The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system

Somatostatin-containing neurons in the rat central nervous system were localized by immunocytochemical methods. The detection of somatostatin-like immunoreactivity was facilitated by (1) the use of brains from colchicine-treated rats, (2) the proteolytic pretreatment of sections with pronase and (3) a ‘double-bridge’ immunoperoxidase staining technique. In addition to the known distribution of somatostatin-like immunoreactivity we also observed immunoreactive perikarya in the following regions: the anterior olfactory nucleus, some areas of the preoptic and hypothalamic regions, the claustrum, the periaqueductal gray, the locus ceruleus, the central gray substance, the lateral parabrachial nucleus, the nucleus of the lateral lemniscus, the nucleus ambiguus, the spinal trigeminal nucleus, the nucleus of the solitary tract and various areas of the reticular formation. Immunoreactive neuronal processes were also observed in several major tracts of the brain, including the stria terminalis, the fornix and the medial forebrain bundle.Our results indicate that somatostatin-containing neurons may occur both as interneurons in some areas of the central nervous system and as projection neurons in others. The widespread but selective distribution of these neurons suggests that somatostatin is not only a hypothalamic regulator of neuroendocrine function, but may also function as a major neuromodulator mediating a variety of functions throughout the central nervous system.     

Identification of somatostatin-containing neurons in primary cultures of rat cerebral cortex

The expression of somatostatin-like immunoreactivity was investigated in primary cultures of dissociated cerebral cortical tissue from the rat. A subpopulation of neurons in the cortical cultures exhibited intense staining for somatostatin. These somatostatin-immunoreactive neurons corresponded to 1.25% of the total neuronal population. Stained neurons were typically small with a soma size of 10-20 micron. The majority of somatostatin-containing cells had stellate and bipolar morphology, with the bipolar class predominating.     

Volume changes induced by osmotic stress in freshly isolated rat hippocampal neurons

 The degree to which osmotic stress changes the volume of mammalian central neurons has not previously been determined. We isolated CA1 pyramidal cells and measured cell volume in four different ways. Extracellular osmolarity (π_o) was lowered by omitting varying amounts of NaCl and raised by adding mannitol; the extremes of π_o tested ranged from 134 to 396 mosm/kg. When π_o was reduced, cell swelling varied widely. We distinguished three types of cells according to their response: ”yielding cells” whose volume began to increase immediately; ”delayed response cells” which swelled after a latent period of 2 min or more; and ”resistant cells” whose volume did not change during exposure to hypo-osmotic solution. When π_o was raised, most cells shrank slowly, reaching minimal volume in 15–20 min. We observed neither a regulatory volume decrease nor an increase. We conclude that the water permeability of the membrane of hippocampal CA1 pyramidal neurons is low compared to that of other cell types. The mechanical support of the plasma membrane given by the cytoskeleton may contribute to the resistance to swelling and protect neurons against swelling-induced damage. Received: 16 September 1997 / Received after revision: 22 June 1998 / Accepted: 30 June 1998     

Postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive nerves in the rat urinary bladder

The postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive (NPY-IR and CGRP-IR) nerve fibers in the rat urinary bladder was investigated using whole-mount preparations and cryostat sections. In newborn and 3-day-old rats, many NPY-IR nerve fibers were observed in the subserous and muscle layers. Many NPY-IR nerve cell bodies clustered at branching points of the subserous nerve bundles. Within 4 weeks after birth, these cell bodies drastically decreased in number and spread along the bundles, although the number of NPY-IR nerve fibers increased moderately. In contrast, CGRP-IR nerve fibers in newborn and 3-day-old rats were less developed, and no CGRP-IR nerve cell body was observed in any rat. However, CGRP-IR nerve fiber distribution in the urinary tissues conspicuously increased within 4 weeks after birth. Especially, an increase of the infraepithelial fibers showing a meshwork appearance was prominent in the fundus and corpus of the bladder. The infra- and intraepithelial CGRP-IR nerve meshwork of the ventral wall was more dense than that of the trigone. At 4 weeks, NPY-IR and CGRP-IR nerves were similar to those of the adult rat (8–12 weeks old). The present study suggests a correlation between the development of the peripheral nervous system in the urinary bladder and maturation of micturition behavior in the rat.     

Peripheral pathways for neuropeptide Y- and cholecystokinin-8-immunoreactive nerves innervating the rat ovary

The superior ovarian nerve (SON) or plexus nerve (PN) innervating the rat ovary was transected separately or in combination and the effects of these nerve lesions on intra-ovarian NPY- or CCK-8-immunoreactive nerve fibers was evaluated. Transection of the SON did not affect NPY or CCK-8-immunoreactive nerve fibers. In contrast, section of the PN eliminated nerve fibers immunoreactive for these neuropeptides. This study demonstrates that both NPY- and CCK-8-immunoreactive nerve fibers reach the ovary via the PN.     

Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat

The localization of two small peptides, somatostatin and substance P, has been studied with the indirect immunofluorescence technique. Both peptides were present in small neuronal cell bodies in spinal ganglia, in fibers in the dorsal horn of the spinal cord and in fibers in the intestinal wall. By comparing consecutive sections incubated with antisera to somastostatin and to substance P respectively, it was established that somatostatin, or somatostatin-like immunoreactivity and substance P, or substance P-like immunoreactivity are present in different cells. This is possibly indicated also by a somewhat differential distribution of the immunoreactive fibers in the dorsal horn: the highest concentration of somatostatin-positive fibers was observed in lamina II, whereas abundant substance P-positive fibers were present also in lamina I. Furthermore, numerous substance P-, but no somatostatin-positive fibers, were found around the central canal and in the ventral horns. In the intestinal wall more substance P-positive than somatostatin-positive fibers were seen.The present results indicate that two subpopulations of primary sensory neurons exist, one containing somatostatin, or somatostatin-like immunoreactivity, and the other containing substance P, or substance P-like immunoreactivity.     

Progressive ankylosis (Ank) protein is expressed by neurons and Ank immunohistochemical reactivity is increased by limbic seizures

Ank is a 492-amino acid multipass transmembrane protein involved in the regulation of extracellular inorganic pyrophosphate levels and the control of tissue calcification. Previous Northern blot hybridization experiments revealed that Ank mRNA was expressed in the brain, but there have been no reports describing the anatomical sites or specific cell types in the brain that express Ank protein. In this study, we demonstrate that Ank is expressed primarily in human brain neurons, with the highest levels of expression observed in the thalamus, the III and V cortical layers, the Purkinje cells of the cerebellum, clusters of cells in the dorsal portion of the pons and midbrain, and neurons of the anterior horn of the spinal cord. In primary mouse neuronal cell cultures, Ank is detected on both the cell body and on cell extensions, mainly dendrites. In the rat brain, Ank mRNA is expressed at relatively high levels in the thalamus, midbrain, and spinal cord, and the Ank protein expression pattern is similar to that observed in the human brain. Finally, we observed a significant increase in Ank immunoreactivity in the rat amygdala, the CA-2 and CA-3 layers of the hippocampus, and the cerebral cortex after the induction of seizure activity. Ank regulation of ATP and/or inorganic pyrophosphate release from neurons may function to modulate the membrane excitability and cell death associated with seizure activity.     

Brain-derived neurotrophic factor immunoreactivity in the limbic system of rats after acute seizures and during spontaneous convulsions: temporal evolution of changes as compared to neuropeptide Y

Seizures increase the synthesis of brain-derived neurotrophic factor in forebrain areas, suggesting this neurotrophin has biological actions in epileptic tissue. The understanding of these actions requires information on the sites and extent of brain-derived neurotrophic factor production in areas involved in seizures onset and their spread. In this study, we investigated by immunocytochemistry the changes in brain-derived neurotrophic factor in the hippocampus, entorhinal and perirhinal cortices of rats at increasing times after acute seizures eventually leading to spontaneous convulsions. We also tested the hypothesis that seizure-induced changes in brain-derived neurotrophic factor induce later modifications in neuropeptide Y expression by comparing, in each instance, their immunoreactive patterns. As early as 100 min after seizure induction, brain-derived neurotrophic factor immunoreactivity increased in CA1 pyramidal and granule neurons and in cells of layers II–III of the entorhinal cortex. At later times, immunoreactivity progressively decreased in somata while increasing in fibres in the hippocampus, the subicular complex and in specific layers of the entorhinal and perirhinal cortices. Changes in neuropeptide Y immunoreactivity were superimposed upon and closely followed those of brain-derived neurotrophic factor. One week after seizure induction, brain-derived neurotrophic factor and neuropeptide Y immunoreactivities were similar to controls in 50% of rats. In rats experiencing spontaneous convulsions, brain-derived neurotrophic factor and neuropeptide Y immunoreactivity was strongly enhanced in fibres in the hippocampus/parahippocampal gyrus and in the temporal cortex. In the dentate gyrus, changes in immunoreactivity depended on sprouting of mossy fibres as assessed by growth-associated protein-43-immunoreactivity. These modifications were inhibited by repeated anticonvulsant treatment with phenobarbital.

The dynamic and temporally-linked alterations in brain-derived neurotrophic factor and neuropeptide Y in brain regions critically involved in epileptogenesis suggest a functional link between these two substances in the regulation of network excitability.     

Hypotension activates neuropeptide Y-containing neurons in the rat medulla oblongata.

The present study was designed to determine whether neurons within cardiovascular control nuclei of the rat brainstem that become activated following a hypotensive insult also possess the capacity to utilize neuropeptide Y. Adult male Wistar-Kyoto rats were injected with glyceryl trinitrate (10 mg/kg, i.p.) or vehicle, and 4 h later anaesthetized (pentobarbitone, 60 mg/kg, i.p.) and transcardially perfused. The brains were removed and processed by standard two-colour peroxidase immunohistochemistry. Activated cells were determined by incubation with a primary antibody to Fos protein, which was followed by a second incubation with a primary antibody to neuropeptide Y for double labelling of Fos-positive cells. Compared to vehicle, glyceryl trinitrate-induced hypotension caused a marked induction of Fos protein in the caudal one-third of the nucleus tractus solitarius (bregma -14 to -13.3 mm), which tailed off rapidly in more rostral sections. Following hypotension, significant populations of activated cells were also observed in the rostral and caudal ventrolateral medulla. In the caudal nucleus tractus solitarius and the posterior part of the medial nucleus tractus solitarius, respectively, 15 of 104 and 40 of 120 Fos-positive cells exhibited cytoplasmic neuropeptide Y immunoreactivity following hypotension, compared to seven of 40 and 15 of 40 in vehicle-treated rats, indicating a significant (two- to three-fold) increase in double-labelled cells following systemic glyceryl trinitrate (P < 0.05, unpaired t-test). In contrast, in the anterior part of the medial nucleus tractus solitarius, the number of double-labelled cells did not change following hypotension. An increase in double-labelled cells was also observed in the rostral ventrolateral medulla (2.5-fold increase compared to vehicle) and caudal ventrolateral medulla (5.8-fold increase compared to vehicle) following hypotension. These data indicate that, in the rat, neuropeptide Y-containing neurons are involved in the central response to a hypotensive challenge. The primary regions where neuropeptide Y-containing neurons appear to be activated are the caudal one-third of the nucleus tractus solitarius and the caudal ventrolateral medulla/rostral ventrolateral medulla, which are key nuclei associated with the integration of the baroreceptor heart rate reflex and sympathetic vasomotor outflow.     

Neuronal expression of the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures

In the brain, the efflux transporter P-glycoprotein (Pgp) is predominantly located on the luminal membrane of endothelial cells lining brain microvessels and forming the blood-brain barrier. Many lipophilic drugs, including antiepileptic drugs, are potential substrates for Pgp. Overexpression of Pgp in endothelial cells of the blood-brain barrier has been determined in patients with drug resistant forms of epilepsy such as temporal lobe epilepsy and rodent models of temporal lobe epilepsy and suggested to lead to reduced penetration of antiepileptic drugs into the brain. Expression of Pgp after seizures has also been described in astrocytes, whereas it is not clear whether neurons can express Pgp. In the present study, Pgp expression was studied by immunohistochemistry in rats 24 h after a status epilepticus induced by either pilocarpine or kainate, widely used models of temporal lobe epilepsy. Unexpectedly, in addition to endothelial Pgp staining, intense Pgp staining was found in neurons in the CA3c/CA4 sectors and hilus of the hippocampus formation, but not in other brain regions examined. The neuronal Pgp staining was confirmed by two different Pgp antibodies. Double immunolabeling and confocal microscopy showed that Pgp was colocalized with the neuronal marker neuronal nuclear antigen, but not with the glial marker glial fibrillary acidic protein. No neuronal Pgp staining was seen in control rats. The expression of Pgp in neurons after limbic seizures was substantiated by determining Pgp encoding genes (mdr1a, mdr1b) in neurons by real time quantitative RT-PCR. Increased Pgp expression in hippocampal neurons is likely to affect the action of drugs with intraneuronal targets and, in view of recent evidence from other cell types, could be associated with prevention of apoptosis which is involved in neuronal damage developing after seizures such as produced by pilocarpine.     

Glucose-sensitive neurons in the rat arcuate nucleus contain neuropeptide Y.

Glucose is known to regulate the activity of the hypothalamic feeding centers. Neuropeptide Y (NPY)-containing neurons in the hypothalamic arcuate nucleus (ARC) have been implicated in the stimulation of feeding. We examined the presence of glucose-sensitive neurons in the ARC and their coincidence with NPY-containing neurons. Cytosolic Ca2+ concentration ([Ca2+]i) in single ARC neurons isolated from rat hypothalamus was measured with fura-2 fluorescence imaging; the cells were then stained immunocytochemically with an anti-NPY antiserum. Lowering the glucose concentration from 10 to 1 mM increased [Ca2+]i in 36 out of 180 neurons (20%), the majority of which (34 neurons, 94%) were immunoreactive for NPY. In conclusion, the ARC contains glucose-sensitive NPY-containing neurons. The suggested role of these neurons is to transduce a reduction in the glucose concentration in the brain to the release of NPY and, subsequently, stimulation of feeding.     

Serotonin innervation of neuropeptide Y-containing neurons in the rat arcuate nucleus

Structural non-synaptic appositions between serotonin (5-HT) nerve endings and neuropeptide Y (NPY)-containing neurons were demonstrated in the rat arcuate nucleus by means of a combined radioautographic and immunocytochemical detection of [3H]5-HT uptake sites and NPY-immunoreactivity. Such cellular relationships are proposed to constitute a morphological substrate for putative 5-HT/NPY interactions in neuroendocrine hypothalamus.     

Alterations in hippocampal extracellular amino acids and purine catabolites during limbic seizures induced by folate injections into the rabbit amygdala

The effects on hippocampal extra- and intracellular amino acids of focal injection of folic acid into the amygdala in the rabbit were studied with brain dialysis. Folate seizures were accompanied by pronounced elevations of extracellular alanine and phosphoethanolamine. The increase of extracellular alanine was related to an enhanced level of this amino acid in total hippocampal tissue, whereas phosphoethanolamine was unaltered in tissue biopsies. Folate seizures did not significantly affect extracellular aspartate and extracellular glutamate was only slightly elevated (50-75% over baseline values). The tissue concentration of glutamate remained at control levels during the seizures and tissue aspartate was decreased by 28%. Extracellular glutamine decreased rapidly after folate injection with a concomitant increase of total hippocampal glutamine. Neither extra- nor intracellular taurine was affected by folate epilepsy. The experiments also encompassed measurements of the extracellular purine catabolites inosine, hypoxanthine and xanthine. Folic acid-induced epilepsy produced changes in these compounds indicative of moderately accelerated degradation of adenosine 5'-triphosphate. The findings support the view of glutamate as a mediator of epilepsy-related brain damage. It is, however, questionable if the small enhancement of extracellular glutamate is enough to provoke neuronal necrosis associated with folate epilepsy.     

Acute and delayed restraint stress-induced changes in nitric oxide producing neurons in limbic regions

RATIONALE: Microinjection into the dentate gyrus of the hippocampus of N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nitric oxide synthase (NOS) inhibitor, induces antinociceptive effect 5 days after a single restraint episode. The mechanisms of this stress-antinociceptive modulatory effect have not been investigated but may involve plastic changes in the hippocampal formation (HF). OBJECTIVE: The objective of the present study was to investigate possible mechanisms of the stress-modulating effect on antinociception induced by NOS inhibition in the hippocampus. We analyzed the effects of restraint stress on neuronal NOS (nNOS) expression and nicotinamide adenine dinucleotide phosphate-diaphorase histochemical activity (NADPH-d) in the HF and related brain regions. METHODS: Male Wistar rats (n=6-11/group) were submitted to a single (acute stress) or repeated (5 days) episodes of 2-h restraint. Control animals remained in their home cages being all animals daily handled during this period. In the fifth day, animals received unilateral microinjection of l-NAME (150 nmol/0.2 microl) or saline (control) into the dentate gyrus of the dorsal hippocampus (DG). Immediately before and after drug microinjection tail-flick reflex latency or hotplate licking reaction was measured. Animals were killed i. immediately; ii. 5 days after acute stress; or iii. after repeated stress. NADPH-d and nNOS expression were quantified in the HF, caudate-putamen, secondary somatosensorial, entorhinal and piriform cortices and amygdaloid complex. RESULTS: Five days after one or five restraint episodes l-NAME microinjection into the DG elicited antinociceptive effect (analysis of variance [ANOVA], P<0.05). Acute restraint stress induced a significant increase in the density of neurons expressing NADPH-d and nNOS in the amygdaloid nuclei. nNOS expression increased also in the DG and piriform cortex. Five days after a single or repeated restraint stress there was an additional increase in NADPH-d- and nNOS-positive neurons in CA1, CA3, and entorhinal cortex. No changes were seen in non-limbic regions such as the caudate-putamen and secondary somatosensorial cortex. CONCLUSION: The results confirm that the dorsal hippocampus participates in the modulation of stress consequences. They also show that a single stress episode causes acute changes in nitric oxide system in the amygdala complex and delayed modifications in the HF. The delayed (5 days) antinociceptive effect of NOS inhibition in the HF after a single restraint episode suggests that those latter modifications may have functional consequences. It remains to be tested if the acute amygdala and delayed hippocampal changes are causally related.     

Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons

The distribution and coexistence of neuropeptide Y (NPY) and somatostatin (SOM) were evaluated in rat and human cerebral cortex and in the rat hypothalamic arcuate nucleus (n) using double immunofluorescent staining in which primary antisera were raised in different species. The results of the study indicate extensive coexistence of NPY and SOM in both rat and human cortex but only occasional coexistence in the rat arcuate n.     

Age-related change of neuropeptide Y-immunoreactive neurons in the rat anterior olfactory nucleus

Neuropeptide Y (NPY) is located in the olfactory system, including the olfactory bulb, and is thought to be one of the main neurotransmitters for olfaction. Thus, we examined age-related changes of NPY-immunoreactive (IR) neurons in the rat anterior olfactory nucleus (AON) at various aging stages over a period of 2 years; postnatal months 1 (PM 1), PM 6, PM 12 and PM 24. NPY-IR neurons in the AON were present in the lateral and medial subdivisions at PM 1 and at PM 6 were distributed in all subdivisions of the AON. Prior to PM 12, the NPY-IR neurons showed a tendency to change from bipolar cells with short processes into multipolar cells with long processes. Moreover, the population of NPY-IR neurons and nerve fibers in the AON increased in proportion to age. In particular, the number of NPY-IR neurons increased about 6-fold between PM 1 and PM 3. At PM 24, the number of NPY-IR neurons was much smaller than that at PM 12 and somal size had decreased. It is therefore suggested that the dramatic increase in the number and size of the NPY-IR neurons between PM 1 and PM 3 may be associated with sexual maturation and that the decrease in the number and cell size of the NPY-IR neurons at PM 24 may underlie age-related changes in the olfactory process.     

Phosphorylated and non-phosphorylated neurofilament proteins: distribution in the rat hippocampus and early changes after kainic acid induced seizures

The regional distribution of neurofilament proteins in the rat hippocampus and their early changes after kainic acid induced seizures were investigated immunocytochemically with antibodies against light weight neurofilament, phosphorylated and non-phosphorylated heavy weight neurofilament. The light weight and non-phosphorylated heavy weight neurofilaments were distributed more unevenly than the phosphorylated neurofilament. The perikarya and processes of pyramidal cells in the CA3 field contained the highest light weight and non-phosphorylated heavy weight neurofilaments, while the perikarya of granule cells contained only few light weight neurofilament and the perikarya of CAI pyramidal cells were even devoid of immunoreactivity of both light and heavy weight neurofilaments. The fiber staining of the light weight and non-phosphorylated heavy weight neurofilaments, especially the former, was less in the CAI field and molecular layer of dentate gyrus. The phosphorylated neurofilament immunoreactivity was identified only in axons. Mossy fibers, the axons of granule cells, contained the light weight and phosphorylated heavy weight neurofilaments, but not the non-phosphorylated neurofilament. Seven days after the kainic acid induced seizures, the phosphorylated neurofilament staining was greatly reduced in the CAI and inner molecular layer of the dentate gyrus, probably resulting from the axonal degeneration of the Schaffer collaterals and the commissural/associational fibers. Furthermore, the non-phosphorylated neurofilament appeared in the mossy fibers of the CA3 stratum lucidum, which normally do not express such immunoreactivity. The results indicate that the neurofilaments are altered following the neuronal degeneration and post lesional plasticity caused by the kainic acid administration. Therefore, the examination of various phosphorylated neurofilaments may offer a comprehensive understanding of major hippocampul pathways, axonal plasticity and the possible roles of neurofilaments in the hippocampus following excitotoxic insults.     

Synaptic relationships between induced neuropeptide Y-like immunoreactive terminals and cuneothalamic projection neurons in the rat cuneate nucleus following median nerve transection

Previous studies have demonstrated that following complete median nerve transection (CMNT), neuropeptide Y-like immunoreactive (NPY-LI) fibers were dramatically increased and predominantly expressed in the ventral portion of the middle cuneate nucleus (CN), reaching maximum numbers at four weeks. Ultrastructurally, NPY-LI terminals made axodendritic synapses, but the postsynaptic elements are unknown. In the present study, using retrograde tract-tracing of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) and NPY immunocytochemistry we examined the synaptic relationships between cuneothalamic projection neurons (CTNs) and NPY-LI terminals in the rat CN following CMNT. The injury-induced NPY-LI fibers were distributed throughout the rostrocaudal extent of the CN. Further, the greatest number of HRP-labeled CTNs was observed in the ventral portion of the middle CN. Ultrastructurally, the NPY-LI terminals made asymmetric axodendritic synaptic contact with the HRP-labeling CTN dendrites. These data suggest that injury-induced NPY may modulate the excitability of CTNs, and thus, play a role in the transmission of neuropathic sensation following median nerve injury.