Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: involvement of basic fibroblast growth factor

Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT_1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT_1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive (‘priming’) dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65–100% increase in the [³H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT_1-1 neurons were co-cultured for 5 days in vitro (DIV), the [³H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT_1-1 neuron–glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.     

Neurochemical identification of A13 dopaminergic neuronal projections from the medial zona incerta to the horizontal limb of the diagonal band of Broca and the central nucleus of the amygdala

Studies utilizing fluorescent histochemical techniques first revealed that A₁₃ dopaminergic (DA) perikarya located in medial zona incerta (MZI) project to various regions within the hypothalamus; accordingly, these DA neurons were designated the ‘incertohypothalamic’ DA neuronal system. More recently, it has been shown that the anterograde neuronal tract tracer Phaseolus vulgaris leucoagglutinin, after injection into MZI, is identified in nerve terminals outside of the hypothalamus: for example, in horizontal limb of the diagonal band of Broca (HDB) and central nucleus of the amygdala (cAMY). The purpose of the present study was to determine, using neurochemical techniques, if A₁₃ DA neurons project to the HDB and cAMY. Concentrations of dopamine and one of its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in HDB and cAMY following: (1) electrical stimulation of MZI, (2) electrolytic lesion or knife ablation of MZI, and (3) administration of gamma-hydroxybutyric acid (GHBA) into MZI. For comparison, similar measurements were made in nucleus accumbens (N. Acc.), a terminal region of A₁₀ DA neurons in the ventral tegmental area (VTA). Electrical stimulation of MZI increased DOPAC concentrations in HDB and cAMY, whereas electrolytic or ablative lesions of MZI decreased dopamine concentrations in both of these regions. By contrast, neither stimulation nor lesion of MZI had any effect on DOPAC or dopamine concentrations in N. Acc. Intracerebral injection of GHBA into MZI increased dopamine concentrations in MZI and HDB, but not in cAMY or N. Acc. Intracerebral administration of GHBA into VTA increased dopamine concentrations in HDB and N. Acc., but not in MZI or cAMY. These results suggest that A₁₃ DA neurons project to HDB and cAMY but not to N. Acc., and HDB receives projections from both A₁₀ and A₁₃ DA neurons.     

LHRH neurons in the medial septal-diagonal band-preoptic area do not project directly to the hippocampus: a double-labeling immunohistochemical study.

While neurons containing immunoreactive luteinizing hormone-releasing hormone (LHRH) are scattered primarily in the medial septal-diagonal band of Broca-medial preoptic area (mS-dbB-PO) complex, autoradiographic studies have demonstrated dense concentrations of LHRH receptors in the hippocampus. The route by which LHRH is transported to its hippocampal receptors is unknown. The present study was designed to test the hypothesis that LHRH-containing neurons in the mS-dbB-PO complex project to hippocampal sites containing LHRH receptors, thereby serving as a source of innervation to these receptors. Large (0.10 microliters) or small (0.02 microliters) volumes of the retrograde tracer wheat germ agglutinin (WGA) were injected unilaterally into four separate hippocampal locations in six ovariectomized female rats. In an additional five females, a 0.15 microliter volume of the retrograde tracer fluorogold (FG) was similarly injected. After a five day survival period, the animals were sacrificed. Vibratome sections of the brain were stained for both WGA and LHRH with a dual immunohistochemical technique. Since FG is a fluorescent chromagen, brains of animals injected with FG only required processing for LHRH immunofluorescence. As a positive control, some sections containing retrogradely labeled cells filled with either WGA or FG were processed for choline acetyltransferase (CHAT) immunoreactivity. The WGA and FG injections covered targeted hippocampal sites and neurons containing retrogradely transported WGA or FG were found in abundance in the mS-dbB-PO complex. In accord with previous reports, many CHAT-positive and fewer LHRH-positive neurons were found in this complex. Approximately 5-10% of the CHAT-positive neurons also contained WGA or FG; however, no neurons were found to co-localize LHRH and either of the retrograde tracers. The results indicate that LHRH neurons in the mS-dbB-PO complex do not project directly to hippocampal sites containing LHRH receptors.     

Medial amygdaloid suppression of predatory attack behavior in the cat: I. Role of a substance P pathway from the medial amygdala to the medial hypothalamus

The medial amygdala is known to powerfully suppress predatory attack behavior in the cat, but the mechanisms underlying such modulation remain unknown. The present study tested the hypothesis that medial amygdaloid suppression of predatory attack is mediated, in part, by a pathway from the medial amygdala to the medial hypothalamus which utilizes substance P as a neurotransmitter. Stimulating electrodes were implanted into the medial amygdala and cannula electrodes were implanted into both the medial and lateral hypothalamus. Predatory attack behavior was elicited by electrical stimulation of the lateral hypothalamus. In the first phase of the study, paired trials compared attack latencies of single stimulation of the lateral hypothalamus with those following dual stimulation of the lateral hypothalamus and medial amygdala. Attack latencies were significantly elevated following dual stimulation of the medial amygdala and lateral hypothalamus. In the second phase of the study, dose and time dependent decreases in response suppression were noted following the infusion of the substance P (NK₁) receptor antagonist, CP96,345 (in doses of 0.05, 0.5 and 2.5 nmol) into the medial hypothalamus. In third phase of the study, the effects of microinjections of the substance P receptor agonist, [Sar⁹,Met(O₂)¹¹]-substance P (in doses of 0.5, 1.0 and 2.0 nmol), directly into the medial hypothalamus upon lateral hypothalamically elicited predatory attack behavior were determined. Microinfusion of this drug elevated attack response latencies in a dose- and time-dependent manner. In addition, pretreatment with CP96,345 into the medial hypothalamus blocked the suppressive effects of subsequent delivery of [Sar⁹,Met(O₂)¹¹]-substance P into the same medial hypothalamic site. Other parts of the study demonstrated the presence of: (1) high densities of substance P receptors in the ventromedial hypothalamus, and (2) neurons that are positively labeled for substance P that project from the medial amygdala to the ventromedial hypothalamus as demonstrated by retrograde labeling with Fluoro-Gold. These findings provide support for the hypothesis that medial amygdaloid suppression of lateral hypothalamically elicited predatory attack behavior includes a substance P pathway from the medial amygdala to the medial hypothalamus. The findings further suggest that stimulation of the medial amygdala activates substance P receptors in the medial hypothalamus, thus triggering an inhibitory mechanism from the medial to the lateral hypothalamus, resulting in suppression of predatory attack behavior.     

Melanin-concentrating hormone and neuropeptide EI projections from the lateral hypothalamic area and zona incerta to the medial septal nucleus and spinal cord: a study using multiple neuronal tracers

The projection pathways of neurons containing melanin-concentrating hormone (MCH) and neuropeptide EI (NEI), two peptides colocalized in the lateral hypothalamic area (LHA) of the rat, were mapped using the retrogradely transported fluorescent dyes, true blue (TB) and diamidino yellow (DY). TB and DY were injected into the medial septum/diagonal band complex (MS/DBC) and the thoracic level of the spinal cord (SpCd), respectively. Brains from rats receiving only one or both tracer injections were immunohistochemically stained for MCH in the spinal cord and NEI in the forebrain. In the MS/DBC, NEI-immunoreactive (-ir) fibers are concentrated in the MS and in the vertical and horizontal limbs of the DBC. In the SpCd, MCH-ir fibers are concentrated primarily in lamina X. Of the diencephalic NEI-ir neurons, 37.15% project to the MS/DBC and reside in the rostromedial zona incerta (ZIm), in the LHA_t and LHA_p, and in the perifornical region. Of the diencephalic MCH-ir neurons, 20.2% project to the SpCd and reside in the LHA_t and LHA_p. In addition, 2.2% of the MCH-ir cells and 8.7% of the NEI-ir cells in the hypothalamus were labeled with both retrograde tracers and thus project to both the MS/DBC and SpCd. These dual projection neurons are located mainly in the LHA_t and LHA_p. Anterograde injections of the tracer Phaseolus vulgaris leucoagglutinin into the LHA_t and ZIm corroborated our findings in the retrograde studies. Potential autonomic and behavioral roles of the NEI and MCH systems in the MS/DBC and the SpCd are discussed.     

Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, β-endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area

The role of corticotropin-releasing factor (CRF) and opiocortin neuronal systems and a possible functional relationship between the two in the control of luteinizing hormone-releasing hormone (LH-RH) activity in the medial preoptic area (MPOA) for the regulation of lordosis behaviour were assessed in ovariectomised oestrogen-progesterone-treated female rats. Lordosis behaviour (assessed as the lordosis quotient) triggered by male mounting was significantly inhibited by either CRF or β-endorphin infused into the MPOA in animals treated with normal doses of oestradiol benzoate (OEP) (5 μg) and progesterone (500 μg). Saline-treated animals exhibited high levels of lordosis. The inhibition of lordosis produced by either CRF or β-endorphin could be reversed by LH-RH microinfusions into the MPOA. While naloxone pretreatment of the MPOA site prevented the inhibitory effects of β-endorphin, neither the opiate antagonist nor anti-β-endorphin-γ-globulin (even in high concentrations) infused into the MPOA was effective in completely preventing the inhibition of lordosis produced by CRF. These findings suggest that the inhibition of LH-RH neuronal activity and lordosis behaviour by CRF may be due to a direct action and may not be the result of activation of β-endorphin release. The possibility that the two peptidergic systems may act in a synergistic fashion is supported by the data showing that combined CRF-β-endorphin treatment in the MPOA completely abolished lordosis. This is further supported by the finding that CRF totally abolished lordosis in animals pretreated with anti-corticotropin (ACTH-γ-globulin although this result could suggest that CRF could preferentially stimulate the release of ACTH in the MPOA. Conversely, naloxone, anti-β-endorphin-γ-globulin, anti-CRF-γ-globulin and ACTH infused into the MPOA produced high levels of lordosis in female rats normally showing low levels of lordosis by treatment with low doses of OEB (2 μg) and normal doses of progesterone (500 μg). In each case the facilitation could be blocked by pretreatment of the MPOA site with a potent antagonist analogue of LH-RH. The results indicate the key role of LH-RH in the action of endogenous CRF, β-endorphin and ACTH in the regulation of lordosis behaviour. Each of these substances may act directly on the LH-RH neurone. The postulated presynaptic relationship between CRF and β-endorphin neuronal systems that seem to exist in the mediobasal hypothalamus and the central gray may be less predominant in the MPOA. The results, however, provide clear further evidence for the potent effects of CRF and opiocortin peptides in the regulation of LH-RH and reproduction.     

Evidence in favour of a direct input from the ventromedial nucleus to gonadotropin-releasing hormone neurones in the ewe: an anterograde tracing study

The mechanism by which oestrogen activates the gonadotropin releasing hormone (GnRH) neurones to induce the preovulatory luteinizing hormone (LH) surge is not understood. Previous work in the ewe has suggested that the primary site of action for oestradiol in stimulating the GnRH neurones was in the region of the ventromedial nucleus (VMN) within the mediobasal hypothalamus (MBH). In the present study, we used anterograde tracing techniques in the ewe to investigate whether direct neuronal projections may exist from neurones located in the region of the VMN to the GnRH neurones. Following the injection of biotinylated dextran amine into the VMN of four ewes, anterogradely labelled fibres were found located principally within the ipsilateral diagonal band of Broca (DBB), septum, preoptic and anterior hypothalamic areas, and periventricular, paraventricular, dorsomedial and arcuate nuclei of the MBH. Dual-labelling for GnRH revealed that fibres containing anterograde tracer were adjacent to the soma and/or dendrites of approximately 50% of all ipsilateral GnRH neurones located throughout the DBB and hypothalamus. Few anterogradely labelled fibres were detected within the median eminence. Although such studies cannot define the presence of direct synaptic connections between VMN neurones and the GnRH cells, these observations support further the hypothesis that oestrogen-sensitive VMN neurones represent a direct transsynaptic input to the GnRH cell bodies which are involved in the generation of the LH surge in the ewe.     

A putative role for extracellular ATP: Facilitation ofcopper uptake and of copper stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants

We have previously shown that extracellular copper stimulates the release of the luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA), that chelated copper (Cu) but not ionic Cu is the active form of the metal, and that there is a direct correlation between the ligand specificity for⁶⁷Cu uptake and Cu action. In this study, we examined the possibility that extracellular ATP can serve as a ligand facilitating Cu action on the LH-RH neuron. Hypothalamic slices or MEA explants of adult male rats were used. It was found that ATP facilitates⁶⁷Cu uptake by hypothalamic slices when Cu:ATP molar ratio was 1:2000 but not 1:2. Keeping the [Cu] constant (150 μM) and varying [ATP], ATP facilitation of Cu stimulation of LH-RH release from MEA explants was found to be a saturable function of [ATP]; maximal facilitation occurred with 2.5 mM ATP. When the nucleotide phosphate specificity for facilitation of Cu action was assessed, ADP, ATP, α,β-methylene-ATP (the non-hydrolyzable analogue of ATP) and GTP were equally effective, whereas AMP and adenosine were ineffective. These results indicate that extracellular ATP can facilitate Cu action on the LH-RH neuron and they are consistent with two mechanisms: (1) ATP facilitating Cu uptake and hence, Cu action and (2) ATP facilitating Cu action via an interaction with a purinergic receptor.     

Projections from the nociceptive area of the central nucleus of the amygdala to the forebrain: a PHA-L study in the rat

The lateral capsular division (CeLC) of the central nucleus (Ce) of the amygdala, in the rat, has been shown to be the main terminal area of a spino(trigemino)-parabrachio-amygdaloid nociceptive pathway [Bernard & Besson (1990) J. Neurophysiol. 63, 473-490; Bernard et al. (1992) J. Neurophysiol. 68, 551-569; Bernard et al. (1993) J. Comp. Neurol. 329, 201-229]. The projections to the forebrain from the CeLC and adjacent regions were studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin (PHA-L) restricted in subdivisions of the Ce and the basolateral amygdaloid nucleus anterior (BLA). Our data showed that the entire CeLC projects primarily and extensively to the substantia innominata dorsalis (SId). The terminal labelling is especially dense in the caudal aspect of the SId. The other projections of the CeLC in the forebrain were dramatically less dense. They terminate in the bed nucleus of the stria terminalis (BST) and the posterior hypothalamus (pLH). No (or only scarce) other projections were found in the remaining forebrain areas. The Ce lateral division (CeL) and the Ce medial division (CeM), adjacent to the CeLC, also project to the SId with slightly lower density labelling. However, contrary to the case of the CeLC, both the CeL and the CeM extensively project to the ventrolateral subnucleus of the BST (BSTvl) with a few additional terminals found in other regions of the lateral BST. Only the CeM projects densely to both the interstitial nucleus of the posterior limb of the anterior commissure and the caudal most portion of the pLH. The projections of the BLA are totally different from those of the Ce as they terminate in the dorsal striatum, the accumbens nucleus, the olfactory tubercle, the nucleus of olfactory tract and the rostral pole of the cingulate/frontal cortex. This study demonstrates that the major output of the nociceptive spino(trigemino)-parabrachio-CeLC pathway is to the SId. It is suggested that the CeLC-SId pathway could have an important role in anxiety, aversion and genesis of fear in response to noxious stimuli.     

Identification of dopamine, gonadotrophin-releasing hormone-I, and vasoactive intestinal peptide neurones activated by electrical stimulation to the medial preoptic area of the turkey hypothalamus: a potential reproductive neuroendocrine circuit

The neural and neurochemical substrates regulating reproduction in birds remain vaguely defined. The findings that electrical stimulation in the medial preoptic area (ES/MPOA) or intracerebroventricular infusion of dopamine (DA) stimulated luteinising hormone (LH) and prolactin (PRL) release in female turkeys, led to the suggestion that ES/MPOA might help to clarify the DA circuitry regulating LH and PRL. We used c-fos mRNA and tyrosine hydroxylase immunoreactivity as measured by double in situ hybridisation/immunocytochemistry (ISH/ICC) to determine which group/subgroup of DA neurones was activated following unilateral ES/MPOA. To establish that the reproductive neuroendocrine system was activated, double ISH/ICC was also conducted on c-fos/gonadotrophin-releasing hormone-I (GnRH-I) and c-fos/vasoactive intestinal peptide (VIP). Changes in circulating LH and PRL were determined by radioimmunoassay. Unilateral ES/MPOA (100 microA, right side) of anaesthetised laying turkeys for 30 min increased circulating LH and PRL levels. It also induced c-fos mRNA expression on the ipsilateral side by all GnRH-I neurones within the septopreoptic region, implying that GnRH-I neurones in this region share similar circuitry. VIP neurones within the nucleus infundibularis were the only VIP group to show c-fos mRNA expression, suggesting their involvement in ES/MPOA induced PRL release. c-fos mRNA expression was also observed in a subgroup of DA neurones in the nucleus mamillaris lateralis (ML). To our knowledge, the present study is the first to show that activation of DAergic cells in the ML is associated with the activation of GnRH-I and VIP neurones and the release of LH and PRL. It is likely that ES/MPOA activated VIP/GnRH-I neurones via activation of DA neurones in the ML, as this was the only DA subgroup that showed c-fos mRNA expression.     

Direct synaptic connections of axons from superior colliculus with identified thalamo-amygdaloid projection neurons in the rat: Possible substrates of a subcortical visual pathway to the amygdala

The aim of the present study was to identify synaptic contacts from axons originating in the superior colliculus with thalamic neurons projecting to the lateral nucleus of the amygdala. Axons from the superior colliculus were traced with the anterograde tracers Phaseolus vulgaris leucoagglutinin or the biotinylated and fluorescent dextran amine “Miniruby.” Thalamo-amygdaloid projection neurons were identified with the retrograde tracer Fluoro-Gold. Injections of Fluoro-Gold into the lateral nucleus of the amygdala labeled neurons in nuclei of the posterior thalamus which surround the medial geniculate body, viz. the suprageniculate nucleus, the medial division of the medial geniculate body, the posterior intralaminar nucleus, and the peripeduncular nucleus. Anterogradely labeled axons from the superior colliculus terminated in the same regions of the thalamus. Tecto-thalamic axons originating from superficial collicular layers were found predominantly in the suprageniculate nucleus, whereas axons from deep collicular layers were detected in equal density in all thalamic nuclei surrounding the medial geniculate body. Double-labeling experiments revealed an overlap of projection areas in the above-mentioned thalamic nuclei. Electron microscopy of areas of overlap confirmed synaptic contacts of anterogradely labeled presynaptic profiles originating in the superficial layers of the superior colliculus with retrogradely labeled postsynaptic profiles of thalamo-amygdaloid projection neurons. These connections may represent a subcortical pathway for visual information transfer to the amygdala. J. Comp. Neurol. 403:158–170, 1999. © 1999 Wiley-Liss, Inc.     

Evidence for a direct neuronal pathway from the suprachiasmatic nucleus to the gonadotropin-releasing hormone system: Combined tracing and light and electron microscopic immunocytochemical studies

The timing and occurrence of the preovulatory luteinizing hormone (LH) surge in the female rodent are critically dependent on the integrity of the suprachiasmatic nucleus (SCN). Destruction of the SCN leads to a cessation of the ovarian cycle, whereas implantation of estrogen in ovariectomized rats results in daily LH surges. The anatomical substrate for these effects is not known. Previous studies involving lesions of the SCN have suggested the presence of a direct vasoactive intestinal polypeptide (VIP)-containing pathway to gonadotropin-releasing hormone (GnRH) neurons. To further investigate the direct connection between the SCN and the GnRH system, we have used tract-tracing with the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PhaL) in combination with an immunocytochemical staining for GnRH in light and electron microscopic studies. Small, unilateral PhaL deposits, especially when they were placed in the rostral ventrolateral portion of the SCN, revealed a bilateral projection to the preoptic area, where PhaL-immunoreactive fibers were regularly found in close apposition to GnRH neurons. Ultrastructural studies showed synaptic interaction of PhaL-containing fibers with GnRH-immunoreactive (IR) cell bodies, thus demonstrating a direct SCN-GnRH connection. Taken together, these data provide evidence for the existence of a monosynaptic pathway from the SCN to the GnRH system in the hypothalamus of the female rat. We suggest that this pathway may contain at least VIP as a putative transmitter and may play a role in the circadian regulation of the estrous cycle in the female rat. J. Comp. Neurol. 384:569–579, 1997. © 1997 Wiley-Liss, Inc.     

Properties of a projection pathway from the medial preoptic nucleus to the midbrain periaqueductal gray of the rat and its role in the regulation of cardiovascular function

In this study we examined (1) the effect of stimulation of the MPO on the firing activity of neurons in the PAG, (2) the role of glutamic acid in this interaction and, (3) whether reversible blockade of neuronal activity in the PAG by lidocaine can alter the effect of stimulation of the MPO on arterial blood pressure. Single pulse stimulation of the MPO produced a biphasic response in232 cells and inhibited332 cells. Train electrical stimulation excited2154 cells and inhibited1254 cells. The latencies to the onset of the excitatory and the inhibitory effects were not different, but the duration of the excitatory effect was slightly longer than that of the inhibitory effect. Chemical stimulation of the MPO excited1797 cells and inhibited1697 cells. The latency to onset of the excitatory response to stimulation of the MPO was longer but the duration was shorter than that of the inhibitory response. In 83% of the animals (2935), stimulation of the MPO produced a decrease in mean arterial pressure (MAP). The duration of the response was 196.9 ± 20.9 s and the average decrease in the MAP was 18.2 ± 1.4 mmHg. Application of KYN blocked the excitatory response to stimulation of the MPO in816 cells and the inhibitory response of310 cells. Injection of lidocaine into the PAG by itself had no effect on the arterial blood pressure. However, in all animals (n = 10) lidocaine totally or significantly reduced the magnitude of the blood pressure change produced by stimulation of the MPO in a reversible manner. These studies electrophysiologically confirm a pathway between the MPO and the PAG that is, in part, under glutamatergic control. In addition, our results demonstrate that stimulation of the MPO produces a distinctive depressor effect that is mediated through the PAG.     

Stimulus-response profile analysis: a comprehensive,quantitative approach to the study of sensory coding and information processing

In order to understand the coding and information processing capabilities of mechanosensory neurons, it is necessary to examine stimulus-response relationships under a wide variety of stimulus conditions, using a comprehensive set of quantitative analytic procedures. We employ the methodology of stimulus-response profile analysis, which is based on 4 principles: (1) the use of a broad-based battery of quantitatively controlled mechanical stimuli; (2) maintenance of comprehensive records of experiments; (3) detailed, quantitative analysis of single-unit responses; and (4) the application of these principles uniformly and consistently to all units studied.In addition to conventional graphical portrayals of single-unit activity, we employ a set of quantitative response indices, each of which represents a particular aspect of a unit's overall responsiveness. The distribution of a response index for the entire sample of neurons examined in a particular population offers insight into the manner in which a specific stimulus feature is represented within that population. The distributions of response indices obtained from different neural populations can be compared statistically in order to evaluate interpopulation differences in stimulus-response relationships. This comprehensive, quantitative approach is capable of demonstrating significant, although subtle, interpopulation differences which are not revealed by more cursory, qualitative methods.     

Neurotensin antagonist acutely and robustly attenuates locomotion that accompanies stimulation of a neurotensin-containing pathway from rostrobasal forebrain to the ventral tegmental area

Neurotensin exerts complex effects on the mesolimbic dopamine system that alter motivation and contribute to neuroadaptations associated with psychostimulant drug administration. Activation of abundant neurotensin receptors in the ventral tegmental area (VTA) enhances dopamine neuron activity and associated release of dopamine in the nucleus accumbens (Acb) and cortex. In view of recent anatomical studies demonstrating that 70% of all neurotensin-containing neurons projecting to the VTA occupy the lateral preoptic area-rostral lateral hypothalamus (LPH) and lateral part of the medial preoptic area (MPOA), the present study examined functionality in the LPH-MPOA neurotensinergic pathway in the rat. Disinhibition (resulting ultimately in stimulation-like effects) of LPH-MPOA neurons with microinjected bicuculline (50 or 100 ng in 0.25 microL) produced locomotor activation that was considerably attenuated by systemic administration of the neurotensin antagonist SR 142948 A (0.03 and 0.1 mg/kg). In contrast, locomotion elicited in this manner was completely blocked by SR 142948 A infused directly into the VTA (5.0 and 15.0 ng in 0.25 microL). Baseline locomotion was unaffected by systemic or intra-VTA administration of SR 142948 A and LPH-MPOA-elicited locomotion was unaffected by infusion of SR 142948 A into the substantia nigra pars compacta and sites rostral and dorsal to the VTA. Locomotion was not elicited by infusions of bicuculline into the lateral hypothalamus at sites caudal to the LPH-MPOA, where neurotensin neurons projecting to the VTA are fewer. The results demonstrate the capacity of a neurotensin-containing pathway from LPH-MPOA to VTA to modulate locomotion. This pathway may be important in linking hippocampal and mesolimbic mechanisms in normal behaviour and drug addiction.     

Projections from the lateral nucleus to the basal nucleus of the amygdala: a light and electron microscopic PHA-L study in the rat.

A recent study, carried out in the monkey brain demonstrated a hitherto undescribed projection from the lateral to the basal nucleus of the amygdaloid complex. In the present study, we used light and electron microscopic techniques to determine whether a similar connection exists in the rat brain and to define what type(s) of synaptic contacts are produced by fibers of this projection. Injections of the lectin tracer Phaseolus vulgaris leucoagglutinin (PHA-L) were placed into several levels of the lateral nucleus and the distribution of fibers in the basal (basolateral) nucleus was evaluated. All lateral nucleus injections resulted in labeled fibers in the basal nucleus, though the density and distribution of labeled fibers depended on the position of the injection site within the lateral nucleus. In general, the heaviest labeling of the basal nucleus was observed after injections at midrostrocaudal levels of the lateral nucleus, especially when the injection was located ventrally. Fibers originating from cells labeled by these injections were observed throughout much of the rostrocaudal extent of the basal nucleus. Rostrally situated injections resulted in substantially lower levels of labeled fibers in the basal nucleus. Injections placed caudally in the lateral nucleus resulted in light to medium levels of labeled fibers in the basal nucleus; the terminal field in these cases did not extend as far rostrally as after the rostral and midlevel injections. Electron microscopic analysis of PHA-L labeled fibers revealed that they contributed synapses to the basal nucleus. The majority of PHA-L labeled terminals formed asymmetric contacts on dendritic spines or shafts; a smaller number of PHA-L labeled terminals formed symmetrical synapses.     

Evidence for a GABAergic projection from the central nucleus of the amygdala to the brainstem of the macaque monkey: a combined retrograde tracing and in situ hybridization study

The central nucleus of the amygdala is interconnected with a variety of visceral and autonomic nuclei of the brainstem. These include the parabrachial nucleus, the nucleus of the solitary tract, the nucleus ambiguus and the dorsal motor nucleus of the vagus. Despite repeated attempts, neurochemical characterization of the major subcortical connections of the central nucleus has not yet been accomplished. Based on earlier immunohistochemical and in situ hybridization evidence indicating the presence of numerous GABAergic neurons in the macaque monkey central nucleus, we predicted that a sizeable portion of the descending projections may be GABAergic. We tested this hypothesis using a novel double labelling method with gold conjugated WGA-apoHRP as a retrograde tracer and in situ hybridization for detecting the mRNA that encodes the enzyme glutamic acid decarboxylase (GAD67) as a marker for GABAergic cells. Following WGA-apoHRP-gold injections into the brainstem, a large number of retrogradely labelled cells was observed in the medial and lateral divisions of the central nucleus. Of the retrogradely labelled cells observed in the medial division of the central nucleus, approximately half were double-labelled for GAD67 mRNA; about 30% double labelling was observed in the lateral division. These data support the view that a sizeable component of the central nucleus projection to the brainstem is GABAergic.