Fundus Striati Research Articles

Applicability of image analysis of neurons and neuroglia of fundus striati using cell profiler software

Analysis of volumetric and morphological neuronal data has been of keen interest to neurologists and neuroscientists because of its implications in pathological conditions such as schizophrenia autism obsessive compulsive disorder etc One such part of human brain which has been explored in recent years is fundus striati a part of basal ganglia bridging caudate nucleus and putamen A versatile technique to measure neurons of fundus striati is the use of Cell profiler software The present study was undertaken to study and analyze images of fundus striati using cell profiler as an automated image analysis technique A qualitative cross sectional study was done using eighty one images of histological sections of hematoxylin and eosin stained micro thick tissue sections of fundus striati Freely downloadable Cell Profiler software was installed specific pipeline for neuronal assay was drawn and modules were used to analyze images Neuronal nuclei were identified as primary objects and cytoplasm was identified by the software as secondary objects Using specific modules neurons and branching points of their neurites in images of fundus striati were measured in pixel units Mean Intensity units of neurons of fundus striati as measured by intensity module of Cell profiler was pixel units Results of the present study can be extrapolated to correlate with pathological conditions associated with emotional and behavioral disorders involving fundus striati Cell profiler is cost effective software which is beneficial to identify and measure neurons of fundus striati

Parvalbumin-immunoreactive neurons and GABAergic neurons of the basal forebrain project to the rat basolateral amygdala

The basal forebrain (BF) contains a diffuse array of cholinergic and non-cholinergic neurons that project to the cerebral cortex and basolateral nuclear complex of the amygdala (BLC). Previous studies have shown that the GABAergic subpopulation of non-cholinergic corticopetal BF neurons selectively innervates cortical interneurons. Although several investigations in both rodents and primates have indicated that some BF neurons projecting to the BLC are non-cholinergic, there have been no studies that have attempted to identify the neurochemical phenotype(s) of these neurons. The present study combined Fluorogold retrograde tract tracing with immunohistochemistry for two markers of BF GABAergic neurons, parvalbumin (PV) or glutamic acid decarboxylase (GAD), to determine if a subpopulation of BF GABAergic cells projects to the BLC. Injections of Fluorogold confined to the rat BLC, and centered in the basolateral nucleus, produced extensive retrograde labeling in the ventral pallidum and substantia innominata regions of the BF. Although the great majority of retrogradely labeled neurons were not double-labeled, about 10% of these neurons, located mainly along the ventral aspects of the fundus striati and globus pallidus, exhibited immunoreactivity for PV or GAD. The results of this investigation contradict the long-held belief that there is no extra-amygdalar source of GABAergic inputs to the BLC, and indicate that the cortex-like BLC, in addition to the cortex proper, receives inhibitory inputs from the basal forebrain.

Distribution of tuberoinfundibular peptide of 39 residues and its receptor, parathyroid hormone 2 receptor, in the mouse brain

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as a potent parathyroid hormone 2 receptor (PTH2R) agonist. Existing anatomical data also support the suggestion that TIP39 is the PTH2R's endogenous ligand, but a comprehensive comparison of TIP39 and PTH2R distributions has not been performed. In the present study, we compared the distributions of TIP39 and PTH2R on adjacent mouse brain sections. In addition, we determined the locations of PTH2R-expressing cell bodies by in situ hybridization histochemistry and by labeling beta-galactosidase driven by the PTH2R promoter in knockin mice. An excellent correlation was found between the distributions of TIP39-containing fibers and PTH2R-containing cell bodies and fibers throughout the brain. TIP39 and the PTH2R are abundant in medial prefrontal, insular, and ectorhinal cortices, the lateral septal nucleus, the bed nucleus of the stria terminalis, the fundus striati, the amygdala, the ventral subiculum, the hypothalamus, midline and intralaminar thalamic nuclei, the medial geniculate body, the periaqueductal gray, the ventral tegmental area, the superior and inferior colliculi, the parabrachial nuclei, the locus coeruleus, subcoeruleus and periolivary areas, and the nucleus of the solitary tract. Furthermore, even the subregional distribution of TIP39- and PTH2R-immunoreactive fibers in these regions showed remarkable similarities, providing anatomical evidence that TIP39 may act on the PTH2R. Based on these observations and on previous pharmacological data, we propose that TIP39 is an endogenous ligand of the PTH2R and that they form a neuromodulator system, which is optimally positioned to regulate limbic, endocrine, and auditory brain functions. Published 2007 Wiley-Liss, Inc.

Forebrain projections of tuberoinfundibular peptide of 39 residues (TIP39)-containing subparafascicular neurons

Neurons containing tuberoinfundibular peptide of 39 residues (TIP39) constitute a rostro-caudally elongated group of cells in the posterior thalamus. These neurons are located in the rostral part of the subparafascicular nucleus and in the subparafascicular area, caudally. Projections of the caudally located TIP39 neurons have been previously identified by their disappearance following lesions. We have now mapped the projections of the rat rostral subparafascicular neurons using injections of the anterograde tracer biotinylated dextran amine and the retrograde tracer cholera toxin B subunit, and confirmed the projections from more caudal areas previously inferred from lesion studies. Neurons from both the rostral subparafascicular nucleus and the subparafascicular area project to the medial prefrontal, insular, ecto- and perirhinal cortex, nucleus of the diagonal band, septum, central and basomedial amygdaloid nuclei, fundus striati, basal forebrain, midline and intralaminar thalamic nuclei, hypothalamus, subthalamus and the periaqueductal gray. The subparafascicular area projects more densely to the amygdala and the hypothalamus. In contrast, only the rostral part of the subparafascicular nucleus projects significantly to the superficial layers of prefrontal, insular, ectorhinal and somatosensory cortical areas. Double labeling showed that anterogradely labeled fibers from the rostral part of the subparafascicular nucleus contain TIP39 in many forebrain areas, but do not in hypothalamic areas. Injections of the retrograde tracer cholera toxin B subunit into the lateral septum and the fundus striati confirmed that they were indeed target regions of both the rostral subparafascicular nucleus and the subparafascicular area. In contrast, TIP39 neurons did not project to the anterior hypothalamic nucleus. Our data provide an anatomical basis for the potential involvement of rostral subparafascicular neurons in limbic and autonomic regulation, with TIP39 cells being major subparafascicular output neurons projecting to forebrain regions.

EDI BOARD

Projections from the thalamic gustatory nucleus, i.e. the parvicellular part of the posteromedial ventral thalamic nucleus (VPMpc) to the forebrain regions were studied in the rat by the tract-tracing methods with anterograde tracer (biotinylated dextran amine, BDA) and anterograde/retrograde tracer (wheat-germ agglutinin-horseradish peroxidase, WGA-HRP). After BDA injection into the VPMpc, terminal labeling was observed in the insular cortex, amygdaloid complex, and fundus striati. The terminal labeling in the amygdaloid complex was distributed in dorsolateral area of the rostral part of the lateral amygdaloid nucleus and the rostral part of the lateral subdivision of the central amygdaloid nucleus. The terminal labeling in the central amygdaloid nucleus extended to the fundus striati. The retrograde tracing study with WGA-HRP revealed that the projection fibers from the VPMpc to the amygdaloid complex originated from the medial part of the VPMpc and also from the thalamic area medial to the VPMpc. In the rats injected with Fluoro-Gold and WGA-HRP, respectively into the insular cortex and amygdaloid complex, no double-labeled neuronal cell bodies were found in the VPMpc, although neurons labeled singly with Fluoro-Gold were intermingled with those singly labeled with WGA-HRP in the medial part of the VPMpc. The results indicated that VPMpc neurons projecting to the amygdaloid complex constituted a population different from VPMpc neurons projecting to the insular cortex.

Altered neurotensin mrna expression in mice lacking the dopamine transporter

Psychostimulants and antipsychotic drugs increase mRNA expression of the neuropeptide neurotensin (NT) in the striatum and nucleus accumbens. In the present study, we used mice lacking the dopamine transporter (DAT) to investigate the consequences of a chronic hyperdopaminergic state on NT gene expression. NT mRNA expression was examined under basal conditions and after administration of haloperidol or amphetamine using in situ hybridization with a digoxigenin-labeled NT cRNA probe. DAT−/− mice exhibited a striking increase in the number of NT mRNA-expressing perikarya in the substantia nigra and ventral tegmental area, as well as a less pronounced increase in the lateral septum compared with wild-type littermates. No changes were detected in other regions expressing NT mRNA. Acute administration of haloperidol (1 mg/kg) induced a significant increase in the number of NT mRNA-expressing neurons in the dorsomedial and dorsolateral striatum of wild-type mice but failed to stimulate NT gene expression in DAT mutants. In contrast, a higher dose of haloperidol (5 mg/kg) stimulated striatal NT mRNA expression both in DAT+/+ and DAT−/− mice. Amphetamine (10 mg/kg) increased the number of hybridized neurons in the nucleus accumbens shell and fundus striati of wild-type and DAT−/− mice, indicating that the drug acted through a target other than DAT, such as the serotonin or the norepinephrine transporters. The up-regulation of NT mRNA observed in DAT−/− mice may represent an adaptive mechanism in response to constitutive hyperdopaminergia. These results illustrate the profound alterations in the NT system induced by chronic stimulation of DA receptors and underscore the potential clinical relevance of NT/DA interactions in schizophrenia and drug abuse.

Neurons containing tuberoinfundibular peptide of 39 residues project to limbic, endocrine, auditory and spinal areas in rat

Accumulating evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) may be the endogenous ligand of the parathyroid hormone 2 receptor. The vast majority of TIP39-containing neurons are localized in two regions, the subparafascicular area at the thalamic-midbrain junction, and the medial paralemniscal nucleus in the rostral pons. In contrast to the restricted localization of TIP39-containing cell bodies, TIP39-containing fibers have a widespread distribution. TIP39 neurons were lesioned electrolytically to determine the origin of TIP39-containing fibers within different parts of the rat CNS. Following bilateral lesions of the medial subparafascicular area including the subparafascicular nucleus, TIP39-immunoreactive fibers almost completely disappeared from forebrain regions including the anterior limbic cortical areas, the shell and cone portions of the nucleus accumbens, the lateral septum, the bed nucleus of the stria terminalis, the amygdaloid nuclei, the fundus striati, the subiculum, the thalamic paraventricular nucleus, and the hypothalamic paraventricular, dorsomedial and arcuate nuclei. Unilateral lesions of the medial and the lateral subparafascicular area demonstrated that the projections are ipsilateral and that medial lesions produce higher reductions in the density of TIP39 fibers except in the amygdala and the hypothalamus. Following lesions of the medial paralemniscal nucleus, TIP39-immunoreactive fibers disappeared from the medial geniculate body, the periaqueductal gray, the deep layers of the superior colliculus, the external cortex of the inferior colliculus, the cuneiform nucleus, the nuclei of the lateral lemniscus, the lateral parabrachial nucleus, the locus coeruleus, the subcoeruleus area, the medial nucleus of the trapezoid body, the periolivary nuclei, and the spinal cord, suggesting that these regions receive TIP39-containing fibers from the medial paralemniscal nucleus, and unilateral lesions demonstrated that the projections are ipsilateral. The projections of the TIP39-containing cells in the subparafascicular area suggest their involvement in limbic and endocrine functions, while the projections of the TIP39-containing cells in the medial paralemniscal nucleus suggest their involvement in auditory and nociceptive functions.

Diet-induced obesity increases μ opioid receptor binding in specific regions of the rat brain

Mu (μ) opioid agonists preferentially increase the intake of highly palatable food. Here we investigated changes in μ opioid binding in feeding- and reward-related brain regions in rats given a palatable diet for 17 weeks. Diet feeding induced variable obesity, and rats were stratified into ‘high-weight gain’ (HWG: weight increase, 513–695 g; n=12) and ‘low-weight gain’ (LWG: range: 396–502 g; n=11) groups. Chow-fed controls ( n=9) gained 324–487 g during this time. Body fat mass and plasma leptin and insulin were significantly higher in LWG than in controls and even higher in HWG. μ-Receptor binding (measured in brain slices using [ 3H]-DAMGO ( d-Ala 2, N-Me-Phe 4,Gly-ol 5) and quantitative autoradiography) was significantly increased in specific forebrain regions of diet-fed rats. In the fundus striati, dorsal endopiriform nucleus and medial preoptic area (MPA), binding was similarly increased (30–40%; P<0.05 vs. controls) in the HWG and LWG groups. Increases in μ binding paralleled weight gain in the basolateral amygdala and basomedial amygdala, being ∼20% above controls ( P<0.001) in LWG and ∼30% higher in HWG ( P<0.05 vs. LWG). The medial habenula showed significantly higher binding (by ∼40%) in HWG, with no significant changes in LWG. In all these areas (except the MPA), binding was significantly correlated with plasma leptin and insulin. We suggest that increased μ binding reflects decreased release of endogenous μ opioid peptides. This orexigenic system therefore seems unlikely to drive appetite for palatable food. Indeed, the μ opioid system in reward-related areas may be inhibited in dietary obesity, probably by increased plasma leptin and insulin, and this may represent a failed homeostatic attempt to limit overeating and eventually obesity.

Immunocytochemical evidence for the striatal nature of the rat lateral part of interstitial nucleus of the posterior limb of the anterior commissure (IPAC)

The present study focuses on the basal forebrain region originally designated as fundus striati, but currently known as ‘interstitial nucleus of the posterior limb of the anterior commissure’ (IPAC). Using multiple immunofluorescence of the calcium-binding proteins parvalbumin and calbindin, the GABA A receptor α1-subunit, vasoactive intestinal polypeptide (VIP), met 5-enkephalin (MENK) and glutamic acid decarboxylase (GAD), it was shown that VIP-immunostained axons, which are typical for major parts of the extended amygdala, densely innervate only the medial part of IPAC, while they are absent in the lateral part. On the other hand, large-sized GABAergic, parvalbumin- and GABA A receptor α1-subunit-immunoreactive neurons, which are densely covered by separate GAD- and MENK-immuno reactive terminals and a type of medium-sized α1-subunit-monolabelled cells, occur in the dorsal striatum and in the adjacent lateral part of IPAC as well. Large-sized neurons double labelled for parvalbumin and the GABA A receptor α1-subunit are also widely distributed in the neighbouring ventral pallidum. Neurons of this type are absent, however, in the medial part of IPAC and other extended amygdala subunits. Our findings confirm the recent suggestion of a morphofunctional dichotomy of IPAC (Comp. Neurol. 439 (2001) 104), as only the medial part reveals characteristics as typical for extended amygdala, while its lateral part exhibits cytochemical peculiarities of striatal tissue. Therefore, the term ‘lateral part of IPAC’ should be replaced by the term ‘putaminal fundus (fundus putaminis)’ according to recently published designations of corresponding striatal constituents (Atlas of the Human Brain, 2002, Academic Press, San Diego, CA; J. Chem. Neuroanat. 23 (2002) 75).

An anterograde and retrograde tract-tracing study on the projections from the thalamic gustatory area in the rat: distribution of neurons projecting to the insular cortex and amygdaloid complex.

Projections from the thalamic gustatory nucleus, i.e. the parvicellular part of the posteromedial ventral thalamic nucleus (VPMpc) to the forebrain regions were studied in the rat by the tract-tracing methods with anterograde tracer (biotinylated dextran amine, BDA) and anterograde/retrograde tracer (wheat-germ agglutinin-horseradish peroxidase, WGA-HRP). After BDA injection into the VPMpc, terminal labeling was observed in the insular cortex, amygdaloid complex, and fundus striati. The terminal labeling in the amygdaloid complex was distributed in dorsolateral area of the rostral part of the lateral amygdaloid nucleus and the rostral part of the lateral subdivision of the central amygdaloid nucleus. The terminal labeling in the central amygdaloid nucleus extended to the fundus striati. The retrograde tracing study with WGA-HRP revealed that the projection fibers from the VPMpc to the amygdaloid complex originated from the medial part of the VPMpc and also from the thalamic area medial to the VPMpc. In the rats injected with Fluoro-Gold and WGA-HRP, respectively into the insular cortex and amygdaloid complex, no double-labeled neuronal cell bodies were found in the VPMpc, although neurons labeled singly with Fluoro-Gold were intermingled with those singly labeled with WGA-HRP in the medial part of the VPMpc. The results indicated that VPMpc neurons projecting to the amygdaloid complex constituted a population different from VPMpc neurons projecting to the insular cortex.

Neurocircuitries of the basal ganglia studied in organotypic cultures: focus on tyrosine hydroxylase, nitric oxide synthase and neuropeptide immunocytochemistry

The nigrostriatal and mesolimbic systems of the rat were reconstructed using an organotypic culture model, whereby neonatal brain tissue was grown in vitro for approximately one month. The nigrostriatal system comprised of tissue from the substantia nigra, the dorsal striatum and the frontoparietal cortex, while the mesolimbic system included the ventral tegmental area, ventral striatum (including the fundus striati, accumbens nucleus, olfactory tubercle, lateral septum, ventral pallidum and piriform cortex) and cingulate cortex. These regions were also cultured alone or in pairs. The cultures were monitored in vitro, and after one month fixed in a formalin–picric acid solution, and processed for immunohistochemistry using antibodies raised against tyrosine hydroxylase, nitric oxide synthase, preprocholecystokinin, glutamate decarboxylase, neuropeptide Y, dopamine- and cyclic AMP-regulated phosphoprotein-32 and glial fibrillary acidic protein. The tissue survived in single, double or triple cultures, although differences were found depending upon the source and combination of cultured region. Neurons had localization and shape as in vivo. Local networks were especially prominent in the mesencephalon, where both tyrosine hydroxylase-positive axons spread from the “substantia nigra” to the rest of the tissue, and where nitric oxide synthase-positive networks also surrounded tyrosine hydroxylase-positive neurons. Glutamate decarboxylase-positive nerve terminals formed dense networks around tyrosine hydroxylase-positive neurons. In the striatum, nitric oxide synthase and dopamine- and cyclic AMP-regulated phosphoprotein-32 neurons were surrounded by tyrosine hydroxylase-positive nerve terminals. The nigral and ventral tegmental area dopamine neurons projected to striatal and cortical structures, but the projection from the ventral tegmental area to the cingulate cortex was more prominent. With regard to co-existence, preprocholecystokinin-like immunoreactivities was found in many tyrosine hydroxylase-positive neurons and neuropeptide Y- and nitric oxide synthase-like immunoreactivity co-existed in striatal and cortical tissues. In general terms, the chemical neuroanatomy in the cultures was similar to that described earlier in vivo. Nitric oxide synthase staining was particularly intense. Taken together, the organotypic model captures many of the morphological and neurochemical features seen in vivo, providing a valuable model for studying neurocircuitries of the brain in detail, where `normal' and `pathological' conditions can be simulated.

Historadioautographic localisation of oxytocin and vasopressin binding sites in the central nervous system of the merione ( Meriones shawi)

The distribution of vasopressin and oxytocin binding sites in the central nervous system of the merione ( Meriones shawi), a rodent adapted to desert life, was studied by means of conventional film radioautography at macroscopic scale and historadioautography at cellular level using radioiodinated ligands highly selective for either oxytocin or type V1a vasopressin receptors. Both types of binding sites exhibited the same selectivity for endogenous peptides as in the rat. Distribution of oxytocin binding sites was similar in some structures (limbic system, spinal cord) to that described in the rat and in other rodents. Vasopressin binding sites were much more widely distributed in the merione than in the rat brain. In addition to locations common to most rodents (lateral septum and suprachiasmatic nucleus), in merione vasopressin binding sites occurred in several areas known to express oxytocin binding sites in the rat (olfactory system, hypothalamus). Colocalisation of vasopressin and oxytocin binding sites, which occurred in the CA1 and CA2 fields of Ammon’s horns of the hippocampus, the caudate-putamen and the fundus striati of the merione, has so far not been reported in any other rodent.

Axonal expression sites of tyrosine hydroxylase, calretinin- and calbindin-immunoreactivity in striato-pallidal and septal nuclei of the rat brain: a double-immunolabelling study

Besides the dopaminergic afferent projection system, calbindin (CALB)- and calretinin (CR)-immunoreactive fibres of intrinsic and extrinsic origin represent the most abundant axonal categories in the rat striatal and lateral septal areas. The question arises whether or not they may represent separate populations, or whether they form subgroups which co-express more than one of these antigens. Therefore, the present study is focused on the distribution patterns of the axons single-immunolabelled by the catecholaminergic marker tyrosine hydroxylase (TH), and on TH-immunoreactive axons displaying also CR- and/or CALB-immunoreactivity in double-immunostained sections. Striking differences were found between the patch and matrix compartments of the caudate–putamen (CP). Whereas the vast majority of TH-immunoreactive fibres in the patches and a patch-associated subcallosal layer co-expressed CR but not CALB, fibres mono-labelled by the TH-immunoreactivity were predominant in the matrix. The matrix-like regions of the core of nucleus accumbens (CACC), fundus striati (FS), the striatal cell bridges (CB) and the striatal part of olfactory tubercle (OTU) coincided in this respect with the matrix in CP. The absence of CR-immunoreactivity was also characteristic of the TH-immunoreactive fibres in the patch-like areas of the accumbal core, although a high number of separate CR-immunoreactive axons were present. In the shell of nucleus accumbens (SACC) which receives a rich catecholaminergic innervation, fibres co-expressing either one of the calcium-binding proteins were absent. The islands of Calleja (CJI) displaying a strongly TH-immunoreactive centre and a periphery of lower staining intensity, showed only a low number of TH-immunoreactive fibres co-expressing CR or CALB. The broad shell-like band of TH-immunoreactive axons between medial and lateral part of the septum was single-stained with the TH-immunoreactivity. In contrast, the TH-positive fibres forming basket-like arrangements around some neurons in the dorsal lateral septal nucleus co-expressed also CR, but not CALB. The results are discussed in view of the recent concepts of basal forebrain organization and the cytochemical characteristics of mesencephalic dopaminergic nuclei giving rise to the vast majority of the striatal and septal TH-immunoreactive fibre supply, in order to correlate the known projection patterns with the content of calcium-binding proteins in TH-immunolabelled fibres and presumed cells of origin. The TH-immunoreactive fibres in the striatal patches displaying CR- but not CALB-immunoreactivity may originate mainly from neurons in the ventral tier of pars compacta (SNC) and from the pars reticulata of substantia nigra (SNR) which show identical cytochemical properties. Axons in the matrix of CP and the accumbal core as well as in the islands of Calleja single-labelled by the TH-immunoreactivity or additionally containing CALB and CR may originate from neurons in the dorsal tier of mesencephalic nuclei like SN, pars compacta and ventral tegmental area. CR-containing TH-immunoreactive basket-like axon terminations in the dorsal lateral septal nucleus are likely to originate either from mesencephalic nuclei or from the supramammillary region.

Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia.

The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (</=15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (</=2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine.

In situ hybridization analysis of preprotachykinin-A and -B mRNA levels in short-term sodium depletion

Tachykinins inhibit salt appetite when applied intracranially in a number of brain regions and may function as endogenous inhibitors of sodium intake. To test the hypothesis that induced increases in salt appetite might involve disinhibition via a reduction in endogenous tachykinin expression, we used a semi-quantitative in situ hybridization analysis to investigate changes in brain areas expressing preprotachykinin-A (PPT-A) and preprotachykinin-B (PPT-B) mRNAs of rats after 1 day of sodium depletion (1d Na dep). PPT-A mRNA levels were detected in neurons of the olfactory tubercle (Tu), the nucleus of the olfactory tubercle (LOT), the dorsal and ventral caudate-putamen (d-CPu and v-CPu), the bed nucleus of the stria terminalis (BNST), the medial preoptic area (mPOA), the habenula (Hb) and the postero-dorsal part of the amygdala (MePD). PPT-B mRNA levels were measured in fundus striati (FStr), d-CPu, v-CPu, BNST, mPOA, dorsomedial hypothalamic nucleus (DMD), arcuate nucleus (Arc), central amygdaloid nucleus (CeL), basolateral amygdaloid nucleus (BLV), LOT, Hb and basal nucleus of Meynert (B). 1d Na dep reduced by 33–61% the mean number of PPT-A grains/cell in Tu, LOT, d-CPu, BNST, mPOA, Hb and MePD compared to control animals. Levels of PPT-B mRNA were not reduced as much by 1d Na dep, although statistically significant reductions of 26, 34 and 17% were found in v-CPu, BNST and B, respectively. These findings, therefore, support the hypothesis that endogenous tachykinins exert an inhibitory influence over sodium appetite.

α 2B Adrenoceptor mRNA expression during rat brain development

The expression of α 2B adrenoceptor mRNA in developing and adult rat brain was examined, using in situ hybridization with 35S-labeled riboprobes. In the adult we have detected more widespread expression than previously reported, with moderate to strong hybridization signals in the fundus striati, olfactory tubercles, septum, thalamus and Purkinje cells of the cerebellum. In addition, there was low expression in the endopiriform nucleus, claustrum, cortex, caudate-putamen and spinal trigeminal nucleus of the brain stem. During embryonic development, there was intense, transient mRNA expression in the developing vascular plexus and vasculature which disappeared by birth. In most brain areas which exhibit mRNA expression in the adult, expression started during late embryonic development; with the exception of the thalamus, where expression was differentially regulated in sensory and non-sensory thalamic nuclei starting at the end of the first postnatal week. In addition, there was transiently upregulated expression in the caudate-putamen and cerebellar Purkinje cells during late embryonic and early postnatal development, respectively. This transient expression correlates with the time of neuronal migration and differentiation in these structures and complements the developmental expression of α 2A and α 2C adrenoceptors. These results suggest that α 2B adrenoceptors may play a role in angiogenesis and in mediating neurotrophic functions of norepinephrine in some brain areas.

Chronic cocaine increases neurotensin gene expression in the shell of the nucleus accumbens and in discrete regions of the striatum

The effects of chronic cocaine administration on neurotensin (NT) mRNA expression were investigated in the rat brain using in situ hybridization. Adult Wistar rats were injected daily with cocaine (15 mg/kg i.p.) or saline for 10 days. One hour after the last injection, the brains were removed and coronal sections of the nucleus accumbens and striatum processed for in situ hybridization using a 35S-labeled NT mRNA oligonucleotide probe. Repeated administration of cocaine induced a specific increase in the expression of NT mRNA in the shell of the nucleus accumbens whereas no changes were observed in the core compartment. In addition, cocaine enhanced the expression of the NT gene in neurons confined to the posterior dorsomedial striatum, but did not alter this same region in the anterior striatum. A strong increase in NT mRNA expression was also observed in rats treated with cocaine in the ventrolateral region of the striatum, the fundus striati. No modifications were seen in the dorsolateral or ventromedial striatum, the lateral septum, or the olfactory tubercle. These findings demonstrate that cocaine affects NT mRNA expression in discrete populations of neurons confined to the shell of the nucleus accumbens and dorsomedial and ventrolateral striatum of the rat. The shell of the nucleus accumbens is a limbic area considered the locus of the reinforcing and locomotor activating properties of cocaine while the dorsal striatum is implicated in the regulation of motor output, and appears to be involved in the stereotypies induced by cocaine. The specific increases in NT gene expression induced by chronic cocaine suggest that these changes could be physiologically relevant for the behavioral effects of psychostimulant drugs.

Prevention of kainic acid-induced limbic seizures and Fos expression by the GABA-A receptor agonist muscimol.

Fos oncoprotein expression has been shown to be a sensitive marker for sequential neuronal activation in response to a specific stimulus. The present study investigated the effect of the gamma-aminobutyric acid (GABA)-A receptor agonist muscimol on kainic acid (KA)-induced limbic seizures and Fos expression in the rat forebrain. One hour after KA injection, a substantial Fos expression was observed in the hippocampal dentate gyrus, whereas only a low level of Fos induction was seen in CA1-3 fields. Six hours post-injection a prominent increase of Fos expression occurred in most forebrain structures, including the whole hippocampus. Following 0.5 mg/kg muscimol treatment a remarkable decrease of Fos expression occurred but only in the caudate putamen and core of the accumbens nucleus. Treatment with 1 mg/kg muscimol led to further significant decreases of Fos expression in CA1-3 pyramidal neurons and the disappearance of Fos induction in the cerebral cortex above the rhinal fissure, reticular thalamic nucleus, claustrum, fundus striati, ventral pallidum, septal nucleus, lateral habenular nucleus, and lateral amygdaloid nucleus. When 2 mg/kg muscimol was injected, animals exhibited "absence seizures' instead of limbic seizures, and Fos expression in the hippocampus was effectively blocked. These results suggest that a reduction of GABAergic inhibition plays a crucial role not only in limbic seizure genesis in the dentate gyrus, but also in the seizure spread mechanism in many brain structures, among which the hippocampal CA1-3 fields are most markedly involved, less marked in the cerebral cortex and some other structures, and least marked in the caudate putamen and core of the accumbens nucleus.

Involvement of the caudal striatum in auditory processing: c- fos response to cortical application of picrotoxin and to auditory stimulation

The topographical organization of corticostriatal connections have been postulated to follow a longitudinal pattern, each cortical area projecting on a longitudinal strip stretching along the whole rostro-caudal axis of the striatum. However, compared to the rostral striatal region, the caudal striatum exhibits distinct features in terms of connectivity and neuronal phenotype. The induction of c- fos expression in the striatum by cortical activation or sensory stimulation may throw more light on these functional corticostriatal relationships. In the present study, we examined the effects of cortical activation by local application of picrotoxin on the Fos-immunoreactivity (Fos-IR) in the striatum of the mouse, with special reference to the caudal part of the striatum. Activation of the auditory cortex induced a dense ipsilateral Fos-IR restricted to the caudal striatum, i.e., in the caudo-medial striatum and in the caudal part of fundus striati, and a very sparse labelling in the medial region of the rostral striatum. Conversely, activation of both sensori-motor and visual cortices only resulted in Fos-IR in the main rostral part of the striatum, without response in the caudal extremity of the striatum. On the other hand, visual or auditory stimulation in awake animals failed to induce c- fos expression in the striatum. However, using quantitative in-situ hybridization for c- fos mRNA, we found that auditory, but not visual stimulation significantly potentiated the c- fos response to the D1 agonist SKF 38393 (2 mg/kg, i.p.) in the caudal part of the striatum. These functional observations suggest that, despite a more widespread cortico-striatal connection pattern deduced from tracing experiments, the strongest functional projections from the auditory system mainly converge onto a restricted part of the caudal striatum, according to a connection pattern that is reminiscent of the transverse segmentation proposed in early lesioning studies of corticostriatal projections.

Efferent projections of the anterior perirhinal cortex in the rat

Because convulsive seizures develop very rapidly from kindling sites in the anterior perirhinal cortex, we studied perirhinal efferents by using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PhAL). PhAL injections into the anterior perirhinal cortex labelled a prominent network of fibers within the frontal cortex that was most dense within layers I and II and layer VI. As individual PhAL injection sites within the perirhinal cortex were restricted to one or two adjacent laminae, we were able to determine that layer V was the main source of the perirhinofrontal projection. This was confirmed by frontal cortex injections of the retrograde tracer Fluorogold (FG). Other cortical areas with densely labelled fibers following perirhinal PhAL injections included the agranular insular, infralimbic, orbital, parietal, and entorhinal cortices. Moderate to mild fiber labelling was also noted in the posterior piriform, temporal and occipital cortices, and the claustrum. Subcortical labelling was seen in the nucleus accumbens; fundus striati; basal and lateral amygdala nuclei; the "acoustic thalamus"; and the central grey. Several of these cortical and subcortical projections were bilateral. The different laminar origin of these perirhinal efferents is discussed. These results confirmed our prediction of extensive direct projections from the anterior perirhinal cortex to the frontal cortex in the rat. The significance of this projection is discussed with special reference to the anatomical basis of convulsive limbic seizures.