J.D. Leah

Selective expression of Jun proteins following axotomy and axonal transport block in peripheral nerves in the rat : evidence for a role in the regeneration process

Expression of the protein products of the immediate-early genes (IEGs), members of the fos, jun and krox families (Jun, Fos, and Krox, resp.) was investigated in the spinal cord and sensory ganglia (DRG) of normal rats; and following transection of, block of axonal transport in, or electrical stimulation of their peripheral axons. The nuclei of many moto- and DRG neurons showed a faint basal immunoreactivity (IR) for Jun proteins, but not for Fos or Krox proteins. There was a strong and selective induction of Jun-IR in moto- and DRG neurons after peripheral nerve transection or crush, or colchicine- or vinblastine-induced block of axonal transport. The Jun-IR induced by nerve transection disappeared after nerve regeneration. In contrast, Jun, Fos and Krox proteins were all induced transynaptically in spinal dorsal horn neurons following electrical stimulation of the C-fibers in the afferent nerves. Thus in differentiated neurons in vivo these IEG proteins can be expressed either independently or concomitantly depending on the type of stimulus.

Expression of c-JUN, JUN B and JUN D proteins in rat nervous system following transection of vagus nerve and cervical sympathetic trunk.

Expression of c-JUN, JUN B and JUN D proteins was investigated in axotomized neurons following transection of the vagus nerve and the cervical sympathetic trunk in the rat. Vagotomy induced the expression of c-JUN and JUN D in the nodose ganglion, dorsal motor nucleus of the vagus nerve and nucleus ambiguus, whereas JUN B was not expressed in these areas, c-JUN and JUN D appeared after 10 h in the nodose ganglion and after 24 h in the dorsal motor nucleus of the vagus nerve with a maximum of immunoreactivity after 48 h. The c-JUN protein remained expressed at an increased level up to 100 days, whereas the immunoreactivity of JUN D declined after five days. Crush of the vagus nerve initially evoked an intense expression of c-JUN and JUN D, but in the course of regeneration the expression of c-JUN and JUN D had returned to more basal levels after 100 days. Similar to vagotomy, application of colchicine and vinblastine on to the intact vagus nerve induced expression of c-JUN and JUN D. On the other hand, application of lidocaine prior to vagotomy did not prevent the expression of these proteins. Transection of the cervical sympathetic trunk induced expression of c-JUN and JUN D, but not of JUN B, in the preganglionic sympathetic neurons of thoracic spinal cord. In these neurons, expression of c-JUN was still enhanced after 60 days whereas JUN D had returned to basal level. One hour after vagotomy, c-JUN and JUN B were transynaptically expressed in the area of central termination of sensory vagal neurons and declined within 10 h to basal levels. JUN D showed a late onset of expression, it appeared after 5 h and persisted for 60 days in this area. We postulate that the expression of c-JUN and JUN D in axotomized neurons is induced by deprivation of a target-derived suppressor.

Krox20 may play a key role in the stabilization of long-term potentiation

Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 required stimulation sufficient to induce LTP and was prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increased within 20 min of tetanization, was maximal between 1 and 8 h, and was still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast with the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 was of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation.

Expression of immediate early gene proteins following axotomy and inhibition of axonal transport in the rat central nervous system.

The expression of the immediate early gene-encoded proteins c-Jun, Jun B, Jun D, c-Fos, Fos B and Krox-24 in central neurons following transection of, or inhibition of, axonal transport in their axons was investigated in the rat using immunocytochemistry. Transection of the medial forebrain bundle, which produces an essentially complete axotomy of neurons in the ipsilateral mammillary nucleus, substantia nigra pars compacta, ventral tegmental area and parafascicularis, induced the expression of c-Jun, Jun D and, to a lesser extent, Krox-24, in these nuclei. Microinjection of colchicine into the medial forebrain bundle to chemically inhibit axonal transport similarly induced the expression of these proteins in these areas. The expression of the proteins was first evident 24 h after transection, reached a maximum at 48 h and was still present after 10 days. However, after 30 days the proteins were absent from the substantia nigra, ventral tegmentum and parafascicularis, and were still present only in the mammillary nuclei. The other immediate early genes, Jun B, c-Fos and Fos B, were never expressed above the basal levels seen in untreated rats. Transection of the corpus callosum and the hippocampal commissure, which produces only a partial axotomy of neurons in the cerebral cortex and hippocampus, respectively, did not induce the expression of any of the genes in these neurons. Microinjection of colchicine or vinblastine to produce a localized inhibition of axonal transport in the cerebral cortex, hippocampus, thalamus and cerebellum also induced the expression of c-Jun, Jun D and, again to a lesser extent, Krox-24, in neurons surrounding the injection site. In contrast to this selective expression, administration of the neuronal excitant metrazole induced the expression of all six immediate early gene proteins in central nervous system neurons. These results demonstrate that transection of, or inhibition of, transport in the axons of central neurons induces a particular pattern of expression of transcriptionally operating immediate early genes that may be related to the regenerative competency of the neurons.

Differential expression of immediate early genes after hippocampal long-term potentiation in awake rats

The pattern of expression of fos and jun family immediate early genes following the induction of long-term potentiation (LTP) was investigated in the dentate gyrus of awake rats. Rapid, transient increases in the levels of c-jun and jun-B mRNA and protein, and in the levels of Fos-related proteins (FRAs), occurred in the dentate gyrus after LTP-inducing tetanization of the perforant path. A delayed, and more prolonged induction occurred for jun-D mRNA and protein. The induction of c-Jun, Jun-B, Jun-D and Fos-related proteins was prevented by administration of an N-methyl-D-aspartate receptor antagonist, which also blocked LTP induction, and by pentobarbital, which reduced but did not block LTP. These findings show that differential expression of fos and jun gene family members occurs in a distinct pattern following LTP in awake rats. The responsive genes may participate in the biochemical cascade leading to the long-term stabilization of synaptic modifications.

c-JUN-like immunoreactivity in the CNS of the adult rat: Basal and transynaptically induced expression of an immediate-early gene

An immunocytochemical study of dorsal root ganglia, spinal cord and medulla oblongata was performed with antisera against the c-jun proto-oncogene encoded protein. The c-JUN-like immunoreactivity was restricted to the cell nucleus. In the CNS of untreated rats a basal c-JUN-like immunoreactivity was present in the nuclei of two types of neurons: motor and autonomic. Labelled nuclei could be seen in many motoneurons of the ventral horn of the entire length of spinal cord and the lower medulla oblongata, as well as in the area of the nucleus hypoglossus, the dorsal motor nucleus of nucleus vagus, nucleus ambiguus, nucleus facialis, nucleus abducens and motor nucleus of nucleus trigeminus. Additionally, labelled nuclei were found in the preganglionic sympathetic and preganglionic parasympathetic cells of the nucleus intermediolateralis and nucleus intercalatus in the spinal cord. In the medulla oblongata we found a cluster of cells with c-JUN-like immunoreactivity in an area between the dorsomedial part of the oral nucleus spinalis trigeminalis and the lateral border of the knee of facial nerve. Additionally, a second cluster of c-JUN-like immunoreactivity cells was visible between the ventromedial part of the oral nucleus spinalis trigeminalis and the lateral border of the rostral nucleus facialis. Examination of the characteristics of all cell groups with a basal c-JUN-like immunoreactivity in the spinal cord and lower brainstem revealed an overlapping distribution with cholinergic cell groups. Basal c-JUN-like immunoreactivity was also seen in the dorsal root ganglion cells. We examined the factors which can effect the expression of the c-JUN protein. Maximal expression of c-JUN-like immunoreactivity was observed after electrical stimulation of primary afferents. Stimulation of sciatic nerve at a strength sufficient to recruit A delta- and C-fibres produced c-JUN-like immunoreactivity in many nuclei of the ipsilateral dorsal horn of the lumbar spinal cord. c-JUN-like immunoreactivity was first detectable at 30 min following the end of stimulation, reached a maximum after 1 h, remained unchanged for another 1 h and declined to the basal level after 16 h. The distribution of c-JUN-like immunoreactivity in the lumbar cord coincided with the region of termination of sciatic nociceptive afferents. Contralateral c-JUN-like immunoreactivity appeared after 4 h. After noxious mechanical stimulation of the plantar hindpaw c-JUN-like immunoreactivity occurred in the spinal area of termination of nociceptive afferents of the tibial nerve. Noxious stimulation did not provoke additional c-JUN-like immunoreactivity in dorsal root ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Potentiated expression of FOS protein in the rat spinal cord following bilateral noxious cutaneous stimulation.

A noxious mechanical or chemical stimulus to the ventral skin of one hindpaw induced the expression of FOS proteins ipsilaterally in the spinal dorsal horn neurons in the rat. The number of FOS-labelled cells reached a maximum at 2-3 h, and decayed to basal levels within 6 h after the stimulus. When a first noxious stimulus was applied to the contralateral hindpaw 1-1.5 h prior to this stimulus, the number of FOS-labelled cells increased, over all laminae, to 153% (mechanical) and 164% (chemical) compared to the number produced by a single stimulus. This effect of a prior stimulus in increasing the number of FOS-labelled cells produced by a contralateral stimulus persisted for several hours after the first stimulus. The results are interpreted as a sensitization of dorsal horn neurons induced by peripheral noxious stimuli, which is manifest at the molecular biological level.

Prolonged and selective induction of Fos-related antigen(s) in striatal neurons after 6-hydroxydopamine lesions of the rat substantia nigra pars compacta ☆

Unilateral 6-hydroxydopamine (6-OHDA) lesions of the rat substantia nigra lead to a large widespread and long-lasting (greater than 3 months) increased expression of Fos-related antigen(s) (FRAs) in striatal neurons ipsilateral to the side of the lesion. However, Fos and Jun expression were only very slightly increased in a few scattered neurons in the dopamine-denervated striatum. These results demonstrate that FRAs are induced long-term in striatal neurons following dopamine-depletion. This increased production of FRAs may be related to neuropeptide and/or D2 dopamine receptor upregulation that also occurs in the dopamine-denervated striatum.

Brainstem peptidergic neurons projecting to the medial and lateral thalamus and zona incerta in the rat.

The presence of neuropeptides in brainstem neurons that project to the medial and lateral thalamus and zona incerta has been studied in the rat. Brainstem neurons were retrogradely labeled from the medial and lateral thalamus and the zona incerta by colloidal gold-WGA-HRP and, after silver intensification of the retrograde label, their content of immunoreactivity for nine different neuropeptides was determined after colchicine administration. The medial thalamus and zona incerta both received a large peptidergic input and the lateral thalamus a smaller input from neurons in several brainstem nuclei. These were principally from the locus coeruleus, parabrachial nucleus, the dorsal raphe and the dorsal tegmentum. The principal input to the medial thalamus arose from neurotensin, neuropeptide Y and galanin neurons in the locus coeruleus, neurotensin neurons in the dorsal tegmentum, dynorphin neurons in the parabrachial nucleus and dorsal tegmentum, galanin neurons in the dorsal raphe, substance P neurons in the lateral and dorsal periaqueductal grey and calcitonin gene-related peptide neurons in the nucleus paragigantocellularis. The principal peptidergic input to the zona incerta was from dynorphin neurons in the nucleus of the solitary tract, bombesin neurons in the lateral reticular nucleus, calcitonin gene-related peptide and cholecystokinin neurons in the dorsal tegmentum, substance P, bombesin and galanin neurons in the locus coeruleus, dynorphin and substance P neurons in the lateral periaqueductal grey and cholecystokinin neurons in the substantia nigra, ventral tegmental nucleus and raphe linearis. The principal peptidergic input to the lateral thalamus came from calcitonin gene-related peptide and cholecystokinin neurons in the dorsal tegmentum, calcitonin gene-related peptide and galanin neurons in the locus coeruleus; substance P, neuropeptide Y, galanin and calcitonin gene-related peptide neurons in the dorsal raphe, substance P neurons in the lateral periaqueductal gray, galanin neurons in the nucleus interpedunculus and cholecystokinin neurons in the raphe linearis. In all these cases, from 25% to virtually all of the projection neurons in the brainstem nucleus could contain immunoreactivity to the neuropeptide. A lesser, but significant peptidergic input to the thalamus and zona incerta also arose from the trigeminal nucleus, the substantia nigra, the nucleus of the solitary tract, the lateral reticular nucleus, the interpeduncular nucleus, the raphe linearis, the paragigantocellularis, the inferior olive and ventral tegmental area. Overall, the neuropeptides most frequently present in the projection neurons were substance P, calcitonin gene-related peptide, galanin and cholecystokinin. Bombesin, neuropeptide Y, neurotensin and dynorphin were less common; and enkephalin was present in only a small percentage of projection neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Jun, Fos and Krox in the thalamus after C-fiber stimulation: coincident-input-dependent expression, expression across somatotopic boundaries, and nucleolar translocation

Expression of the inducible transcription factors Jun, Fos and Krox is commonly used to map neurons in the brain that are activated by sensory inputs. However, some neurons known to be electrically excited by such inputs do not always express these factors. In particular, stimulation of hindlimb sensory nerve C-fibers induces expression of c-Fos in the medial thalamus (the mediodorsal, intermediodorsal, centrolateral and centromedial), but not in the lateral thalamus (the ventroposterolateral, ventroposteromedial and posterior group). We hypothesized that c-Fos expression might only occur in these lateral areas after more complex stimulation patterns, or that only other transcription factors can be induced in these areas by such stimuli. Thus we examined the effects of single, repeated and coincident C-fiber inputs on expression of six inducible transcription factors in the medial, lateral and reticular thalamus of the rat. A weak C-fiber input caused by noxious mechanical stimulation of the skin of one hindpaw did not induce expression of c-Fos, FosB, Krox-20 or Krox-24; but it did reduce the basal expressions of c-Jun and JunD in both the medial and lateral areas. An intense input produced by electrical stimulation of all the C-fibers in one sciatic nerve also failed to induce expression of c-Fos, FosB, Krox-20 or Krox-24 in the medial or lateral areas. However, in the medial thalamus it increased c-Jun and reduced the basal expression of JunD, whereas in the lateral thalamus it had no effect on c-Jun but again reduced the basal expression of JunD. With repeated stimulation, i.e. when the noxious stimulus was applied to the contralateral hindpaw 6 h after the sciatic stimulation, there was again no induction of c-Fos, FosB or Krox-20 in the medial thalamus; but there was an increase in c-Jun and Krox-24, and a decrease in JunD levels. In the lateral thalamus the repeated stimulation again failed to induce c-Fos, but the expressions of FosB, c-Jun and Krox-24 were increased, and that of JunD was again reduced. With coincident stimulation, i.e. when a stimulus was applied to each hindpaw simultaneously, c-Fos and Krox-24 remained absent; but there was a marked induction of FosB and Krox-20, a strong repression of c-Jun, and no effect or a reduction of the basal levels of JunD. This coincident stimulation also caused FosB to appear in the nucleolus of many thalamic neurons. MK-801, but not L-NAME, blocked all these changes. In summary, noxious stimulation affects the expression of all transcription factors in the medial, lateral and reticular thalamus in a complex manner depending upon the inducible transcription factor considered, the thalamic nucleus, and the stimulation paradigm. The expression of some transcription factors uniquely after simultaneous inputs suggests they act as coincidence detectors at the gene level.