Nectin-2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula.

The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and ß-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, ß-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.

Connections of the laterodorsal tegmental nucleus with the habenular-interpeduncular-raphe system.

The laterodorsal tegmental nucleus (LDTg) is a hindbrain cholinergic cell group thought to be involved in mechanisms of arousal and the control of midbrain dopamine cells. Nowadays, there is increasing evidence that LDTg is also engaged in mechanisms of anxiety/fear and promotion of emotional arousal under adverse conditions. Interestingly, LDTg appears to be connected with other regulators of aversive motivational states, including the lateral habenula (LHb), medial habenula (MHb), interpeduncular nucleus (IP), and median raphe nucleus (MnR). However, the circuitry between these structures has hitherto not been systematically investigated. Here, we placed injections of retrograde or anterograde tracers into LDTg, LHb, IP, and MnR. We also examined the transmitter phenotype of LDTg afferents to IP by combining retrograde tracing with immunofluorescence and in situ hybridization techniques. We found LHb inputs to LDTg mainly emerging from the medial division of the LHb (LHbM), which also receives axonal input from LDTg. The bidirectional connections between IP and LDTg displayed a lateralized organization, with LDTg inputs to IP being predominantly GABAergic or cholinergic and mainly directed to the contralateral IP. Moreover, we disclosed reciprocal LDTg connections with structures involved in the modulation of hippocampal theta rhythm including MnR, nucleus incertus, and supramammillary nucleus. Our findings indicate that the habenula is linked with LDTg either by direct reciprocal projections from/to LHbM or indirectly via the MHb-IP axis, supporting a functional role of LDTg in the regulation of aversive behaviors, and further characterizing LHb as a master controller of ascending brainstem state-setting modulatory projection systems.

Transforming Sensory Cues into Aversive Emotion via Septal-Habenular Pathway.

Emotions evoked by environmental cues are important for animal survival and life quality. However, neural circuits responsible for transforming sensory signals to aversive emotion and behavioral avoidance remain unclear. Here, we found that medial septum (MS) mediates aversion induced by both auditory and somatosensory stimuli. Ablation of glutamatergic or GABAergic MS neurons results in impaired or strengthened aversion, respectively. Optogenetic activation of the two cell types results in place avoidance and preference, respectively. Cell-type-specific screening reveals that glutamatergic MS projections to the lateral habenula (LHb) are responsible for the induction of aversion, which can be antagonized by GABAergic MS projections to LHb. Additionally, the sensory-induced place avoidance is facilitated by enhanced locomotion mediated by glutamatergic MS projections to the preoptic area. Thus, MS can transmit innately aversive signals via a bottom-up multimodal sensory pathway and produce concurrent emotional and motional effects, allowing animals to efficiently avoid unfavorable environments.

Neuropeptidomics of the Rat Habenular Nuclei.

Conserved across vertebrates, the habenular nuclei are a pair of small symmetrical structures in the epithalamus. The nuclei functionally link the forebrain and midbrain by receiving input from and projecting to several brain regions. Each habenular nucleus comprises two major asymmetrical subnuclei, the medial and lateral habenula. These subnuclei are associated with different physiological processes and disorders, such as depression, nicotine addiction, and encoding aversive stimuli or omitting expected rewarding stimuli. Elucidating the functions of the habenular nuclei at the molecular level requires knowledge of their neuropeptide complement. In this work, three mass spectrometry (MS) techniques-liquid chromatography (LC) coupled to Orbitrap tandem MS (MS/MS), LC coupled to Fourier transform (FT)-ion cyclotron resonance (ICR) MS/MS, and matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS-were used to uncover the neuropeptide profiles of the rodent medial and lateral habenula. With the assistance of tissue stabilization and bioinformatics, a total of 262 and 177 neuropeptides produced from 27 and 20 prohormones were detected and identified from the medial and lateral habenula regions, respectively. Among these neuropeptides, 136 were exclusively found in the medial habenula, and 51 were exclusively expressed in the lateral habenula. Additionally, novel sites of sulfation, a rare post-translational modification, on the secretogranin I prohormone are identified. The results demonstrate that these two small brain nuclei have a rich and differentiated peptide repertoire, with this information enabling a range of follow-up studies.

Anterior and posterior parts of the rat ventral tegmental area and the rostromedial tegmental nucleus receive topographically distinct afferents from the lateral habenular complex.

That activation of the reward system involves increased activity of dopaminergic (DA) neurons in the ventral tegmental area (VTA) is widely accepted. In contrast, the lateral habenular complex (LHb), which is known as the center of the anti-reward system, directly and indirectly inhibits DA neurons in the VTA. The VTA, however, is not a homogenous entity. Instead, it displays major functional differences between its anterior (aVTA) and posterior (pVTA) regions. It is not precisely known, whether habenular input to the aVTA, pVTA, and the newly recognized rostromedial tegmental nucleus (RMTg) are similarly or differently organized. Consequently, the present investigation addressed the connections between LHb and aVTA, pVTA, and RMTg using retrograde and anterograde tracing techniques in the rat. Our experiments disclosed strictly reciprocal and conspicuously focal interconnections between LHbM (LHbMPc/LHbMC) and PN, as well as between RLi and LHbLO. In addition, we found that LHb inputs to the aVTA are dorsoventrally ordered. Dorsal parts of the aVTA receive afferents from LHbL and LHbM, whereas ventral parts of the aVTA are preferentially targeted by the LHbM. LHb afferents to the pVTA are distinct from those to the RMTg, given that the RMTg is primarily innervated from the LHbL, whereas pVTA receives afferents from LHbM and LHbL. These data indicate the existence of two separate pathways from the LHb to the VTA, a direct and an indirect one, which may subserve distinct biological functions.

Afferent and efferent connections of the interpeduncular nucleus with special reference to circuits involving the habenula and raphe nuclei.

The habenula is an epithalamic structure differentiated into two nuclear complexes, medial (MHb) and lateral habenula (LHb). Recently, MHb together with its primary target, the interpeduncular nucleus (IP), have been identified as major players in mediating the aversive effects of nicotine. However, structures downstream of the MHb-IP axis, including the median (MnR) and caudal dorsal raphe nucleus (DRC), may contribute to the behavioral effects of nicotine. The afferent and efferent connections of the IP have hitherto not been systematically investigated with sensitive tracers. Thus, we placed injections of retrograde or anterograde tracers into different IP subdivisions or the MnR and additionally examined the transmitter phenotype of major IP and MnR afferents by combining retrograde tract tracing with immunofluorescence and in situ hybridization techniques. Besides receiving inputs from MHb and also LHb, we found that IP is reciprocally interconnected mainly with midline structures, including the MnR/DRC, nucleus incertus, supramammillary nucleus, septum, and laterodorsal tegmental nucleus. The bidirectional connections between IP and MnR proved to be primarily GABAergic. Regarding a possible topography of IP outputs, all IP subnuclei gave rise to descending projections, whereas major ascending projections, including focal projections to ventral hippocampus, ventrolateral septum, and LHb originated from the dorsocaudal IP. Our findings indicate that IP is closely associated to a distributed network of midline structures that modulate hippocampal theta activity and forms a node linking MHb and LHb with this network, and the hippocampus. Moreover, they support a cardinal role of GABAergic IP/MnR interconnections in the behavioral response to nicotine.

Nicotine regulates activity of lateral habenula neurons via presynaptic and postsynaptic mechanisms.

There is much interest in brain regions that drive nicotine intake in smokers. Interestingly, both the rewarding and aversive effects of nicotine are probably critical for sustaining nicotine addiction. The medial and lateral habenular (LHb) nuclei play important roles in processing aversion, and recent work has focused on the critical involvement of the LHb in encoding and responding to aversive stimuli. Several neurotransmitter systems are implicated in nicotine's actions, but very little is known about how nicotinic acetylcholine receptors (nAChRs) regulate LHb activity. Here we report in brain slices that activation of nAChRs depolarizes LHb cells and robustly increases firing, and also potentiates glutamate release in LHb. These effects were blocked by selective antagonists of α6-containing (α6*) nAChRs, and were absent in α6*-nAChR knockout mice. In addition, nicotine activates GABAergic inputs to LHb via α4ß2-nAChRs, at lower concentrations but with more rapid desensitization relative to α6*-nAChRs. These results demonstrate the existence of diverse functional nAChR subtypes at presynaptic and postsynaptic sites in LHb, through which nicotine could facilitate or inhibit LHb neuronal activity and thus contribute to nicotine aversion or reward.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-µM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.

Prolonged expression of c-Fos protein in the lateral habenular nucleus of the Japanese monkey (Macaca fuscata) after eye enucleation.

To elucidate the effect of traumatic stress on the lateral habenular nucleus, we investigated the time course of the expression of c-Fos protein in this nucleus of the Japanese monkey (Macaca fuscata) after enucleation of one eye using c-Fos protein immunocytochemistry. c-Fos protein-like immunoreactive neurons were significantly increased; the increase started 1 h after the enucleation and remained high for 3-9 h in the lateral habenular nucleus on both sides. These results suggest that the prolonged expression of c-Fos protein occurred in the lateral habenular nucleus after traumatic stress through multiple transsynaptic activations.

Neuronal and astrocyte expression of nicotinic receptor subunit beta4 in the adult mouse brain.

Neuronal nicotinic acetylcholine receptor (nAChR) expression and function are customized in different brain regions through assembling receptors from closely related but genetically distinct subunits. Immunohistochemical analysis of one of these subunits, nAChRbeta4, in the mouse brain suggests an extensive and potentially diverse role for this subunit in both excitatory and inhibitory neurotransmission. Prominent immunostaining included: 1) the medial habenula, efferents composing the fasciculus retroflexus, and the interpeduncular nucleus; 2) nuclei and ascending tracts of the auditory system inclusive of the medial geniculate; 3) the sensory cortex barrel field and cell bodies of the ventral thalamic nucleus; 4) olfactory-associated structures and the piriform cortex; and 5) sensory and motor trigeminal nuclei. In the hippocampus, nAChRbeta4 staining was limited to dendrites and soma of a subset of glutamic acid dehydrogenase-positive neurons. In C57BL/6 mice, but to a lesser extent in C3H/J, CBA/J, or CF1 mice, a subpopulation of astrocytes in the hippocampal CA1 region prominently expressed nAChRbeta4 (and nAChRalpha4). Collectively, these results suggest that the unique functional and pharmacological properties exerted by nAChRbeta4 on nAChR function could modify and specialize the development of strain-specific sensory and hippocampal-related characteristics of nicotine sensitivity including the development of tolerance.

Morphologic and cytochemical criteria for the identification and delineation of individual subnuclei within the lateral habenular complex of the rat.

The lateral habenular complex is part of the habenular nuclei, a distinct structure in the dorsal diencephalon of all vertebrates. In contrast to the bewildering diversity of behaviors, in which the lateral habenular complex is thought to be involved, there is an astonishing lack of information concerning its cellular organization, its neuronal circuits, and the neurophysiological mechanisms, which may provide the physiological and molecular basis for its diverse biological functions. This problem may be due to an unexpected heterogeneity of the lateral habenular complex. Recently, a detailed subnuclear organization has been described (Andres et al. [1999] J Comp Neurol 407:130-150), which provides the base for a subsequent physiological and behavioral analysis of this area. Available criteria, however, can be applied to semithin sections only. To facilitate further investigations, the present work aimed to elaborate novel morphologic and immunocytochemical criteria that can be applied to conventional cryostat or Vibratome sections to allow identification and delineation of subnuclei of the lateral habenular complex. Consequently, the regional, cellular, and subcellular localization of approximately 30 different neuroactive molecules was investigated. Of these candidate molecules, gamma-aminobutyric acid-B receptor protein, Kir3.2 potassium channel protein, tyrosine hydroxylase, and neurofilament heavy chain proved to be suitable markers. Our observation suggests that the habenular subnuclei express distinct immunocytochemical characteristics. These features may be used to identify and delineate the subnuclei on conventional cryostat or Vibratome sections. From our results, it is expected that the further functional analysis of the lateral habenular complex will be facilitated considerably.

Isolation and characterization of two teleost melanopsin genes and their differential expression within the inner retina and brain.

Melanopsin is a newly discovered photopigment that is believed to be involved in the regulation of circadian rhythms in tetrapods. Here we describe the characterization of the first two teleost melanopsins (opn4a and opn4b) isolated from Atlantic cod (Gadus morhua). These two teleost genes belong to a subgroup of melanopsins that also include members from Xenopus, chicken, and Takifugu. In situ hybridization revealed that opn4a and opn4b are differentially expressed within the retina and brain. In the larval and adult retina, both melanopsins are expressed in a subset of cells in the inner retina, resembling amacrine and ganglion cells. In addition, opn4a is expressed in the horizontal cells, indicating a separate task for this gene. In the brain, the two melanopsins are separately expressed in two major retinal and extraretinal photosensitive integration centers, namely, the suprachiasmatic nucleus (opn4a) and the habenula (opn4b). The expression of opn4a in the suprachiasmatic nucleus in cod is similar to the melanopsin expression found in Xenopus. This suggests a conserved role for this opsin and an involvement in mediation of nonvisual photoreceptive tasks, such as entraining circadian rhythms and/or hypophysiotrophic systems. The differential expression of opn4b in the habenula suggests that this gene plays a role similar to that of opn4a, in that it is also situated in an area that integrates photic inputs from the pineal as well as other brain regions. Thus, the habenula may be an additional region that mediates photic cues in teleosts.

Mapping of olfactory memory circuits: region-specific c-fos activation after odor-reward associative learning or after its retrieval.

Although there is growing knowledge about intracellular mechanisms underlying neuronal plasticity and memory consolidation and reconsolidation after retrieval, information concerning the interaction among brain areas during formation and retrieval of memory is relatively sparse and fragmented. Addressing this question requires simultaneous monitoring of activity in multiple brain regions during learning, the post-acquisition consolidation period, and retrieval and subsequent reconsolidation. Immunoreaction to the immediate early gene c-fos is a powerful tool to mark neuronal activation of specific populations of neurons. Using this method, we are able to report, for the first time, post-training activation of a network of closely related brain regions, particularly in the frontal cortex and the basolateral amygdala (BLA), that is specific to the learning of an odor-reward association. On the other hand, retrieval of a well-established associative memory trace does not seem to differentially activate the same regions. The amygdala, in particular, is not engaged after retrieval, whereas the lateral habenula (LHab) shows strong activation that is restricted to animals having previously learned the association. Although intracellular mechanisms may be similar during consolidation and reconsolidation, this study indicates that different brain circuits are involved in the two processes, at least with respect to a rapidly learned olfactory task.

Distribution of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) receptor mRNAs in the rat brain.

The precise involvement of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) receptors in the pleiotropic actions of 5-HT remain incompletely known. To gain insights into their physiological function(s), localization of mRNAs encoding these subtypes was carried out using in situ hybridization on rat brain sections. Localization was heterogeneous. For example, 5-ht(5A) mRNA was widely expressed while 5-ht(5B) mRNA was predominantly expressed in habenula, hippocampus and inferior olive. 5-ht(6) mRNA was abundant in olfactory tubercles and caudate putamen, and highest levels of 5-HT(7) mRNA were observed in multiple thalamic nuclei. These data suggest that these receptors may have distinct functional roles within the serotonergic system.

Thalamic distribution of zinc-rich terminal fields and neurons of origin in the rat.

Several cortico-cortical and limbic-related circuits are enriched in zinc, which is considered as an important modulator of glutamatergic transmission. While heavy metals have been detected in the thalamus, the specific presence of zinc has not been examined in this region. We have used two highly sensitive variations of the Timm method to study the zinc-rich innervation in the rat thalamus, which was compared to the distribution of acetylcholinesterase activity. The origin of some of these zinc-rich projections was also investigated by means of retrograde transport after intracerebral infusions of sodium selenium (Na2SeO3). The overall zinc staining in the thalamus was much lower than in the neocortex, striatum or basal forebrain; however, densely stained terminal fields were observed in the dorsal tip of the reticular thalamic nucleus, the anterodorsal and lateral dorsal thalamic nuclei and the zona incerta. In addition, moderately stained zinc-rich terminal fields were found in the rostral intralaminar nuclei, nucleus reuniens and lateral habenula. Intracerebral infusions of Na2SeO3 in the lateral dorsal nucleus resulted in retrogradely labeled neurons that were located in the postsubiculum, and also in the pre- and parasubiculum. These results are the first to establish the existence of a zinc-rich subicular-thalamic projection. Similar infusions in either the intralaminar nuclei or the zona incerta resulted in labeling of neurons in several brainstem structures related to the reticular formation. Our results provide morphological evidence for zinc modulation of glutamatergic inputs to highly selective thalamic nuclei, arising differentially from either cortical limbic areas or from brainstem ascending activation systems.

Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.

Distribution and properties of GABA(B) antagonist [3H]CGP 62349 binding in the rhesus monkey thalamus and basal ganglia and the influence of lesions in the reticular thalamic nucleus.

GABA(B) receptors are believed to be associated with the efferents of the nucleus reticularis thalami, which is implicated in the regulation of activity in the thalamocortical-corticothalamic circuit and plays a role in absence seizures. Yet, the distribution of GABA(B) receptors in the thalamus has only been studied in the rat, and there is no comparable information in primates. The potent GABA(B) receptor antagonist [3H]CGP 62349 was used to study the distribution and binding properties of the receptor in control monkeys and those with small ibotenic acid lesions in the anterodorsal segment of the nucleus reticularis thalami. Eight-micrometer-thick cryostat sections of the fresh frozen brains were incubated in the presence of varying concentrations of the ligand. Autoradiographs were analysed using a quantitative image analysis technique, and binding parameters were calculated for select thalamic nuclei as well as basal ganglia structures present in the same sections. The overall number of GABA(B) binding sites in the monkey thalamus and basal ganglia was several-fold higher than previously reported values for the rat. In the thalamus, the receptors were distributed rather uniformly and the binding densities and affinities were high (Bmax range of 245.5-437.9 fmol/ mg of tissue, Kd range of 0.136-0.604 nM). In the basal ganglia, the number of binding sites and the affinities were lower (Bmax range of 51.1-244.2 fmol/mg of tissue; K(d) range of 0.416-1.394 nM), and the differences between nuclei were more pronounced, with striatum and substantia nigra pars compacta displaying the highest binding densities. Seven days post-lesion, a 20-30% decrease in Bmax values (P < 0.05) was found in the nuclei receiving input from the lesioned nucleus reticularis thalami sector (the mediodorsal nucleus and densicellular and magnocellular parts of the ventral anterior nucleus) without changes in affinity. No significant changes were detected in any other structures. The results of the lesioning experiments suggest that a portion of thalamic GABA(B) receptors is in a presynaptic location on the nucleus reticularis thalami efferents. The overall distribution pattern in the thalamus also suggests a partial association of GABA(B) receptors with corticothalamic terminals presynaptically.

Developmental changes in nicotinic receptor mRNAs and responses to nicotine in the suprachiasmatic nucleus and other brain regions.

Our previous studies demonstrated that nicotine induces c-fos expression in the suprachiasmatic nucleus (SCN) of the rat during a narrow developmental window occurring in the perinatal period. We have extended these observations by showing that c-fos cannot be induced in the adult SCN by nicotine even during the subjective night, when phase shifts do occur. In contrast to the SCN, significant induction of c-fos and NGFI-A was observed in the medial habenula and paraventricular nucleus at all circadian times. In the fetal rat SCN we show that NGFI-A and junB are also induced by nicotine, but not c-jun. To investigate whether changes in nicotinic acetylcholine receptor (nAChR) expression in the SCN may underlie this change in sensitivity during the perinatal period, we examined nAChR mRNAs across this developmental period. By Northern analyses, alpha2, alpha3 and alpha4 subunit mRNAs are relatively abundant in the fetal SCN but decline substantially in the adult. alpha7 mRNA increases substantially while beta2 mRNA is relatively abundant throughout development. We also examine expression in the whole mouse brain beginning at embryonic day 11. Many mRNA sizes for nAChR subunits in both the rat and mouse are characterized here for the first time by Northern analyses and some show very large changes in expression across development. In particular, a small 1.4 kb alpha2-related mRNA is highly expressed during early development, perhaps indicating an important novel function for this subunit.

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

The present study examined the effects of ageing on preprotachykinin-A (PPT-A) mRNA levels in discrete regions of the rat brain. Semiquantitative analysis of silver grains revealed a 16% statistically significant decrease in PPT-A mRNA in the shell of the nucleus accumbens (AcbSh), a 27.6% statistically significant lower level of PPT-A mRNA in the olfactory tubercle (Tu), a 19.2% and 31. 5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate-putamen (d-CPu) (v-CPu), respectively, a 30% statistically significant lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% statistically significant decrease in PPT-A mRNA in the habenula (Hb) and a 30% statistically significant decrease of PPT-A mRNA levels in the postero-dorsal part of the medial amygdala (MePD). No changes in PPT-A mRNA levels were found in the nucleus accumbens, core (AcbC), in the islands of Calleja (Icj), and in the medial preoptic area (mPOA). These results show that ageing of the central nervous system (CNS) is associated with widespread changes in tachykinin gene expression, suggesting that alteration in the tachykinergic system may have implications in the physio-pathology of the elderly.