James E. Krause

The cloned vanilloid receptor VR1 has attracted recent attention as a molecular integrator of painful stimuli on primary sensory neurons. The existence of vanilloid-sensitive neurons in the brain is, however, controversial. In this study, we have used an antibody and a complementary RNA probe to explore the distribution of neurons that express VR1 in rat and in certain areas of human brain. In the rat, we observed VR1-expressing neurons throughout the whole neuroaxis, including all cortical areas (in layers 3 and 5), several members of the limbic system (e.g., hippocampus, central amygdala, and both medial and lateral habenula), striatum, hypothalamus, centromedian and paraventricular thalamic nuclei, substantia nigra, reticular formation, locus coeruleus, cerebellum, and inferior olive. VR1-immunopositive cells also were found in the third and fifth layers of human parietal cortex. Reverse transcription-PCR performed with rat VR1-specific primers verified the expression of VR1 mRNA in cortex, hippocampus, and hypothalamus. In the central nervous system, neonatal capsaicin treatment depleted VR1 mRNA from the spinal nucleus of the trigeminal nerve, but not from other areas such as the inferior olive. The finding that VR1 is expressed not only in primary sensory neurons but also in several brain nuclei is of great importance in that it places VRs in a much broader perspective than pain perception. VRs in the brain (and putative endogenous vanilloids) may be involved in the control of emotions, learning, and satiety, just to name a few exciting possibilities.

Synthetic oligonucleotides were used to screen a rat striatal cDNA library for sequences corresponding to the tachykinin peptides substance P and neurokinin A. The cDNA library was constructed from RNA isolated from the rostral portion of the rat corpus striatum, the site of striatonigral cell bodies. Two types of cDNAs were isolated and defined by restriction enzyme analysis and DNA sequencing to encode both substance P and neurokinin A. The two predicted preprotachykinin protein precursors (130 and 115 amino acids in length) differ from each other by a pentadecapeptide sequence between the two tachykinin sequences, and both precursors possess appropriate processing signals for substance P and neurokinin A production. The presence of a third preprotachykinin mRNA of minor abundance in rat striatum was established by S1 nuclease protection experiments. This mRNA encodes a preprotachykinin of 112 amino acids containing substance P but not neurokinin A. These three mRNAs are derived from one rat gene as a result of differential RNA processing; thus, this RNA processing pattern further increases the diversity of products that can be generated from the preprotachykinin gene.

A sensitive solution-hybridization assay was used to investigate the expression of genes encoding insulin-like growth factors I and II (IGF-I and -II) in the rat central nervous system (CNS). mRNAs for both IGFs are synthesized throughout the CNS of adult rats but exhibit distinct regional differences for each growth factor. IGF-I mRNA is 8-10 times more abundant in the cervical-thoracic spinal cord and in the olfactory bulb than in whole brain and is enriched 3-fold in the midbrain and cerebellum. IGF-II mRNA is minimally enriched in the medulla-pons and cerebellum but is 3-5 times less abundant in the midbrain and corpus striatum than in total brain. During CNS development the content of IGF-I and IGF-II mRNAs is highest at embryonic day 14 and declines by a factor of 3-4 at birth, to values found in adult brain. Embryonic neurons and glia synthesize IGF-I mRNA during short-term cell culture; only glia produce IGF-II mRNA. These observations show that IGF-I and IGF-II are differentially expressed in the developing and adult CNS and suggest that each growth factor may play a unique role in the mammalian nervous system.

The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.

Substance P is a member of the tachykinin peptide family and participates in the regulation of diverse biological processes. The polymerase chain reaction and conventional library screening were used to isolate a complementary DNA (cDNA) encoding the rat substance P receptor from brain and submandibular gland. By homology analysis, this receptor belongs to the G protein-coupled receptor superfamily. The receptor cDNA was expressed in a mammalian cell line and the ligand binding properties of the encoded receptor were pharmacologically defined by Scatchard analysis and tachykinin peptide displacement as those of a substance P receptor. The distribution of the messenger RNA for this receptor is highest in urinary bladder, submandibular gland, striatum, and spinal cord, which is consistent with the known distribution of substance P receptor binding sites. Thus, this receptor appears to mediate the primary actions of substance P in various brain regions and peripheral tissues.