptors

Modulation of nociceptive transmission at the brainstem involves several neurochemical systems. The precise location and specific characteristics of nociceptive neurons activated in each system was never reported. In this study, the presence of GABAB, µ‐opioid, and neurokinin‐1 (NK‐1) receptors in brainstem nociceptive neurons was investigated by double‐immunocytochemical detection of each receptor and noxious‐evoked induction of the c‐fos proto‐oncogene. Noxious cutaneous mechanical stimulation significantly increased the proportions of neurons double‐labelled for Fos and GABAB receptors in several brainstem regions, namely, the reticular formation of the caudal ventrolateral medulla (VLMlat and VLMrf), lateral reticular nucleus, spinal trigeminal nucleus, pars caudalis (Sp5C), nucleus of the solitary tract, dorsal reticular nucleus, ventral reticular nucleus, raphe obscurus nucleus and dorsal parabrachial nucleus (DPB). For µ‐opioid receptors, the proportions of double‐labelled neurons in noxious‐stimulated animals were higher than in controls only in the VLMlat, VLMrf, Sp5C, DPB and A5 noradrenergic cell group. As for the NK‐1 receptor, no significant differences were found between control and stimulated animals. According to these results, neurons expressing GABAB, µ‐opioid and NK‐1 receptors at several pain control centres of the brainstem are differentially involved in processing nociceptive mechanical input. The data provide the definition of new supraspinal targets for selective modulation of nociceptive neurons in order to define better strategies of pain control.

Projection sites of superficial and deep spinal dorsal horn cells in the nucleus tractus solitarii of the rat.

By using anterograde transport of biotin dextran amine injected into the cervical spinal dorsal horn, we have shown that fibres from superficial and deep dorsal horn project to the nucleus tractus solitarii via two distinct pathways. Afferent fibres from the superficial lamina (I-III) were found to course in the dorsal funiculus and terminate bilaterally in the caudal zone of the nucleus tractus solitarii (NTS), mainly within the commissural subnucleus. In contrast, afferents from the deeper dorsal horn laminae (IV-V) were found to course in the dorsolateral fasciculus and terminate ipsilaterally, mostly in the lateral areas of the caudal nucleus tractus solitarii. Similar, but more extensive patterns of labelled fibres were produced by injections into the white matter of the dorsal funiculus and dorsolateral fasciculus, respectively. These observations suggest that the caudal NTS not only serves as a location of visceral afferent convergence and integration, but may also be a receptive area for monosynaptic projections from dorsal horn neurons receiving sensory afferent inputs. Such projections may represent pathways through which NTS neurons are influenced by nociceptive and non-nociceptive information from the dorsal horn and thereby can co-ordinate the appropriate autonomic response, including adjustments in cardiorespiratory reflex output.