Jens Hannibal

Pituitary adenylate cyclase-activating peptide in the rat central nervous system: an immunohistochemical and in situ hybridization study.

In the present study the localization of pituitary adenylate cyclase‐activating peptide (PACAP)‐expressing cell bodies and PACAP projections were mapped in the adult rat brain and spinal cord by using immunohistochemistry and in situ hybridization histochemistry. A widespread occurrence of PACAP‐containing cell bodies was found, with the greatest accumulation in several hypothalamic nuclei and in several brainstem nuclei, especially the habenular nuclei, the pontine nucleus, the lateral parabrachial nucleus (LPB), and the vagal complex. PACAP was also present in cell bodies in the olfactory areas, in neocortical areas, in the hippocampus, in the vestibulo‐ and cochlear nuclei, in cell bodies of the intermediolateral cell column of the spinal cord and in Purkinje cells of the cerebellum, in the subfornical organ, and in the organum vasculosum of the lamina terminalis. An intense accumulation of PACAP‐immunoreactive (‐IR) nerve fibers was observed throughout the hypothalamus, in the amydaloid and extended amygdaloid complex, in the anterior and paraventricular thalamic nuclei, in the intergeniculate leaflet, in the pretectum, and in several brainstem nuclei, such as the parabrachial nucleus, the sensory trigeminal nucleus, and the nucleus of the solitary tract. PACAP‐IR nerve fibers were also found in the area postrema, the posterior pituitary and the choroid plexus, and the dorsal and ventral horn of the spinal cord. The widespread distribution of PACAP in the brain and spinal cord suggests that PACAP is involved in the control of many autonomic and sensory functions as well as higher cortical processes. J. Comp. Neurol. 453:389–417, 2002.

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.

Dissociation between Light-Induced Phase Shift of the Circadian Rhythm and Clock Gene Expression in Mice Lacking the Pituitary Adenylate Cyclase Activating Polypeptide Type 1 Receptor

The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1−/−) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type andPAC1−/− mice. However, in contrast to the phase advance seen in wild-type mice,PAC1−/− mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest thatmPer1, mPer2, and c-fosare of less importance for light-induced phase shifts at night.

Pituitary adenylate cyclase activating peptide expression in the rat dorsal root ganglia : up-regulation after peripheral nerve injury

Pituitary adenylate cyclase activating peptide (PACAP) is expressed in a population of capsaicin-sensitive primary sensory neurons of small to medium size in the rat. In the present report we have examined the effect of sciatic nerve injury (unilateral transection) on PACAP expression (immunocytochemistry, radioimmunoassay, in situ hybridization and northern blot analysis) in dorsal root ganglia at the lumbar level and on immunoreactive PACAP in the spinal cord and in the sciatic nerve stump. For comparison, calcitonin gene-related peptide was examined. In dorsal root ganglia of the intact side immunoreactive PACAP and PACAP messenger RNA were localised to a population of nerve cell bodies of small to medium size. In dorsal root ganglia on the injured side, PACAP-immunoreactive nerve cell bodies were more numerous and PACAP messenger RNA was considerably more abundant as studied 14 days after sciatic nerve transection. By contrast, calcitonin gene-related peptide-containing nerve cell bodies were numerous and rich in calcitonin gene-related peptide messenger RNA in dorsal root ganglia on the intact side, while after transection both the number of immunoreactive nerve cell bodies and their content of messenger RNA were markedly reduced. There were indications of axotomy-induced expression of PACAP messenger RNA in larger neurons. In the dorsal horn of the spinal cord on the intact side PACAP and calcitonin gene-related peptide-immunoreactive fibres were densely accumulated in the superficial layers. On the transected side the densities of both PACAP and calcitonin gene-related peptide-immunoreactive nerve fibres were reduced in the medial part. The data obtained indicate a marked up-regulation of PACAP in sensory neurons following peripheral nerve injury. Since PACAP depresses a C-fibre evoked flexion reflex, this may have implications for sensory transmission. Further, in view of the known promoting effects of PACAP on neuronal survival and differentiation and non-neuronal cell growth as well as its proinflammatory effects a role of PACAP in the neuronal and periaxonal tissue restoration after injury is not inconceivable.

Target areas innervated by PACAP-immunoreactive retinal ganglion cells

The retinohypothalamic tract (RHT) originates from a subset of retinal ganglion cells (RGCs). The cells of the RHT co-store the neurotransmitters PACAP and glutamate, which in a complex interplay mediate light information to the circadian clock located in the suprachiasmatic nuclei (SCN). These ganglion cells are intrinsically photosensitive probably due to expression of melanopsin, a putative photoreceptor involved in light entrainment. In the present study we examined PACAP-containing retinal projections to the brain using intravitreal injection of the anterograde tracer cholera toxin subunit B (ChB) and double immunostaining for PACAP and ChB. Our results show that the PACAP-containing nerve fibres not only constituted the major projections to the SCN and the intergeniculate leaflet of the thalamus but also had a large terminal field in the olivary pretectal nucleus. The contralateral projection dominated except for the SCN, which showed bilateral innervation. PACAP-containing retinal fibres were also found in the ventrolateral preoptic nucleus, the anterior and lateral hypothalamic area, the subparaventricular zone, the ventral part of the lateral geniculate nucleus and the nucleus of the optic tract. Retinal projections not previously described in the rat also contained PACAP. These new projections were found in the lateral posterior nucleus, the posterior limitans nucleus, the dorsal part of the anterior pretectal nucleus and the posterior and medial pretectal nuclei. Only a few PACAP-containing retinal fibres were found in the superior colliculus. Areas innervated by PACAP-immunoreactive fibres also expressed the PACAP-specific PAC1 receptor as shown by in situ hybridization histochemistry. The findings suggest that PACAP plays a role as neurotransmitter in non-imaging photoperception to target areas in the brain regulating circadian timing, masking, regulation of sleep-wake cycle and pupillary reflex.

Expression of pituitary adenylate cyclase-activating polypeptide in dorsal root ganglia following axotomy: time course and coexistence.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been demonstrated in sensory neurons. In the present study on rat 17.5% of all neurons, mainly of small size, contained PACAP in normal dorsal root ganglia (DRGs). Transection of the sciatic nerve induced a rapid and strong upregulation in PACAP peptide and mRNA levels which could be seen already after 15 h. After 3 days more than 51.5% of neurons of different sizes expressed PACAP. However, the intensity of PACAP-LI in the DRG neurons declined after 10 days. Thirty days after axotomy, 56.7% of the DRG neurons still expressed PACAP, but with a low intensity, in fact even lower than in normal controls. No VIP- or NPY-positive neurons were observed in normal or axotomized DRGs at 15 h. However a distinct increase in VIP and NPY levels were seen 3 days after the lesion, and their levels were considerably higher after 30 days. PACAP was often present in neurons expressing VIP, NPY and/or galanin. Thus, 3 days after injury, PACAP was present in 84.4%, 95.7%, and 76.8% of the VIP-, NPY-, and galanin-positive neurons, respectively. PACAP was also found in nerve fibers in control sciatic nerves. After nerve ligation, accumulation of PACAP was seen mainly proximal to the injury but also distally, suggesting both anterograde and retrograde transport of the peptide. Also a moderate increase (about 20%) in PACAP levels was found in the superficial spinal dorsal horn 3 days after nerve transection. Taken together, our results suggest that PACAP is involved in the response to nerve injury. The very high levels of expression in different populations of DRG neurons after axotomy, and its different time course as compared to galanin, NPY and VIP indicate that it may play a complementary and/or different role than these peptides in the adaptation to nerve injury, especially in its early phase.

Melanopsin retinal ganglion cells are resistant to neurodegeneration in mitochondrial optic neuropathies

Mitochondrial optic neuropathies, that is, Leber hereditary optic neuropathy and dominant optic atrophy, selectively affect retinal ganglion cells, causing visual loss with relatively preserved pupillary light reflex. The mammalian eye contains a light detection system based on a subset of retinal ganglion cells containing the photopigment melanopsin. These cells give origin to the retinohypothalamic tract and support the non-image-forming visual functions of the eye, which include the photoentrainment of circadian rhythms, light-induced suppression of melatonin secretion and pupillary light reflex. We studied the integrity of the retinohypothalamic tract in five patients with Leber hereditary optic neuropathy, in four with dominant optic atrophy and in nine controls by testing the light-induced suppression of nocturnal melatonin secretion. This response was maintained in optic neuropathy subjects as in controls, indicating that the retinohypothalamic tract is sufficiently preserved to drive light information detected by melanopsin retinal ganglion cells. We then investigated the histology of post-mortem eyes from two patients with Leber hereditary optic neuropathy and one case with dominant optic atrophy, compared with three age-matched controls. On these retinas, melanopsin retinal ganglion cells were characterized by immunohistochemistry and their number and distribution evaluated by a new protocol. In control retinas, we show that melanopsin retinal ganglion cells are lost with age and are more represented in the parafoveal region. In patients, we demonstrate a relative sparing of these cells compared with the massive loss of total retinal ganglion cells, even in the most affected areas of the retina. Our results demonstrate that melanopsin retinal ganglion cells resist neurodegeneration due to mitochondrial dysfunction and maintain non-image-forming functions of the eye in these visually impaired patients. We also show that in normal human retinas, these cells are more concentrated around the fovea and are lost with ageing. The current results provide a plausible explanation for the preservation of pupillary light reaction despite profound visual loss in patients with mitochondrial optic neuropathy, revealing the robustness of melanopsin retinal ganglion cells to a metabolic insult and opening the question of mechanisms that might protect these cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) in the gastrointestinal tract of the rat: distribution and effects of capsaicin or denervation

Abstract The expression of pituitary adenylate cyclase activating polypeptide (PACAP) was studied in the gastrointestinal tract (GI-tract) of normal rats using radioimmunoassay, chromatography, immunocytochemistry, and in situ hybridization. PACAP-38, PACAP-27, and PACAP-related peptide were demonstrated in all parts of the GI-tract, PACAP-38 being the predominant form confirmed by chromatography. PACAP-immunoreactive nerve fibers and nerve cell bodies were found in the myenteric ganglia throughout the GI-tract. PACAP-containing nerve cell bodies were also demonstrated in the submucous ganglia of the small and large intestine. The synthesis of PACAP in intrinsic neurons was confirmed by in situ hybridization. Double immunostaining showed that PACAP is present in calcitonin gene-related peptide-containing sensory nerve fibers as well as in vasoactive intestinal polypeptide (VIP)- or VIP/gastrin-releasing peptide (GRP)-containing (intramural) nerve fibers in the upper GI-tract and in anally projecting, intrinsic VIP-and VIP/nitric oxide syntase-containing nerve cell bodies and nerve fibers in the small and large intestine. Neonatal treatment with capsaicin significantly reduced the concentration of PACAP-38 in the esophagus, stomach, and colon. Extrinsic denervation decreased the PACAP-38 concentration in the stomach, while no change was observed in the small intestine. These results indicate that PACAP- immunoreactive nerve fibers in the GI-tract originate from both intrinsic (enteric) and extrinsic (presumably sensory) sources suggesting that PACAP may have diverse gastrointestinal functions.

The effects of axotomy and preganglionic denervation on the expression of pituitary adenylate cyclase activating peptide (PACAP), galanin and PACAP type 1 receptors in the rat superior cervical ganglion

The effects of axotomy, chemical sympathectomy and preganglionic denervation on the expression of the neuropeptides, pituitary adenylate cyclase-activating peptide (PACAP), galanin (GAL), and the PACAP type 1 receptor in the rat superior cervical ganglion (SCG) were investigated by immunocytochemistry, in situ hybridization and receptor autoradiography. An antibody recognizing the rat vesicular acetylcholine transporter (VAChT) was used for the detection of preganglionic cholinergic fibers. In the normal SCG, PACAP-immunoreactivity (-IR) was present in numerous, basket-forming, preganglionic nerve fibers, while very few SCG neurons expressed PACAP. GAL-IR was restricted to occasional neurons, and a few nerve fibers, most of which were, in addition, PACAP-IR. PACAP type 1 receptors were expressed in all nerve cell bodies. Axotomy resulted in a rapid and prominent upregulation of PACAP in a large number of nerve cell bodies. There was a large increase also in GAL expression in many nerve cell bodies. In contrast, there was a marked decline in PACAP type 1 receptor expression. Chemical sympathectomy by administration of the catcholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), gave rise to similar changes. Preganglionic denervation led to the disappearance of PACAP- and VAChT-IR baskets and to the upregulation of PACAP and GAL expression in neurons located close to the entrance of the sympathetic chain, whereas PACAP type 1 receptor expression was not affected. PACAP and GAL were coexpressed in most neurons after axotomy and chemical sympathectomy. Taken together, these results indicate that disruption of target contact and/or the infliction of an injury to the axons of the sympathetic neurons, rather than the preganglionic output, regulates the expression of PACAP, GAL and the PACAP type 1 receptor.

Characterization and synaptic connectivity of melanopsin‐containing ganglion cells in the primate retina

Melanopsin is a photopigment expressed in retinal ganglion cells, which are intrinsically photosensitive and are also involved in retinal circuits arising from rod and cone photoreceptors. This circuitry, however, is poorly understood. Here, we studied the morphology, distribution and synaptic input to melanopsin‐containing ganglion cells in a New World monkey, the common marmoset (Callithrix jacchus). The dendrites of melanopsin‐containing cells in marmoset stratify either close to the inner nuclear layer (outer stratifying), or close to the ganglion cell layer (inner stratifying). The dendritic fields of outer‐stratifying cells tile the retina, with little overlap. However, the dendritic fields of outer‐stratifying cells largely overlap with the dendritic fields of inner‐stratifying cells. Thus, inner‐stratifying and outer‐stratifying cells may form functionally independent populations. The synaptic input to melanopsin‐containing cells was determined using synaptic markers (antibodies to C‐terminal binding protein 2, CtBP2, for presumed bipolar synapses, and antibodies to gephyrin for presumed amacrine synapses). Both outer‐stratifying and inner‐stratifying cells show colocalized immunoreactive puncta across their entire dendritic tree for both markers. The density of CtBP2 puncta on inner dendrites was about 50% higher than that on outer dendrites. The density of gephyrin puncta was comparable for outer and inner dendrites but higher than the density of CtBP2 puncta. The inner‐stratifying cells may receive their input from a type of diffuse bipolar cell (DB6). Our results are consistent with the idea that both outer and inner melanopsin cells receive bipolar and amacrine input across their dendritic tree.