Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

Effect of Peripheral Axotomy on Expression of Neuropeptide Y Receptor mRNA in Rat Lumbar Dorsal Root Ganglia

Using in situ hybridization, the expression of the mRNA for a neuropeptide Y (NPY) receptor, was studied in lumbar (L) 4 and 5 dorsal root ganglia (DRGs) of normal rats and at various intervals after unilateral sciatic nerve transection. Twenty percent of all normal DRG neurons were NPY receptor mRNA-positive, and the majority of these neurons were of the small type, with only a few labelled medium-sized and large neurons. In L5 normal ganglia NPY receptor mRNA colocalized with substance P, calcitonin gene-related peptide and galanin mRNAs in small neurons, but not in medium-sized or large neurons containing these peptides. NPY receptor mRNA was not observed in somatostatin or nitric oxide synthase mRNA-positive neurons. Sciatic nerve transection induced a marked decrease in NPY receptor mRNA levels. However, in parallel there was a transient increase in the number of NPY receptor mRNA-positive small neuron profiles, but the intensity of labelling was mostly very low, although a few strongly labelled, small neuron profiles were also encountered. In addition, axotomy caused a marked increase in the number of NPY receptor mRNA-positive large neuron profiles in the ipsilateral DRGs, and they constituted 15–20% of counted DRG neuron profiles and 45–65% of counted large neuron profiles, 7–28 days after axotomy. In L5 DRGs, ipsilateral to the axotomy, NPY receptor mRNA colocalized with NPY mRNA in many large and some medium-sized neuron profiles, with galanin mRNA in some small, medium-sized and large neuron profiles and with vasoactive intestinal polypeptide mRNA in some small and medium-sized neuron profiles and a few large profiles. Occasionally, NPY receptor mRNA was observed in nitric oxide synthase mRNA-positive small neurons. In the dorsal horn, NPY receptor mRNA-positive small neurons were concentrated in lamina II at L4 and L5 levels, and were scattered in deeper laminae. No marked changes were observed ipsilateral to the axotomy. No NPY receptor mRNA-positive cells were found in the normal rat gracile nucleus, or in this nucleus after axotomy. These results show that a NPY receptor may be a prejunctional receptor in primary afferent neurons and play a role in the modulation of somatosensatory information, both in normal and lesioned primary afferent DRG cells. However, axotomy induced a distinct shift in NPY receptor mRNA expression from small to large neurons, indicating that sensitivity to NPY is switched from one modality to another. Thus, not only several sensory neuropeptides, as shown in previous studies, but at least also one of the peptide receptors change their expression dramatically in response to axotomy, suggesting complex adaptive responses.     

Altered Calcitonin Gene-related Peptide, Substance P and Enkephalin Immunoreactivities and Receptor Binding Sites in the Dorsal Spinal Cord of the Polyarthritic Rat

The dorsal horn of the spinal cord, which forms the locus of first synapses in pain pathways, is an important site of interaction between calcitonin gene-related peptide (CGRP), substance P and enkephalin—the neuropeptides considered to be especially involved in the regulation of pain perception. Since adjuvant-induced arthritic rats provide a suitable model for peripheral inflammation and hyperalgesia, the possible alterations of immunoreactive CGRP, substance P and enkephalin as well as the binding sites for [¹²⁵I]hCGRPα, [¹²⁵I]substance Plneurokinin-1, (NK1) and [¹²⁵I]FK-33-824/μ-opioid receptors were studied in the dorsal horn of the spinal cord receiving projections from the inflamed limbs. In arthritic rats compared to control animals, a bilateral increase in CGRP- and substance P-immunoreactive fibres and the presence of enkephalin-immunoreactive cell bodies were noted in the dorsal horn of the spinal cord. As for receptors, while a significant decrease in [¹²⁵I]hCGRPα and [¹²⁵I]substance P/NK1 binding sites was observed in selective layers, no measurable alteration in [¹²⁵I]FK-33-824/μ-opioid binding sites was noted in any regions of the arthritic rat dorsal horn compared to the unaffected control rats. Following unilateral section of the peripheral nerve prior to induction of arthritis, CGRP- and substance P-immunoreactive fibres were markedly depleted and no enkephalin-positive neurons were observed in the ipsilateral dorsal horn. Analysis of receptor binding sites in denervated arthritic rats, however, exhibited differential responses, i.e. a significant increase in [¹²⁵I]hCGRPα, a marked decrease in [¹²⁵I]FK-33-824/μ-opioid and apparently no alteration in [¹²⁵I]substance P/NK1 receptor binding sites were observed in the ipsilateral dorsal horn compared to the intact contralateral side. These results taken together provide anatomical evidence for a concerted role of these peptides in the regulation of adjuvant-induced hyperalgesia accompanying peripheral inflammation.     

Distribution of Galanin mRNA Containing Cells and Galanin Receptor Binding Sites in Human and Rat Hypothalamus

The distribution of cells containing galanin mRNA and that of galanin receptor binding sites were investigated using in situ hybridization histochemistry and receptor autoradiography in male rat hypothalamus and in postmortem hypothalamic tissues from control human brains. Oligonucleotide probes labelled with 32P were used for hybridization experiments. The specificity of the hybridization signal was ascertained using several probes, competition assays and Northern blot analysis. High levels of hybridization were found in the paraventricular, supraoptic and arcuate nuclei of rat and human hypothalamus. Human intermediate nuclei and scattered cells of the posterior perifornical nucleus also contained galanin mRNA. Galanin mRNA was also found in the dorsomedial nucleus of the rat. The distribution of galanin receptor sites was investigated by receptor autoradiography using 125I-labelled porcine galanin. The specificity of the binding was assessed by competition with different neuropeptides. While galanin blocked the binding at nanomolar concentrations, the other neuropeptides examined were ineffective at 10-7 M concentrations. The highest densities of galanin binding sites were seen in the preoptic area, ventromedial and lateral nuclei, of rat and human hypothalamus. In contrast, very low densities of binding sites were observed in the paraventricular, supraoptic and arcuate nuclei. Our results show that the distribution of neurons expressing galanin is complementary to that of galanin receptors in the rat and human hypothalamus. This suggests that receptors for galanin are not located on the cell bodies of galaninergic neurons, but are probably presynaptic on or postsynaptic to the processes of these cells.     

Prominent Expression of bFGF in Dorsal Root Ganglia after Axotomy

Using quantitative in situ hybridization and immunohistochemistry the expression of acidic and basic fibroblast growth factors (aFGF, bFGF) in dorsal root ganglia (DRGs) was examined. Around 5% of the small neurons expressed bFGF mRNA in normal DRGs. Nerve injury induced a very dramatic and rapid up-regulation in bFGF mRNA levels, and around 80% of all DRG neurons expressed bFGF mRNA 3 days after axotomy. A distinct increase in bFGF-like immunoreactivity (LI) was also detected as early as 15 h after axotomy. The elevation of bFGF mRNA and protein levels declined after 1 week. bFGF mRNA was also up-regulated in non-neuronal cells following axotomy. Normally bFGF-LI was mainly localized in the nuclei of DRG neurons and in some non-neuronal cells. After nerve section, bFGF-LI was in addition found in the cytoplasm, and many more bFGF-positive non-neuronal cells were observed. By means of confocal microscopy analysis of axotomized DRGs, some bFGF-LI could be detected in vesicle-like structures in the cytoplasm as well as in the nucleoli, in addition to the nuclear location. Application of leukaemia inhibitory factor to the transected sciatic nerve significantly increased the number of bFGF-positive neurons, whereas the bFGF-LI in non-neuronal cells was strongly suppressed. About 70% of the normal DRG neurons expressed aFGF mRNA and aFGF-LI. Axotomy produced a moderate increase in aFGF mRNA levels, but no detectable effect on protein levels. Taken together, the results show that bFGF may be involved in the neuronal response to injury and suggest a role in neuronal survival and regeneration in axotomized DRG neurons.     

Effect of Sustained Physiological Hyperinsulinaemia on Hypothalamic Neuropeptide Y and NPY mRNA Levels in the Rat

Neuropeptide Y (NPY) synthesized in the arcuato-paraventricular projection in the rat hypothalamus is thought to play an important role in controlling energy homeostasis. The factors that regulate hypothalamic NPY are not known but, amongst others, insulin has been postulated as an inhibitory modulatory agent. To test this hypothesis, normal male rats were given either insulin (2 units/day) or saline via subcutaneous osmotic minipumps for 3 days. Euglycaemia was maintained by a concomitant glucose infusion in insulin-infused rats which had peripheral insulin levels 5–8 times higher than saline-infused controls. Hyperinsulinaernic rats ate 42% less than controls, but their total energy intake (food intake plus glucose infusion) was higher than that of controls, and they gained more weight than controls during the experimental period. Hyperinsulinaemia had no significant effect on hypothalamic NPY mRNA or NPY levels in the arcuate nucleus. NPY concentrations in the paraventricular nucleus were, however, significantly increased by 73% in hyperinsulinaemic rats, but were closely similar to controls in all other areas. Insulin may act as a satiety factor in that hyperinsulinaemic rats ate less, but the fact that these animals had increased total energy intake and gained excessive weight suggests that insulin may not function as an overall regulator of energy balance. In addition, physiological hyperinsulinaemia does not apparently inhibit NPY gene expression in the arcuate nucleus. Due to the lack of effect of hyperinsulinaemia on NPY synthesis in the arcuate nucleus, the elevated NPY concentrations in the paraventricular nucleus could result from a reduction of its release, which would be in keeping with the reduction in food intake.     

Leukaemia Inhibitory Factor Induced in the Sciatic Nerve after Axotomy is Involved in the Induction of Galanin in Sensory Neurons

Dramatic changes occur in neuropeptide expression in sensory and sympathetic neurons following axonal injury. Based on the finding that the cytokine leukemia inhibitory factor (LIF) plays an important role in mediating these changes in sympathetic neurons, its participation in triggering changes in sensory neurons was examined. By the use of transgenic mice in which the LIF gene had been knocked out, LIF was found to contribute to the induction of galanin expression in dorsal root ganglia (DRG) after sciatic nerve lesion. On the other hand, two other neuropeptide changes that occur in DRG under these conditions, the reduction of substance P and induction of neuropeptide Y, were independent of LIF expression. In the sympathetic superior cervical ganglion, transection of the postganglionic nerves close to the ganglion resulted in a rapid induction of LIF mRNA in the ganglion and in the lesioned nerve trunk. In contrast, transection of the sciatic nerve close to or distant from the DRG caused a rapid induction of LIF mRNA in the lesioned nerve, but not in the DRG. DRG were capable of making substantial amounts of LIF mRNA when placed in explant cultures, but, in vivo , only a slight induction was found even when both central and peripheral nerve processes of these sensory neurons were transected. These latter observations suggest that, in contrast to the superior cervical ganglia, the DRG environment inhibits the lesion-induced expression of LIF in vivo , and/or explanted DRG produce stimulatory signals not found in vivo. , Together with the data on the induction of galanin, these observations provide evidence that LIF, generated at a site at some distance from the ganglion, is involved in triggering part of the cell body reaction in sensory neurons.     

Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice

Neuropeptide Y (NPY) exerts anxiolytic- and antidepressant-like effects in rodents that appear to be mediated via Y1 receptors. Gene therapy using recombinant viral vectors to induce overexpression of NPY in the hippocampus or amygdala has previously been shown to confer anxiolytic-like effect in rodents. The present study explored an alternative and more specific approach: overexpression of Y1 receptors. Using a recombinant adeno-associated viral vector (rAAV) encoding the Y1 gene (rAAV-Y1), we, for the first time, induced overexpression of functional transgene Y1 receptors in the hippocampus of adult mice and tested the animals in anxiety- and depression-like behavior. Hippocampal Y1 receptors have been suggested to mediate seizure-promoting effect, so the effects of rAAV-induced Y1 receptor overexpression were also tested in kainate-induced seizures. Y1 receptor transgene overexpression was found to be associated with modest anxiolytic-like effect in the open field and elevated plus maze tests, but no effect was seen on depression-like behavior using the tail suspension and forced swim tests. However, the rAAV-Y1 vector modestly aggravated kainate-induced seizures. These data indicate that rAAV-induced overexpression of Y1 receptors in the hippocampus could confer anxiolytic-like effect accompanied by a moderate proconvulsant adverse effect. Further studies are clearly needed to determine whether Y1 gene therapy might have a future role in the treatment of anxiety disorders.     

Modulation of Neuropathic Pain by Galanin and Neuropeptide Y at the Level of the Medulla in Rats

This study was conducted to examine the role of galanin and neuropeptide Y (NPY) in the modulation of neuropathic pain at the level of the medulla. Under pentobarbital anesthesia, Sprague-Dawley rats were subjected to neuropathic surgery. Intracisternal injections of galanin and NPY were performed 2 weeks after nerve injury and mechanical allodynia was monitored. In an electrophysiological experiment, rats were reanesthetized with urethane and the responses of gracile nucleus neurons to mechanical stimulation were observed. Galanin and NPY were applied microiontophoretically. Intracisternally administered NPY reduced neuropathic pain behaviors in a dose-dependent manner. High doses of galanin inhibited neuropathic pain behaviors. Iontophoretically ejected galanin and NPY inhibited responses of gracile nucleus neurons to mechanical stimulation. These results suggest that galanin and NPY play a role in modulating neuropathic pain in the gracile nucleus of the medulla.     

Neuropeptide Y and [Leu,Pro]neuropeptide Y potentiate potassium-induced noradrenaline release in the paraventricular nucleus of the aged rat

This microdialysis study investigated the effects of NPY and the Y₁ selective agonist [Leu³¹,Pro³⁴]NPY on basal and potassium-stimulated noradrenaline release in the PVN of 18-month-old anaesthetised male Sprague–Dawley rats. Microdialysate noradrenaline, DOPAC and HVA concentrations were measured by HPLC after i.c.v. administration of 2 nmol NPY, [Leu³¹,Pro³⁴]NPY or vehicle. [Leu³¹,Pro³⁴]NPY produced a significant 40% reduction in basal noradrenaline concentration (P<0.05). Aged rats had blunted noradrenaline responses to potassium stimulation, however stimulated noradrenaline release was similar in 18-month-old NPY-treated animals and 3-month-old saline treated age controls (2.8 and 3.2 times resting, respectively). [Leu³¹,Pro³⁴]NPY induced a significantly greater release of noradrenaline in response to KCl (5.0 times resting, P<0.05). Thus, in 18-month-old animals with reduced endogenous hypothalamic NPY content, administration of NPY or [Leu³¹,Pro³⁴]NPY increased potassium-induced noradrenaline release to levels seen in 3-month-old rats. This effect may be mediated by an NPY Y₁ receptor.     

The Effects of Mitotic Inhibition on the Spinal Cord Response to the Superimposed Injuries of Spinal Cord Hemisection and Peripheral Axotomy

The present study was carried out to test the hypothesis that dividing microglia are responsible for the depression of crossed phrenic nerve activity documented at 2 weeks postphrenicotomy in an injury model which superimposes the effects of spinal cord injury on peripheral axotomy. Crossed phenic nerve activity is defined as the respiratory activity recorded from the phrenic nerve during the crossed phrenic phenomenon (CPP) which is a respiratory reflex induced by respiratory stress following an ispsilateral spinal cord hemisection. Young adult female Sprague-Dawley rats were subjected to left intrathoracic phrenicotomies. Cytarabine (Cyt-A, a powerful antimitotic drug) or saline-filled miniosmotic pumps were then implanted into the cisterna magna and 2 weeks were allowed to pass at which time the CPP was induced by a left C2 spinal cord hemisection and transection of the contralateral phrenic nerve. Control studies including bromodeoxyuridine labeling of mitotic cells and a triple immunofluorescent protocol were carried out to verify that microglial cells were the primary cell type undergoing mitosis in the current injury model and that Cyt-A completely inhibited cellular proliferation. Quantitative electrophysiological analysis of crossed phrenic nerve activity showed that there is a statistically significant depression of activity at 2 weeks postphrenicotomy when animals were infused with saline compared to controls. Crossed phrenic nerve activity levels were not significantly different, however, from control levels when 2-week postphrenicotomized rats were infused with Cyt-A. Immunofluorescent studies showed that the majority of cells dividing in response to phrenicotomy were microglia. Furthermore, there were no astrocytes seen dividing at any time. From the results, we conclude that activated microglial cells may be responsible for the depression in crossed phrenic activity normally seen 2 weeks postphrenicotomy. Further, the activation of microglia may be related to the astrocytic response to injury. The activated microglial cell may be acting as a coordinator of various aspects of the injury response. Alternatively, the activation of microglia may be a necessary step in the cascade of multiple events that take place in the spinal cord after injury.     

Distribution of GLP-1 Binding Sites in the Rat Brain: Evidence that Exendin-4 is a Ligand of Brain GLP-1 Binding Sites

The distribution and biochemical properties of glucagon-like peptide (GLP)-1(7–36)amide (GLP-1) binding sites in the rat brain were investigated. By receptor autoradiography of tissue sections, the highest densities of [¹²⁵I]GLP-1 binding sites were identified in the lateral septum, the subfornical organ (SFO), the thalamus, the hypothalamus, the interpenduncular nucleus, the posterodorsal tegmental nucleus, the area postrema (AP), the inferior olive and the nucleus of the solitary tract (NTS). Binding studies with [¹²⁵I][Tyr39]exendin-4, a GLP-1 receptor agonist, showed an identical distribution pattern of binding sites. Binding specificity and affinity was investigated using sections of the brainstem containing the NTS. Binding of [¹²⁵I]GLP-1 to the NTS was inhibited concentration-dependently by unlabelled GLP-1 and [Tyr39]exendin-4 with K ₁ values of 3.5 and 9.4 nM respectively. Cross-linking of hypothalamic membranes with [¹²⁵I]GLP-1 or [¹²⁵I][Tyr39]exendin-4 identified a single ligand-binding protein complex with a molecular mass of 63 000 Da. The fact that no GLP-1 binding sites were detected in the cortex but that they were detected in the phylogenetically oldest parts of the brain emphasizes that GLP-1 may be involved in the regulation of vital functions. In conclusion, the biochemical data support the idea that the central GLP-1 receptor resembles the peripheral GLP-1 receptor. Furthermore, the presence of GLP-1 binding sites in the circumventricular organs suggests that these may be receptors which act as the target for both peripheral blood-borne GLP-1 and GLP-1 in the nervous system.     

Expression of c-Jun as a Response to Dorsal Root and Peripheral Nerve Section in Damaged and Adjacent Intact Primary Sensory Neurons in the Rat

It was previously shown that the immediate early gene, c-jun , was highly expressed over long periods, in both peripheral sensory and motor neurons following axon damage or block of axoplasmic transport. Here we have examined the question of whether the expression of c-Jun protein is related to axon injury per se or to the process of axon growth. We have examined dorsal root ganglion (DRG) cells subjected to different manipulations which are associated with varying degrees of regrowth, as follows: (i) after peripheral nerve section, where it appears that all damaged neurons make some regenerative effort. 1 – 24 days after sciatic nerve section and ligation most cells in L4/L5 DRG were c-Jun-positive; (ii) after section of the central processes of the DRG cells, which then showed a slow and limited regrowth of their axons towards, but not into, the spinal cord. This resulted in a variable, but significant, expression of c-Jun in a small number of DRG cells; (iii) in intact sensory neurons that were offered the opportunity to sprout into adjacent denervated peripheral tissue. The sciatic nerve was ligated and the response of cells in the L3 ganglia (many of which project to the saphenous nerve) was measured. A small but significant number of cells were c-Jun-positive; (iv) in intact sensory neurons that were offered the opportunity to sprout centrally into partialy denervated neuropil of the spinal cord. We examined neurons in the L3 DRG after rhizotomy of the adjacent L4/L5 dorsal roots. Previous work suggests that sensory neurons show at best a very limited growth under these conditions. No significant increase was seen in c-Jun expression in these cases. These results suggest that c-Jun expression is closely correlated with growth and regeneration, and not simply a consequence of neuronal injury.     

BDNF Produces Analgesia in the Formalin Test and Modifies Neuropeptide Levels in Rat Brain and Spinal Cord Areas Associated With Nociception

Previous studies have demonstrated an antinociceptive effect of brain-derived neurotrophic factor (BDNF) following infusion into the midbrain, near the periaqueductal grey and dorsal raphe nuclei. BDNF administration attenuated the behavioural response in the tail-flick and hot-plate tests, two models employing a phasic, thermal high-intensity nociceptive stimulus; the present studies extend our previous findings to include a model of moderate, continuous pain resulting from a chemical stimulus, the formalin test. Midbrain infusion of BDNF decreased the behavioural paw flinch response to subcutaneous formalin injection in both the early and late phases of the test. As our previous studies showed that BDNF-induced analgesia was reversible by naloxone, we have examined the effects of BDNF administration on brain and spinal cord levels of neuropeptides involved in the modulation of nociceptive information, including the endogenous opioid peptides, met-enkephalin and P-endorphin, as well as substance P and neuropeptide Y (NPY). At the site of infusion, within the PAG and dorsal raphe, BDNF increased the level of β-endorphin by 63%, but had no effect on substance P, metenkephalin or NPY levels. In the dorsal spinal cord, substance P (113% increase), β-endorphin (97% increase) and NPY (64% increase) were elevated, although ventral spinal cord levels of these peptides remained unchanged. These studies demonstrate a modulatory effect of BDNF on relevant neuropeptides within areas of the brain and spinal cord involved in the processing of nociceptive information.     

Some Acute Effects of Intracerebroventricular Neuropeptide Y on Insulin Secretion and Glucose Metabolism in the Rat

Acute administration of neuropeptide Y(NPY) into the hypothalamus and cerebral ventricles can stimulate insulin secretion in the absence of available food. However, the relationship of this effect to blood glucose and other hormones which regulate glucose metabolism remains unclear. The purpose of this study was to compare the effects of NPY injected into the third ventricle (ICV) on serum insulin, glucose, glucagon, corticosterone and non-esterified fatty acids. Studies were performed on conscious, unrestrained female rats, not given access to food. ICV NPY, 2 and 5  μg produced an increase in serum insulin and glucagon, while the 5  μg dose only increased plasma glucose transiently and increased non-esterified fatty acids for a longer period. Corticosterone was not affected by ICV NPY. The insulinaemic response to i.v. glucose, 0.5 g/kg was doubled by ICV NPY, 4  μg. The maximal insulin levels were 113±18 for ICV NPY versus 67±8  μU/ml for ICV saline-treated animals. The glycaemic response was not altered. The hypoglycaemic response to i.v. insulin, 0.15 U/kg was significantly attenuated by ICV NPY, 5  μg. We concluded that ICV NPY promotes insulin secretion in the absence of available food and may potentiate the insulinaemic response to hyperglycaemia. Furthermore, possibly through its effects on glucagon and non-esterified fatty acids, ICV NPY may decrease the ability of insulin to control glucose metabolism.     

Localization of Neuropeptide Y Y1 mRNA in the Human Brain: Abundant Expression in Cerebral Cortex and Striatum

Many neurobiological functions have been ascribed to the NPY Y1 receptor subtype, but autoradiographic analysis has failed to detect Y1 binding sites in most human brain areas, in contrast to the rat. We examined the regional distribution of Y1 mRNA-containing cells in the post-mortem human brain to clarify if there is a major species difference in terms of the existence of Y1 receptors in the human telencephalon, in particular the striatum and cortex. In situ hybridization experiments revealed widespread distribution of Y1 mRNA signals in all layers of most limbic and neocortical regions, predominantly in layer IV (most cortical regions) and layer VI. The striatum showed moderate Y1 receptor mRNA expression levels with intensely expressing cells localized to the nucleus accumbens. The highest Y1 receptor mRNA expression was apparent within the dentate gyrus, and the lowest in the subiculum, parahippocampal gyrus, cerebellum, and thalamus. In vitro autoradiography using [¹²⁵I]Leu³¹Pro³⁴-PYY and [¹²⁵I]PYY with NPY (13–36) or Leu³¹ Pro³⁴ NPY; confirmed the presence of low Y1–like binding in the human brain despite abundant Y1 mRNA expression. However, using a rat model of the human autopsy process, it was apparent that the inability to reveal high Y1– versus Y2–like receptors in the human brain was related in part to marked reductions of Y1–like, but not Y2–like, receptors within a 4 h post-mortem delay. Altogether, the results indicate that the Y1 receptor gene is abundant in the human brain and this receptor may have important roles in cognitive, limbic and motor function.     

Cholecystokinin in Mammalian Primary Sensory Neurons and Spinal Cord: In Situ Hybridization Studies in Rat and Monkey

The peptide cholecystokinin (CCK) has been suggested to be involved in nociception, but its exact localization at the level of the spinal cord and in spinal ganglia has been a controversial issue. Therefore the distribution of messenger RNA (mRNA) for CCK was studied by in situ hybridization using oligonucleotide probes on sections of adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve and on sections of untreated monkey trigeminal ganglia, spinal cord and spinal ganglia from all levels. For comparison, calcitonin gene-related peptide (CGRP) mRNA was also studied in the monkey tissue using the same techniques. Peripheral sectioning of the sciatic nerve in the rat resulted in the appearance of detectable CCK mRNA in up to 30% of remaining ipsilateral L4 and L5 dorsal root ganglion neurons 3 weeks after surgery, with a distinct but more limited appearance also in the contralateral ganglia. No cells, or only single cells, could be seen in normal control rat ganglia. In contrast, in the normal monkey, ∼20% of dorsal root ganglion neurons, regardless of spinal level, and 10% of trigeminal ganglia neurons expressed mRNA for CCK. CGRP mRNA was expressed at detectable levels in ∼80% of these monkey dorsal root ganglion neurons. In the monkey spinal cord, CCK mRNA was detected in the dorsal horn and in motoneurons, whereas CGRP mRNA was only seen in motoneurons. The present results suggest that CCK peptides can be involved in sensory processing in the dorsal horn of the spinal cord in normal monkeys and in rats after peripheral nerve injury, adding one more possible excitatory peptide to the group of mediators in the dorsal horn.     

GAP-43 mRNA in Rat Spinal Cord and Dorsal Root Ganglia Neurons: Developmental Changes and Re-expression Following Peripheral Nerve Injury

The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000–3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.     

Cortical Regulation of Striatal Neuropeptide Y (NPY)-Containing Neurons in the Rat

This study examined the functional relationships established by nigral, cortical, and thalamic striatal afferent pathways with neuropeptide Y (NPY)-containing neurons in the rat rostral striatum by coupling selective deafferentation procedures and NPY immunohistochemistry. Previous experiments have shown that after unilateral 6-hydroxydopamine (6-OHDA)-induced degeneration of nigrostriatal dopaminergic neurons, the mean number of NPY-immunoreactive (Ir) neurons per frontal section was increased in the striatum ipsilateral to the lesion side and unaltered in the contralateral striatum. The present topographical analysis of the 6-OHDA lesion effects led us to state that the increase in NPY-Ir neuron density occurs in restricted ventral and medial zones of the ipsilateral striatum. Unilateral ablation of the frontoparietal cerebral cortex by thermocoagulation was moreover shown to elicit, 20 - 30 days later, a significant bilateral increase in the number of striatal NPY-Ir cells. The increase was more marked in the striatum ipsilateral to the hemidecortication where it was similar in amplitude to that induced by the 6-OHDA lesion. The topographical analysis of the cortical lesion effects also revealed an uneven striatal response, but, in contrast to that observed for the 6-OHDA lesion, changes were restricted to dorsolateral areas of the striatum in both brain sides, revealing an apparent complementarity of nigral dopaminergic and cortical influences over striatal NPY neuronal system. Combined unilateral nigral and cortical lesions surprisingly counteracted in a survival time dependent manner the effects of each lesion considered separately. In that condition topographical changes related to the 6-OHDA lesion totally disappeared and those related to the cortical lesion were attenuated but still present. These results suggest that expression of striatal dopamine - NPY interaction is dependent on corticostriatal transmission. Interestingly lesion of thalamic areas projecting to the striatum did not significantly modify the mean number of NPY-Ir neurons determined per section from the whole striatal surface, but selectively increased the NPY neuron density in the mediodorsal region of the striatum, suggesting that the striatal NPY-containing neuronal system is also influenced by thalamostriatal projections.     

The Distribution of &mgr; and delta Opioid Binding Sites Belonging to a Single Cervical Dorsal Root in the Superficial Dorsal Horn of the Rat Spinal Cord: A Quantitative Autoradiographic Study

Numerous studies have demonstrated a dense concentration of opioid receptors in the superficial layers (laminae I - II) of the spinal cord. These receptors are located both pre- and postsynaptically at this level. The purpose of this study was to assess the distribution of opioid receptors belonging to a single (C7) dorsal root. Thus, quantitative autoradiography of &mgr; ([3H]Tyr-d-Ala-Gly-NMe-Phe-Gly-ol; [3H]DAMGO) and delta ([3H]Tyr-d-Thr-Gly-Phe-Leu-Thr; [3H]DTLET) opioid binding sites was performed for several experimental groups: control rats with intact dorsal roots and lesioned rats with a unilateral dorsal rhizotomy of (a) the C7 root alone, (b) the three successive roots rostral and caudal to the spared C7 root, and (c) the seven roots C4 - Th2. By subtracting results of the 'C7 cut' group from the 'intact' group or by subtracting results of the C4 - Th2 cut group from the C7 spaced group, it was possible to measure the distribution of &mgr; and delta opioid binding sites belonging to the C7 root. The combination of these two methods of calculation allowed us to demonstrate a significant distribution over two segments rostral and one segment caudal to the segment of entry. For [3H]DAMGO, the distribution was 10% (P < 0.05) in the C5, 27%, (P < 0.001) in the C6, 38% (P < 0.001) in the C7 and 14% (P < 0.05) in the C8 segment. For [3H]DTLET, the distribution was 11% (P=0.05) in the C5, 27%, (P < 0.01) in the C6, 37% (P < 0.001) in the C7 and 18% (P < 0.05) in the C8 segment. It is also noted that rostral distributions spread more densely and further than the caudal ones.