Ludmila Mackerlova

Rat Brain Vascular Distribution of Interleukin-1 Type-1 Receptor Immunoreactivity : Relationship to Patterns of Inducible Cyclooxygenase Expression by Peripheral Inflammatory Stimuli

Interleukin‐1β (IL‐1β) is thought to act on the brain to induce fever, neuroendocrine activation, and behavioral changes during disease through induction of prostaglandins at the blood–brain barrier (BBB). However, despite the fact that IL‐1β induces the prostaglandin‐synthesizing enzyme cyclooxygenase‐2 (COX‐2) in brain vascular cells, no study has established the presence of IL‐1 receptor type 1 (IL‐1R1) protein in these cells. Furthermore, although COX inhibitors attenuate expression of the activation marker c‐Fos in the preoptic and paraventricular hypothalamus after administration of IL‐1β or bacterial lipopolysaccharide (LPS), they do not alter c‐Fos induction in other structures known to express prostaglandin receptors. The present study thus sought to establish whether IL‐1R1 protein is present and functional in the rat cerebral vasculature. In addition, the distribution of IL‐1R1 protein was compared to IL‐1β‐ and LPS‐induced COX‐2 expression. IL‐1R1‐immunoreactive perivascular cells were mostly found in choroid plexus and meninges. IL‐1R1‐immunoreactive vessels were seen throughout the brain, but concentrated in the preoptic area, subfornical organ, supraoptic hypothalamus, and to a lesser extent in the paraventricular hypothalamus, cortex, nucleus of the solitary tract, and ventrolateral medulla. Vascular IL‐1R1‐ir was associated with an endothelial cell marker, not found in arterioles, and corresponded to the induction patterns of phosphorylated c‐Jun and inhibitory‐factor kappaB mRNA upon IL‐1β stimulation, and colocalized with peripheral IL‐1β‐ or LPS‐induced COX‐2 expression. These observations indicate that functional IL‐1R1s are expressed in endothelial cells of brain venules and suggest that vascular IL‐1R1 distribution is an important factor determining BBB prostaglandin‐dependent activation of brain structures during infection. J. Comp. Neurol. 472:113–129, 2004.

Expression of melanocortin-4 receptor by rat parabrachial neurons responsive to immune and aversive stimuli.

The pontine parabrachial nucleus is a major relay area for visceral and other interoceptive information, and has been implicated in mechanisms underlying anorexia and food aversion during disease. Thus, physiological studies have shown that peripheral immune stimuli, as well as the administration of aversive substances such as lithium chloride, evoke a prominent Fos-expression in the lateral parabrachial nucleus and behavioral experiments have demonstrated that this structure is critical for the acquisition of conditioned taste aversion. The present study examined in rats the relationship between parabrachial neurons activated by systemic administration of bacterial cell-wall lipopolysaccharide or lithium chloride and the melanocortin system, a major regulator of feeding and energy homeostasis that also has been implicated in aversive behavior. Dual-labeling in situ hybridization showed melanocortin-4 receptor expression on neurons in the external lateral parabrachial subnucleus that displayed lipopolysaccharide- or lithium chloride-induced expression of c-fos mRNA. Melanocortin-4 receptor mRNA was also co-expressed with mRNA for calcitonin gene-related peptide in this subnucleus. Taken together with previous observations showing that calcitonin gene-related peptide expressing neurons in the external lateral parabrachial subnucleus are activated by peripheral immune challenge, that lipopolysaccharide-activated external lateral parabrachial subnucleus neurons project to the amygdala, and that the amygdala-projecting neurons in the external lateral parabrachial subnucleus are calcitonin gene-related peptide-positive, the present findings suggest the presence of a melanocortin-regulated calcitonin gene-related peptide-positive pathway from the external lateral parabrachial subnucleus to the amygdala that relays information of importance to forebrain responses to certain aspects of sickness behavior. These observations may thus help explain how melanocortins can reduce feeding and influence conditioned taste aversion during inflammation and other disease conditions.

Ontogenetic aspects of phospholipid-sensitive calcium-dependent protein kinase in guinea pig tissues

Abstract Changes in the activity levels of phospholipid-sensitive Ca2+-dependent protein kinase in various tissues from developing guinea pigs were investigated. The fetal cerebral cortex, cerebellum, midbrain, spleen and kidney contained lower levels of the enzyme activity compared to the corresponding adult tissues. Conversely, higher enzyme levels were noted for the fetal liver and retina. The activity levels in the ileum, heart and lung, on the other hand, were unaltered during development. The ontogeny-related changes in the enzyme were dissimilar to those for cAMP- and cGMP-dependent protein kinases previously reported, suggesting separate functional roles for the newly recognized Ca2+-target enzyme.

Activation of the parabrachio-amygdaloid pathway by immune challenge or spinal nociceptive input: A quantitative study in the rat using Fos immunohistochemistry and retrograde tract tracing

Peripheral nociceptive stimulation results in activation of neurons in the pontine parabrachial nucleus (PB) of rats. Electrophysiological studies have suggested that noxiously activated PB neurons project to the amygdala, constituting a potential pathway for emotional aspects of pain. In the present study we examined this hypothesis by combining retrograde tract tracing with Fos immunohistochemistry. Cholera toxin subunit B was injected into the amygdala of rats. After a minimum of 48 hours the rats were given a subcutaneous injection of 100 μl of 5% formalin into one hindpaw and killed 60–90 minutes later. A dense aggregation of retrogradely labeled neurons was seen in the external lateral PB. Fos‐expressing neurons were present preferentially in the central, dorsal, and superior lateral subnuclei as well as in the lateral crescent area, as described previously. There was little overlap between the retrogradely labeled and Fos‐expressing populations and double‐labeled neurons were rare. In contrast, systemic immune challenge by intravenous injection of bacterial wall lipopolysaccharide resulted in a Fos expression that overlapped the retrograde labeling in the external lateral PB, and many double‐labeled neurons were seen. While these data provide direct functional anatomical evidence that nociceptive information from the hindlimb is relayed to the amygdala via the parabrachial nucleus, the number of parabrachio‐amygdaloid neurons involved is small. Considering the widespread activation of parabrachio‐amygdaloid neurons by a variety of visceral and humoral stimuli, the parabrachio‐amygdaloid pathway thus appears to be more involved in the mediation of information related to viscerally and humorally elicited activity than in transmission of spinal nociceptive inputs. J. Comp. Neurol. 481:210–219, 2005.

Spinal projections to the parabrachial nucleus are substance P-immunoreactive

Substance P plays an important role in nociceptive processing in the spinal cord. Substance P is also present in several supraspinal regions, such as the pontine parabrachial nucleus, a major relay for autonomic regulation. In this study we examined in the cat with an immunogold method the presence of substance P-like immunoreactivity in spinoparabrachial terminals that were labelled by anterograde transport of unconjugated and lectin-conjugated horseradish peroxidase. We found that dense core vesicles in anterogradely labelled terminals were substance P-immunoreactive. Taken together with previous observations that noxious stimuli increase preprotachykinin expression in ascending nociceptive pathways from the spinal dorsal horn, the present finding provides evidence that substance P is involved in nociceptive processing also in the brain stem.

Feeding-related immune responsive brain stem neurons: association with CGRP.

Using dual-labeling in situ hybridization histochemistry, the neurotransmitter expression of immune-responsive neurons in the pontine parabrachial nucleus, a major relay for interoceptive information, was investigated. Intravenous injection of bacterial wall lipopolysaccharide resulted in dense c-fos mRNA expression in the external lateral parabrachial nucleus, and a majority of the c-fos expressing cells also expressed calcitonin gene-related peptide (CGRP) mRNA. In contrast CGRP-posi- tive cells in the adjoining external medial subnucleus were c-fos negative. Taken together with previous hodological and behavioral studies, these data suggest that CGRPergic parabrachial neurons may mediate lipopolysaccharide-induced anorexia by means of their projection to central nucleus of the amygdala.

Preproenkephalin mRNA expression in rat parabrachial neurons: relation to cells activated by systemic immune challenge

By using a dual-labeling immunohistochemical/in situ hybridization technique we examined if enkephalin-expressing neurons in the pontine parabrachial nucleus, a major brain stem relay for ascending visceral and homeostatic information, were activated by systemic immune challenge. While rats subjected to intravenous injection of bacterial wall lipopolysaccharide expressed dense labeling for the immediate-early gene product FOS in parts of the parabrachial nucleus that also demonstrated dense preproenkephalin expression, only a small proportion of the enkephalin-positive neurons were FOS-positive. These data indicate that enkephalins, although implicated in a variety of autonomic responses, are not primarily involved in the transmission of immune-related information from the parabrachial nucleus to its different forebrain and brain stem targets.

Intraocular hypothalamic transplants containing histaminergic neurons: Innervation of host iris and hippocampal cografts

Hypothalamic tissue containing the tuberomammillary nucleus was dissected from fetuses of Embryonic Day 17 and inserted into the anterior chamber of the eye of young adult recipient rats. The growth of hypothalamic grafts was monitored through the translucent cornea and transplants were found to double in size over the 8 first weeks in oculo. After 4 weeks fetal hippocampal formation (Embryonic Day 18) was inserted into the eye chamber in half of the previously grafted animals and placed in contact with the first grafts. Double grafts were allowed to mature for up to 18 weeks before sacrifice. Recipient rats were anesthetized and superfused with carbodiimide and paraformaldehyde, after which transplants were removed, frozen, sectioned on a cryostat, and incubated with histamine antibodies. Immunohistochemical evaluations revealed a large number of histamine-positive nerve cell bodies with processes innervating the entire hypothalamic graft with a dense plexus of varicose fibers. Such histamine-positive fibers were also seen to invade the surrounding host iris in some cases with thick axon bundles as well as with single fibers. When hypothalamic transplants were combined with hippocampal grafts numerous histamine-immunoreactive fibers invaded the hippocampal tissue to form a plexus of varicose terminals throughout the cografts. After 4 weeks in oculo only a sparse histamine-positive innervation of hippocampal grafts was found, while 18-week-old double grafts contained a considerably larger amount of immunoreactive neurites.(ABSTRACT TRUNCATED AT 250 WORDS)

AMPA-selective glutamate receptor subunits and their relation to glutamate- and GABA-like immunoreactive terminals in the nucleus submedius of the rat.

Glutamate plays an important role in supraspinal nociceptive systems. Thus, glutamate is present in the nucleus submedius of the medial thalamus, a major relay for nociceptive information. In this study, immunoreactivity for the four subunits (GluR1-4) of alpha-amino-3-hydroxy-5-methyl-4-isoxasoleproprionate (AMPA) receptors was examined by a preembedding immunohistochemical method in order to evaluate the presence of this glutamate receptor subtype in the nucleus submedius. Combining the preembedding method with a postembedding immunogold technique, we found that AMPA receptor-like immunoreactivity was present postsynaptically to glutamatergic terminals but not to terminals containing gamma-aminobutyric acid (GABA). These findings suggest a role for AMPA receptors in excitatory synaptic transmission in the nucleus submedius of the rat thalamus.

Lipopolysaccharide induces preproenkephalin transcription in hypophysiotropic neurons of the rat paraventricular hypothalamic nucleus suggesting a neuroendocrine role for enkephalins during immune stress

Opioids have impact on stress responses and possess immune modulatory functions. We have previously shown that immune stress elevates the levels of preproenkephalin transcript in a variety of autonomic structures in the rat brain, including the paraventricular hypothalamic nucleus. By using in situ hybridization with an intronic probe recognizing the preproenkephalin heteronuclear RNA combined with retrograde tract tracing, we examined the efferent target of the enkephalinergic neurons in the paraventricular hypothalamic nucleus that display induced transcriptional activity during immune challenge. Rats were first given i.p. injections of the tracer substance Fluoro-Gold, which following this route of administration is taken up only by nerve terminals residing outside the blood-brain barrier, and were then given an i.v. injection of lipopolysaccharide. Neuronal cell bodies retrogradely labeled with Fluoro-Gold were detected by immunohistochemistry, and-using a dual-labeling approach-the same cells were then examined for their expression of preproenkephalin heteronuclear RNA. We found that over 90% of the neurons that expressed preproenkephalin heteronuclear RNA also contained Fluoro-Gold. In addition, approximately 40% of the neurons expressing preproenkephalin heteronuclear RNA co-expressed mRNA for corticotropin-releasing hormone, the main adrenocorticotropic hormone secretagogue. These data show that the paraventricular hypothalamic neurons that display induced preproenkephalin transcription following immune challenge are almost exclusively hypophysiotropic neurons, indicating a role for enkephalin in the hypothalamic control of hormone release during infectious and inflammatory conditions.