Pablo Henny

Dichotomous Organization of the External Globus Pallidus

Summary Different striatal projection neurons are the origin of a dual organization essential for basal ganglia function. We have defined an analogous division of labor in the external globus pallidus (GPe) of Parkinsonian rats, showing that the distinct temporal activities of two populations of GPe neuron in vivo are underpinned by distinct molecular profiles and axonal connectivities. A first population of prototypic GABAergic GPe neurons fire antiphase to subthalamic nucleus (STN) neurons, often express parvalbumin, and target downstream basal ganglia nuclei, including STN. In contrast, a second population (arkypallidal neurons) fire in-phase with STN neurons, express preproenkephalin, and only innervate the striatum. This novel cell type provides the largest extrinsic GABAergic innervation of striatum, targeting both projection neurons and interneurons. We conclude that GPe exhibits several core components of a dichotomous organization as fundamental as that in striatum. Thus, two populations of GPe neuron together orchestrate activities across all basal ganglia nuclei in a cell-type-specific manner.

Projections from basal forebrain to prefrontal cortex comprise cholinergic, GABAergic and glutamatergic inputs to pyramidal cells or interneurons

The present study was undertaken to characterize the pre‐ and postsynaptic constituents of the basal forebrain (BF) projection to the prefrontal cortex in the rat, and determine whether it includes glutamatergic in addition to established γ‐aminobutyric acid (GABA)ergic and cholinergic elements. BF fibres were labelled by anterograde transport using biotin dextran amine (BDA) and dual‐stained for the vesicular transporter proteins (VTPs) for glutamate (VGluT), GABA (VGAT) or acetylcholine (VAChT). Viewed by fluorescence microscopy and estimated by stereology, proportions of BDA‐labelled varicosities were found to be stained for VGluT2 (and not VGluT1 or 3), VGAT or VAChT (representing, respectively, ∼15%, ∼52% and ∼19% within the infralimbic cortex). Each type was present in all, though commonly most densely in deep, cortical layers. Material was triple‐stained for postsynaptic proteins to examine whether BDA+VTP+ varicosities might form excitatory or inhibitory synapses, respectively, labelled by postsynaptic density‐95 kDA (PSD‐95) or gephyrin (Geph). Viewed by confocal microscopy, a majority of BDA+/VGluT2+ varicosities were found to be apposed to PSD‐95+ elements, and a majority of BDA+/VGAT+ varicosities to be apposed to Geph+ elements. Other series were triple‐stained for cell marker proteins to assess whether the varicosities contacted interneurons or pyramidal cells. Viewed by confocal microscopy, BDA‐labelled VGluT2+, VGAT+ and VAChT+ BF terminals were all found in contact with calbindin+ interneurons, whereas VGAT+ BF terminals were also seen in contact with parvalbumin+ interneurons and non‐phosphorylated neurofilament+ pyramidal cells. Through distinct glutamatergic, GABAergic and cholinergic projections, the BF can thus influence cortical activity in a diverse manner.

Discharge Profiles of Identified GABAergic in Comparison to Cholinergic and Putative Glutamatergic Basal Forebrain Neurons across the Sleep-Wake Cycle

Whereas basal forebrain (BF) cholinergic neurons are known to participate in processes of cortical activation during wake (W) and paradoxical sleep (PS or P, also called REM sleep), codistributed GABAergic neurons have been thought to participate in processes of cortical deactivation and slow-wave sleep (SWS or S). To learn the roles the GABAergic neurons might play, in relation to cholinergic and glutamatergic neurons, we juxtacellularly recorded and labeled neurons during natural sleep–wake states in head-fixed rats. Neurobiotin (Nb)-labeled cells were identified immunohistochemically as choline acetyltransferase (ChAT)+, glutamic acid decarboxylase (GAD)+, or ChAT−/GAD−. Of the latter, some were identified as glutamatergic by immunostaining of their terminals with the vesicular glutamate transporter (VGluT2). In contrast to ChAT+ neurons, which all discharged maximally during W and PS, GAD+ neurons comprised multiple sleep–wake subgroups. Some GABAergic neurons discharged maximally during W and PS, as WP-max active cells (36%), and in positive correlation with gamma electroencephalographic (EEG) activity. Some discharged maximally during SWS, as S-max active cells (28%), and in positive correlation with delta EEG activity. Others increased their discharge progressively during sleep to discharge maximally during PS, as P-max active cells (36%), and in negative association with electromyographic (EMG) activity. ChAT−/GAD− cells comprised WP-max (46%), S-max (17%), P-max (17%), and W-max active cells (14%), whose discharge was positively correlated with EMG activity. GABAergic neurons would thus play similar or reciprocal roles to other cholinergic and glutamatergic BF neurons in regulating cortical activity and muscle tone along with behavior across sleep–wake states.

Stereological estimates of the basal forebrain cell population in the rat, including neurons containing choline acetyltransferase, glutamic acid decarboxylase or phosphate-activated glutaminase and colocalizing vesicular glutamate transporters.

The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and comprise GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as approximately 355,000 and the numbers of ChAT-immuno-positive (+) as approximately 22,000, GAD+ approximately 119,000 and PAG+ approximately 316,000, corresponding to approximately 5%, approximately 35% and approximately 90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also has the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, vesicular glutamate transporter (VGluT) 3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or dendrites.

Innervation of Orexin/Hypocretin Neurons by GABAergic, Glutamatergic or Cholinergic Basal Forebrain Terminals Evidenced by Immunostaining for Presynaptic Vesicular Transporter and Postsynaptic Scaffolding Proteins

Orexin/hypocretin (Orx) neurons are critical for the maintenance of waking in association with behavioral arousal and postural muscle tone, since with their loss narcolepsy with cataplexy occurs. Given that basal forebrain (BF) neurons project to the hypothalamus and play important diverse roles in sleep/wake states, we sought to determine whether acetylcholine (ACh), glutamate (Glu), and/or GABA‐releasing BF neurons innervate and could thereby differentially regulate the Orx neurons. From discrete injections of biotinylated dextran amine (BDA, 10,000 MW) into the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) in the rat, BDA‐labeled fibers projected to the lateral hypothalamus (LH), perifornical area (PF), and dorsomedial hypothalamus (DMH), where ∼41%, ∼11%, and 9% of Orx‐positive (+) neurons were respectively contacted in each region. Employing triple fluorescent staining for Orx, BDA, and presynaptic vesicular (V) transporters (T), we found that only 4% of the innervated Orx+ neurons in the LH were contacted by BDA+[VAChT+] terminals, whereas ∼31% and ∼67% were respectively contacted by BDA+[VGluT2+] and BDA+[VGAT+] terminals. In 3D‐rendered and rotated confocal images, we confirmed the latter contacts and examined staining for postsynaptic proteins PSD‐95, a marker for glutamatergic synapses, and gephyrin, a marker for GABAergic synapses, that were located on Orx+ neurons facing BDA‐labeled terminals in ∼20% and ∼50% of contacts, respectively. With such synaptic input, BF glutamatergic neurons can excite Orx neurons and thus act to maintain behavioral arousal with muscle tone, whereas GABAergic neurons can inhibit Orx neurons and thus promote behavioral quiescence and sleep along with muscle atonia. J. Comp. Neurol. 499:645–661, 2006.

GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep

The lateral hypothalamus (LH), where wake‐active orexin (Orx)‐containing neurons are located, has been considered a waking center. Yet, melanin‐concentrating hormone (MCH)‐containing neurons are codistributed therein with Orx neurons and, in contrast to them, are active during sleep, not waking. In the present study employing juxtacellular recording and labeling of neurons with Neurobiotin (Nb) in naturally sleeping–waking head‐fixed rats, we identified another population of intermingled sleep‐active cells, which do not contain MCH (or Orx), but utilize γ‐aminobutyric acid (GABA) as a neurotransmitter. The ‘sleep‐max’ active neurons represented 53% of Nb‐labeled MCH‐(and Orx) immunonegative (−) cells recorded in the LH. For identification of their neurotransmitter, Nb‐labeled varicosities of the Nb‐labeled/MCH− neurons were sought within sections adjacent to the Nb‐labeled soma and immunostained for the vesicular transporter for GABA (VGAT) or for glutamate. A small proportion of sleep‐max Nb+/MCH− neurons (19%) discharged maximally during slow‐wave sleep (called ‘S‐max’) in positive correlation with delta electroencephalogram activity, and from VGAT staining of Nb‐labeled varicosities appeared to be GABAergic. The vast proportion of sleep‐max Nb+/MCH− neurons (81%) discharged maximally during paradoxical sleep (PS, called ‘P‐max’) in negative correlation with electromyogram amplitude, and from Nb‐labeled varicosities also appeared to be predominantly GABAergic. Given their discharge profiles across the sleep–wake cycle, P‐max together with S‐max GABAergic neurons could thus serve to inhibit other neurons of the arousal systems, including local Orx neurons in the LH. They could accordingly dampen arousal with muscle tone and promote sleep, including PS with muscle atonia.

Structural correlates of heterogeneous in vivo activity of midbrain dopaminergic neurons

Dopaminergic neurons of the substantia nigra pars compacta (SNc) exhibit functional heterogeneity that likely underpins their diverse roles in behavior. We examined how the functional diversity of identified dopaminergic neurons in vivo correlates with differences in somato-dendritic architecture and afferent synaptic organization. Stereological analysis of individually recorded and labeled dopaminergic neurons of rat SNc revealed that they received approximately 8,000 synaptic inputs, at least 30% of which were glutamatergic and 40–70% were GABAergic. The latter synapses were proportionally greater in number and denser on dendrites located in the substantia nigra pars reticulata (SNr) than on those located in SNc, revealing the existence of two synaptically distinct and region-specific subcellular domains. We also found that the relative extension of SNc neuron dendrites into the SNr dictated overall GABAergic innervation and predicted inhibition responses to aversive stimuli. We conclude that diverse wiring patterns determine the heterogeneous activities of midbrain dopaminergic neurons in vivo.

Activity of Neurochemically Heterogeneous Dopaminergic Neurons in the Substantia Nigra during Spontaneous and Driven Changes in Brain State

Dopaminergic neurons of the substantia nigra (SN) and ventral tegmental area (VTA) are collectively implicated in motor- and reward-related behaviors. However, dopaminergic SN and VTA neurons differ on several functional levels, and dopaminergic SN neurons themselves vary in their intrinsic electrical properties, neurochemical characteristics and connections. This heterogeneity is not only important for normal function; calbindin (CB) expression by some dopaminergic SN neurons has been linked with their increased survival in Parkinsons disease. To test whether the activity of CB-negative and CB-positive dopaminergic SN neurons differs during distinct spontaneous and driven brain states, we recorded single units in anesthetized rats before, during and after aversive somatosensory stimuli. Recorded neurons were juxtacellularly labeled, confirmed to be dopaminergic, and tested for CB immunoreactivity. During cortical slow-wave activity, the firing of most dopaminergic neurons was slow and regular/irregular and unrelated to cortical slow oscillations. During spontaneous cortical activation, dopaminergic SN neurons fired in a more regular manner, with fewer bursts, but did not change their firing rate. Regardless of brain state, CB-negative dopaminergic neurons fired significantly faster than CB-positive dopaminergic neurons. This difference in firing rate was not mirrored by different firing patterns. Most CB-negative and CB-positive dopaminergic neurons did not respond to the aversive stimuli; of those that did respond, most were inhibited. We conclude that CB-negative and CB-positive dopaminergic neurons exhibit different activities in vivo. Furthermore, the firing of dopaminergic SN neurons is brain state-dependent, and, unlike dopaminergic VTA neurons, they are not commonly recruited or inhibited by aversive stimuli.

Immunohistochemical evidence for synaptic release of glutamate from orexin terminals in the locus coeruleus

Orexin (Orx or hypocretin) is critically important for maintaining wakefulness, since in its absence, narcolepsy with cataplexy occurs. In this role, Orx-containing neurons can exert their influence upon multiple targets through the brain by release of Orx but possibly also by release of other neurotransmitters. Indeed, evidence was previously presented to suggest that Orx terminals could utilize glutamate (Glu) in addition to Orx as a neurotransmitter. Using fluorescence and confocal laser scanning microscopy, we investigated whether Orx varicosities contain the presynaptic markers for synaptic release of Glu or GABA and come into contact with postsynaptic markers for excitatory synapses within the locus coeruleus of the rat brain. We found that a proportion of the Orx+ varicosities were immunostained for the vesicular transporter for Glu, VGluT2. None were immunostained for vesicular glutamate transporter 1 (VGluT1) or VGluT3 or for the vesicular transporter for GABA, vesicular GABA transporter (VGAT). Among the Orx+ varicosities, 4% of all and 28% of large varicosities contained VGluT2. A similar proportion of the large Orx+ varicosities contained synaptophysin (Syp), a presynaptic marker for synaptic vesicles. Orx+ varicosities also contacted elements immunostained for postsynaptic density protein-95 (PSD)-95, a postsynaptic marker for glutamatergic synapses. We thus conclude that synaptic release of Glu occurs from Orx terminals within the locus coeruleus and can thus be important for the engagement of noradrenergic neurons in stimulating and maintaining arousal.

Vesicular glutamate (VGlut), GABA (VGAT), and acetylcholine (VACht) transporters in basal forebrain axon terminals innervating the lateral hypothalamus.

The basal forebrain (BF) is known to play important roles in cortical activation and sleep, which are likely mediated by chemically differentiated cell groups including cholinergic, γ‐aminobutyric acid (GABA)ergic and other unidentified neurons. One important target of these cells is the lateral hypothalamus (LH), which is critical for arousal and the maintenance of wakefulness. To determine whether chemically specific BF neurons provide an innervation to the LH, we employed anterograde transport of 10,000 MW biotinylated dextran amine (BDA) together with immunohistochemical staining of the vesicular transporter proteins (VTPs) for glutamate (VGluT1, ‐2, and ‐3), GABA (VGAT), or acetylcholine (ACh, VAChT). In addition, we applied triple staining for the postsynaptic proteins (PSPs), PSD‐95 with VGluT or Gephyrin (Geph) with VGAT, to examine whether the BDA‐labeled varicosities may form excitatory or inhibitory synapses in the LH. Axons originating from BDA‐labeled neurons in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) descended within the medial forebrain bundle and extended collateral varicose fibers to contact LH neurons. In the LH, the BDA‐labeled varicosities were immunopositive (+) for VAChT (∼10%), VGluT2 (∼25%), or VGAT (∼50%), revealing an important influence of newly identified glutamatergic together with GABAergic BF inputs. Moreover, in confocal microscopy, VGluT2+ and VGAT+ terminals were apposed to PSD‐95+ and Geph+ profiles respectively, indicating that they formed synaptic contacts with LH neurons. The important inputs from glutamatergic and GABAergic BF cells could thus regulate LH neurons in an opposing manner to stimulate vs. suppress cortical activation and behavioral arousal reciprocally. J. Comp. Neurol. 496:453–467, 2006.