René Bernard

Altered expression of glutamate signaling, growth factor and glia genes in the locus coeruleus of patients with major depression

Several studies have proposed that brain glutamate signaling abnormalities and glial pathology have a role in the etiology of major depressive disorder (MDD). These conclusions were primarily drawn from post-mortem studies in which forebrain brain regions were examined. The locus coeruleus (LC) is the primary source of extensive noradrenergic innervation of the forebrain and as such exerts a powerful regulatory role over cognitive and affective functions, which are dysregulated in MDD. Furthermore, altered noradrenergic neurotransmission is associated with depressive symptoms and is thought to have a role in the pathophysiology of MDD. In the present study we used laser-capture microdissection (LCM) to selectively harvest LC tissue from post-mortem brains of MDD patients, patients with bipolar disorder (BPD) and from psychiatrically normal subjects. Using microarray technology we examined global patterns of gene expression. Differential mRNA expression of select candidate genes was then interrogated using quantitative real-time PCR (qPCR) and in situ hybridization (ISH). Our findings reveal multiple signaling pathway alterations in the LC of MDD but not BPD subjects. These include glutamate signaling genes, SLC1A2, SLC1A3 and GLUL, growth factor genes FGFR3 and TrkB, and several genes exclusively expressed in astroglia. Our data extend previous findings of altered glutamate, astroglial and growth factor functions in MDD for the first time to the brainstem. These findings indicate that such alterations: (1) are unique to MDD and distinguishable from BPD, and (2) affect multiple brain regions, suggesting a whole-brain dysregulation of such functions.

Individual neurons in the rat lateral habenular complex project mostly to the dopaminergic ventral tegmental area or to the serotonergic raphe nuclei.

The lateral habenular complex (LHb) is a bilateral epithalamic brain structure involved in the modulation of ascending monoamine systems in response to afferents from limbic regions and basal ganglia. The LHb is implicated in various biological functions, such as reward, sleep–wake cycle, feeding, pain processing, and memory formation. The modulatory role of the LHb is partially assumed by putative spontaneously active LHb neurons projecting to the dopaminergic ventral tegmental area (VTA) and to the serotonergic median (MnR) and dorsal raphe nuclei (DR). All four nuclei form a complex and coordinated network to evoke appropriate responses to reward‐related stimuli. At present it is not known whether individual LHb neurons project to only one or to more than one monoaminergic nucleus. To answer this question, we made dual injections of two different retrograde tracers into the rat VTA and either DR or MnR. Tracers were visualized by immunohistochemistry. In coronal sections, the different retrogradly labeled habenular neurons were quantified and assigned to the corresponding habenular subnuclei. Our results show that 1) the distribution of neurons in the LHb projecting to the three monoamine nuclei is similar and exhibits a great overlap, 2) the vast majority of LHb projection neurons target one monoaminergic nucleus only, and 3) very few, heterogeneously distributed LHb neurons project to both dopaminergic and serotonergic nuclei. These results imply that the LHb forms both separate and interconnected circuits with each monoaminergic nucleus, permitting the LHb to modulate its output to different monoamine systems either independently or jointly. J. Comp. Neurol. 520:2545–2558, 2012.

Upregulation of inward rectifier K+ (Kir2) channels in dentate gyrus granule cells in temporal lobe epilepsy

In humans, temporal lobe epilepsy (TLE) is often associated with Ammons horn sclerosis (AHS) characterized by hippocampal cell death, gliosis and granule cell dispersion (GCD) in the dentate gyrus. Granule cells surviving TLE have been proposed to be hyperexcitable and to play an important role in seizure generation. However, it is unclear whether this applies to conditions of AHS. We studied granule cells using the intrahippocampal kainate injection mouse model of TLE, brain slice patch‐clamp recordings, morphological reconstructions and immunocytochemistry. With progressing AHS and GCD, ‘epileptic’ granule cells of the injected hippocampus displayed a decreased input resistance, a decreased membrane time constant and an increased rheobase. The resting leak conductance was doubled in epileptic granule cells and roughly 70–80% of this difference were sensitive to K+ replacement. Of the increased K+ leak, about 50% were sensitive to 1 mm Ba2+. Approximately 20–30% of the pathological leak was mediated by a bicuculline‐sensitive GABAA conductance. Epileptic granule cells had strongly enlarged inwardly rectifying currents with a low micromolar Ba2+ IC50, reminiscent of classic inward rectifier K+ channels (Irk/Kir2). Indeed, protein expression of Kir2 subunits (Kir2.1, Kir2.2, Kir2.3, Kir2.4) was upregulated in epileptic granule cells. Immunolabelling for two‐pore weak inward rectifier K+ channels (Twik1/K2P1.1, Twik2/K2P6.1) was also increased. We conclude that the excitability of granule cells in the sclerotic focus of TLE is reduced due to an increased resting conductance mainly due to upregulated K+ channel expression. These results point to a local adaptive mechanism that could counterbalance hyperexcitability in epilepsy.

Hypocretin-1 causes G protein activation and increases ACh release in rat pons

The effects of the arousal‐promoting peptide hypocretin on brain stem G protein activation and ACh release were examined using 16 adult Sprague‐Dawley rats. In vitro[35S]GTPγS autoradiography was used to test the hypothesis that hypocretin‐1‐stimulated G protein activation is concentration‐dependent and blocked by the hypocretin receptor antagonist SB‐334867. Activated G proteins were quantified in dorsal raphe nucleus (DR), locus coeruleus (LC) and pontine reticular nucleus oral part (PnO) and caudal part (PnC). Concentration–response data revealed a significant (P < 0.001) effect of hypocretin‐1 (2–2000 nm) in all brain regions examined. Maximal increases over control levels of [35S]GTPγS binding were 37% (DR), 58% (LC), 52% (PnO) and 44% (PnC). SB‐334867 (2 µm) significantly (P < 0.002) blocked hypocretin‐1 (200 nm)‐stimulated [35S]GTPγS binding in all four nuclei. This is the first autoradiographic demonstration that hypocretin‐1 activates G proteins in arousal‐related brain stem nuclei as a result of specific receptor interactions. This finding suggests that some hypocretin receptors in brain stem couple to inhibitory G proteins. In vivo microdialysis was used to test the hypothesis that PnO administration of hypocretin‐1 increases ACh release in PnO. Dialysis delivery of hypocretin‐1 (100 µm) significantly (P < 0.002) increased (87%) ACh release. This finding is consistent with the interpretation that one mechanism by which hypocretin promotes arousal is by enhancing cholinergic neurotransmission in the pontine reticular formation.

Distinct populations of presympathetic-premotor neurons express orexin or melanin-concentrating hormone in the rat lateral hypothalamus

Orexin and melanin‐concentrating hormone (MCH) have been implicated in mediating a variety of different behaviors. These include sleep and wakefulness, locomotion, ingestive behaviors, and fight‐or‐flight response, as well as anxiety‐ and panic‐like behaviors in rodents. Despite such diversity, all these processes require coordinated recruitment of the autonomic and somatomotor efferents. We have previously mapped the locations of presympathetic‐premotor neurons (PSPMNs) in the rat brain. These putative dual‐function neurons send trans‐synaptic projections to somatomotor and sympathetic targets and likely participate in somatomotor‐sympathetic integration. A significant portion of these neurons is found within the dorsomedial (DMH) and lateral hypothalamus (LH), areas of the brain that contain MCH‐ and orexin‐ synthesizing neurons in the central nervous system. Thus, we hypothesized that hypothalamic PSPMNs utilize MCH or orexin as their neurotransmitter. To test this hypothesis, we identified PSPMNs by using recombinant strains of the pseudorabies virus (PRV) for trans‐synaptic tract tracing. PRV‐152, a strain that expresses enhanced green fluorescent protein, was injected into sympathectomized gastrocnemius muscle, whereas PRV‐BaBlu, which expresses β‐galactosidase, was injected into the adrenal gland in the same animals. By using immunofluorescent methods, we determined whether co‐infected neurons express MCH or orexin. Our findings demonstrate that PSPMNs synthesizing either MCH or orexin are present within LH, where they form two separate populations. PSPMNs located around the fornix express orexin, whereas those located around the cerebral peduncle are more likely to express MCH. These two clusters of PSPMNs within LH likely play distinct functional roles in autonomic homeostasis and stress coping mechanisms. J. Comp. Neurol. 505:586–601, 2007.

A glutamatergic projection from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the rat.

Homeostasis describes the fundamental biological ability of individuals to maintain stable internal conditions in a changing environment. Homeostatic reactions include internal adjustments as well as behavioral responses. In vertebrates, behavioral responses are induced by the reward system. This system originates in the ventral tegmental area (VTA) and leads to increased dopamine levels in the forebrain whenever activated. A major inhibitor of VTA activity is the lateral habenula (LHb). This epithalamic structure is able to almost completely suppress dopamine release, either directly or via the rostromedial tegmental nucleus (RMTg), when rewarding expectations are not met. A major input to the LHb arises from the lateral hypothalamic area (LHA), an important regulator of the homeostatic system. Currently, little is known about the effects of the strong hypothalamic projection on the activity of LHb neurons. In the present study, we analyze neurotransmitters and cellular targets of the LHA-LHb projection in the rat. Therefore, anterograde tracing from the LHA was combined with the visualization of neurotransmitters in the LHb. These experiments revealed a mainly glutamatergic projection, probably exerting excitatory effects on the targeted LHb cells. These cellular targets were analyzed in a second step. Anterograde tracing from the LHA in combination with retrograde tracing from the VTA/RMTg region revealed that LHb neurons projecting to the VTA/RMTg region are densely targeted by the LHA projection. Visualization of synaptophysin at these contact sites indicates that the contact sites indeed are synapses. Taken together, the present study describes a strong mainly glutamatergic projection from the LHA that targets VTA/RMTg-projecting neurons in the LHb. These findings emphasize the potential role of the LHb as direct link between homeostatic areas and reward circuitries, which may be important for the control of homeostatic behaviors.

Gene expression profiling of neurochemically defined regions of the human brain by in situ hybridization-guided laser capture microdissection

Laser capture microdissection (LCM) permits isolation of specific cell types and cell groups based upon morphology, anatomical landmarks and histochemical properties. This powerful technique can be used for region-specific dissection if the target structure is clearly delineated. However, it is difficult to visualize anatomical boundaries in an unstained specimen, while histological staining can complicate the microdissection process and compromise downstream processing and analysis. We now introduce a novel method in which in situ hybridization (ISH) signal is used to guide LCM on adjacent unstained sections to collect tissue from neurochemically defined regions of the human postmortem brain to minimize sample manipulation prior to analysis. This approach was validated in nuclei that provide monoaminergic inputs to the forebrain, and likely contribute to the pathophysiology of mood disorders. This method was used successfully to carry out gene expression profiling and quantitative real-time PCR (qPCR) confirmation from the dissected material. When compared to traditional micropunch dissections, our ISH-guided LCM method provided enhanced signal intensity for mRNAs of specific monoaminergic marker genes as measured by genome-wide gene expression microarrays. Enriched expression of specific monoaminergic genes (as determined by microarrays and qPCR) was detected within appropriate anatomical locations validating the accuracy of microdissection. Together these results support the conclusion that ISH-guided LCM permits acquisition of enriched nucleus-specific RNA that can be successfully used for downstream gene expression investigations. Future studies will utilize this approach for gene expression profiling of neurochemically defined regions of postmortem brains collected from mood disorder patients.

Lateral habenular neurons projecting to reward-processing monoaminergic nuclei express hyperpolarization-activated cyclic nucleotid-gated cation channels.

The lateral habenular complex (LHb) is a key signal integrator between limbic forebrain regions and monoaminergic hindbrain nuclei. Major projections of LHb neurons target the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal (DR) and median raphe nuclei (MnR). Both monoaminergic neurotransmitter systems play a central role in reward processing and reward-related decision-making. Glutamatergic LHb efferents terminate on GABAergic neurons in the VTA, the rostromedial tegmental nucleus (RMTg), and the raphe nuclei, thereby suppressing monoamine release when required by the present behavioral context. Recent studies suggest that the LHb exerts a strong tonic inhibition on monoamine release when no reward is to be obtained. It is yet unknown whether this inhibition is the result of a continuous external activation by other brain areas, or if it is intrinsically generated by LHb projection neurons. To analyze whether the tonic inhibition may be the result of a hyperpolarization-activated cyclic nucleotid-gated cation channel (HCN)-mediated pacemaker activity of LHb projection neurons, we combined retrograde tracing in rats with in situ hybridization of HCN1 to HCN4 mRNAs. In fact, close to all LHb neurons targeting VTA or raphe nuclei are equipped with HCN subunit mRNAs. While HCN1 mRNA is scarce, most neurons display strong expression of HCN2 to HCN4 mRNAs, in line with the potential formation of heteromeric channels. These results are supported by quantitative PCR and immunocytochemical analyses. Thus, our data suggest that the tonic inhibition of monoamine release is intrinsically generated in LHb projection neurons and that their activity may only be modulated by synaptic inputs to the LHb.

Expression patterns of corticotropin-releasing factor, arginine vasopressin, histidine decarboxylase, melanin-concentrating hormone, and orexin genes in the human hypothalamus†‡

The hypothalamus regulates numerous autonomic responses and behaviors. The neuroactive substances corticotropin‐releasing factor (CRF), arginine‐vasopressin (AVP), histidine decarboxylase (HDC), melanin‐concentrating hormone (MCH), and orexin/hypocretins (ORX) produced in the hypothalamus mediate a subset of these processes. Although the expression patterns of these genes have been well studied in rodents, less is known about them in humans. We combined classical histological techniques with in situ hybridization histochemistry to produce both 2D and 3D images and to visually align and quantify expression of the genes for these substances in nuclei of the human hypothalamus. The hypothalamus was arbitrarily divided into rostral, intermediate, and caudal regions. The rostral region, containing the paraventricular nucleus (PVN), was defined by discrete localization of CRF‐ and AVP‐expressing neurons, whereas distinct relationships between HDC, MCH, and ORX mRNA‐expressing neurons delineated specific levels within the intermediate and caudal regions. Quantitative mRNA signal intensity measurements revealed no significant differences in overall CRF or AVP expression at any rostrocaudal level of the PVN. HDC mRNA expression was highest at the level of the premammillary area, which included the dorsomedial and tuberomammillary nuclei as well as the dorsolateral hypothalamic area. In addition, the overall intensity of hybridization signal exhibited by both MCH and ORX mRNA‐expressing neurons peaked in distinct intermediate and caudal hypothalamic regions. These results suggest that human hypothalamic neurons involved in the regulation of the HPA axis display distinct neurochemical patterns that may encompass multiple local nuclei. J. Comp. Neurol. 518:4591–4611, 2010.

Evidence for transcriptional factor dysregulation in the dorsal raphe nucleus of patients with major depressive disorder

Extensive evidence implicates dysfunction in serotonin (5-HT) signaling in the etiology of major depressive disorder (MDD). Dorsal raphe nucleus (DR) is a major source of serotonin in the brain, and previous studies have reported within it alterations in 5-HT-related gene expression, protein levels, receptor binding, and morphological organization in mood disorders. In the present study, we utilized in situ hybridization-guided laser capture microdissection to harvest tissue samples from the middle-caudal subregion of the human DR post-mortem from MDD patients and from psychiatrically normal comparison subjects. Extracted RNA was prepared for gene expression profiling, and subsequent confirmation of select targets with quantitative real-time PCR. Our data indicate expression changes in functional gene families that regulate: (1) cellular stress and energy balance, (2) intracellular signaling and transcriptional regulation, and (3) cell proliferation and connectivity. The greatest changes in expression were observed among transcriptional regulators, including downregulation in the expression of TOB1, EGR1, and NR4A2 and their downstream targets. Previous studies have implicated these gene products in the regulation of functional domains impacted by MDD, including cognitive function, affective regulation, and emotional memory formation. These observations indicate altered function of several transcriptional regulators and their downstream targets, which may lead to the dysregulation of multiple cellular functions that contribute to the pathophysiology of MDD. Future studies will require single cell analyses in the DR to determine potential impact of these changes on its cellular functions and related circuits.