Neurochemical properties for defining subdivisions of the mouse medial geniculate body.

The medial geniculate body (MGB) exhibits anatomical and physiological properties that underlie its role in the auditory system. Anatomical properties, including myelo- and cyto-architecture, are used to identify MGB subdivisions. Recently, neurochemical properties, including calcium-binding proteins, have also been employed to define the MGB subdivisions. Because these properties do not show clear boundaries in the MGB and do not involve anatomical connectivity, whether the MGB subdivisions can be defined based on anatomical and neurochemical properties remains unclear. In this study, 11 different neurochemical markers were employed for defining the MGB subdivisions. In terms of anatomical connectivity, immunoreactivities for vesicular transporter demonstrated glutamatergic, GABAergic and glycinergic afferents and provided clues about the boundaries of the MGB subdivisions. On the other hand, the distribution of novel neurochemical markers of the MGB demonstrated distinct boundaries of the MGB subdivisions and resulted in the discovery of a putative homolog of the rabbit internal division of the MGB. Additionally, corticotropin-releasing factor was expressed in the larger neurons in the medial division of the MGB (MGm), particularly in the caudal MGm. Lastly, the analysis of anatomical details by measuring the size and density of vesicular transporters revealed heterogeneity among the MGB subdivisions. Our results demonstrate that the MGB is composed of five subdivisions based on their anatomical and neurochemical properties.

Temporal profiles of neuronal responses to repeated tone stimuli in the mouse primary auditory cortex.

How the auditory system processes temporal information of sound has been investigated extensively using repeated stimuli. Recent studies on how the response of neurons in the primary auditory cortex (A1) changes with the progression of stimulus repetition, have reported response temporal profiles of two categories: "adaptation", i.e., gradual decrease, and "facilitation", i.e., gradual increase. To explore the existence of profiles of other categories and to examine the tone-frequency-dependence of the profile category in single neurons, here we studied the response of mouse A1 neurons to four or five tone-trains; each train comprised 10 identical tone pips, with 0.5-s inter-tone-intervals, and the four or five trains differed only in tone frequency. The response to each tone in a train was evaluated using the peak of the ON response, and how the peak response changed with the tone number in the train, i.e., the response temporal profile, was examined. We confirmed the existence of profiles of both "adaptation" and "facilitation" categories; "adaptation" could be further subcategorized into "slow adaptation" and "fast adaptation" profiles, with the latter being encountered more frequently. Moreover, two new categories of non-monotonic profiles were identified: an "adaptation with recovery" profile and a "facilitation followed by adaptation" profile. Examination of single neurons with trains of different tone frequencies revealed that some A1 neurons exhibited profiles of the same category to tone trains of different tone frequencies, whereas others exhibited profiles of different categories, depending on the tone frequency. These results demonstrate the variety in the response temporal profiles of mouse A1 neurons, which may benefit the encoding of individual tones in a train.

Postnatal development of subfields in the core region of the mouse auditory cortex.

The core region of the rodent auditory cortex has two subfields: the primary auditory area (A1) and the anterior auditory field (AAF). Although the postnatal development of A1 has been studied in several mammalian species, few studies have been conducted on the postnatal development of AAF. Using a voltage-sensitive-dye-based imaging method, we examined and compared the postnatal development of AAF and A1 in mice from postnatal day 11 (P11) to P40. We focused on the postnatal development of tonotopy, the relative position between A1 and AAF, and the properties of tone-evoked responses in the subfields. Tone-evoked responses in the mouse auditory cortex were first observed at P12, and tonotopy was found in both A1 and AAF at this age. Quantification of tonotopy using the cortical magnification factor (CMF; octave difference per unit cortical distance) revealed a rapid change from P12 to P14 in both A1 and AAF, and a stable level from P14. A similar time course of postnatal development was found for the distance between the 4 kHz site in A1 and AAF, the distance between the 16 kHz site in A1 and AAF, and the angle between the frequency axis of A1 and AAF. The maximum amplitude and rise time of tone-evoked signals in both A1 and AAF showed no significant change from P12 to P40, but the latency of the responses to both the 4 kHz and 16 kHz tones decreased during this period, with a more rapid decrease in the latency to 16 kHz tones in both subfields. The duration of responses evoked by 4 kHz tones in both A1 and AAF showed no significant postnatal change, but the duration of responses to 16 kHz tones decreased exponentially in both subfields. The cortical area activated by 4 kHz tones in AAF was always larger than that in A1 at all ages (P12-P40). Our results demonstrated that A1 and AAF developed in parallel postnatally, showing a rapid maturation of tonotopy, slow maturation of response latency and response duration, and a dorsal-to-ventral order (high-frequency site to low-frequency site) of functional maturation.

Synaptic organisation and behaviour-dependent activity of mGluR8a-innervated GABAergic trilaminar cells projecting from the hippocampus to the subiculum.

In the hippocampal CA1 area, the GABAergic trilaminar cells have their axon distributed locally in three layers and also innervate the subiculum. Trilaminar cells have a high level of somato-dendritic muscarinic M2 acetylcholine receptor, lack somatostatin expression and their presynaptic inputs are enriched in mGluR8a. But the origin of their inputs and their behaviour-dependent activity remain to be characterised. Here we demonstrate that (1) GABAergic neurons with the molecular features of trilaminar cells are present in CA1 and CA3 in both rats and mice. (2) Trilaminar cells receive mGluR8a-enriched GABAergic inputs, e.g. from the medial septum, which are probably susceptible to hetero-synaptic modulation of neurotransmitter release by group III mGluRs. (3) An electron microscopic analysis identifies trilaminar cell output synapses with specialised postsynaptic densities and a strong bias towards interneurons as targets, including parvalbumin-expressing cells in the CA1 area. (4) Recordings in freely moving rats revealed the network state-dependent segregation of trilaminar cell activity, with reduced firing during movement, but substantial increase in activity with prolonged burst firing (> 200 Hz) during slow wave sleep. We predict that the behaviour-dependent temporal dynamics of trilaminar cell firing are regulated by their specialised inhibitory inputs. Trilaminar cells might support glutamatergic principal cells by disinhibition and mediate the binding of neuronal assemblies between the hippocampus and the subiculum via the transient inhibition of local interneurons.

Corticofugal GABAergic projection neurons in the mouse frontal cortex.

Cortical projection neurons are classified by hodology in corticocortical, commissural and corticofugal subtypes. Although cortical projection neurons had been regarded as only glutamatergic neurons, recently corticocortical GABAergic projection neurons has been also reported in several species. Here, we demonstrate corticofugal GABAergic projection neurons in the mouse frontal cortex. We employed viral-vector-mediated anterograde tracing, classical retrograde tracing, and immunohistochemistry to characterize neocortical GABAergic projection neurons. Injections of the Cre-dependent adeno-associated virus into glutamate decarboxylase 67 (GAD67)-Cre knock-in mice revealed neocortical GABAergic projections widely to the forebrain, including the cerebral cortices, caudate putamen (CPu), ventral pallidum (VP), lateral globus pallidus (LGP), nucleus accumbens, and olfactory tubercle (Tu). Minor GABAergic projections were also found in the mediodorsal thalamic nucleus, diagonal band of Broca, medial globus pallidus, substantial nigra, and dorsal raphe nucleus. Retrograde tracing studies also demonstrated corticofugal GABAergic projection neurons in the mouse frontal cortex. Further immunohistochemical screening with neurochemical markers revealed the majority of corticostriatal GABAergic projection neurons were positive for somatostatin (SS)-immunoreactivity. In contrast, corticothalamic GABAergic projection neurons were not identified by representative neurochemical markers for GABAergic neurons. These findings suggest that corticofugal GABAergic projection neurons are heterogeneous in terms of their neurochemical properties and target nuclei, and provide axonal innervations mainly to the nuclei in the basal ganglia.

Glutamatergic input from specific sources influences the nucleus accumbens-ventral pallidum information flow.

The nucleus accumbens (NAc) is positioned to integrate signals originating from limbic and cortical areas and to modulate reward-related motor output of various goal-directed behaviours. The major target of the NAc GABAergic output neurons is the ventral pallidum (VP). VP is part of the reward circuit and controls the ascending mesolimbic dopamine system, as well as the motor output structures and the brainstem. The excitatory inputs governing this system converge in the NAc from the prefrontal cortex (PFC), ventral hippocampus (vHC), midline and intralaminar thalamus (TH) and basolateral nucleus of the amygdala (BLA). It is unclear which if any of these afferents innervate the medium spiny neurons of the NAc, that project to the VP. To identify the source of glutamatergic afferents that innervate neurons projecting to the VP, a dual-labelling method was used: Phaseolus vulgaris leucoagglutinin for anterograde and EGFP-encoded adenovirus for retrograde tract-tracing. Within the NAc, anterogradely labelled BLA terminals formed asymmetric synapses on dendritic spines that belonged to medium spiny neurons retrogradely labelled from the VP. TH terminals also formed synapses on dendritic spines of NAc neurons projecting to the VP. However, dendrites and dendritic spines retrogradely labelled from VP received no direct synaptic contacts from afferents originating from mPFC and vHC in the present material, despite the large number of fibres labelled by the anterograde tracer injections. These findings represent the first experimental evidence for a selective glutamatergic innervation of NAc neurons projecting to the VP. The glutamatergic inputs of different origin (i.e. mPFC, vHC, BLA, TH) to the NAc might thus convey different types of reward-related information during goal-directed behaviour, and thereby contribute to the complex regulation of nucleus accumbens functions.

A novel instrumented multipeg running wheel system, Step-Wheel, for monitoring and controlling complex sequential stepping in mice.

Motor control is critical in daily life as well as in artistic and athletic performance and thus is the subject of intense interest in neuroscience. Mouse models of movement disorders have proven valuable for many aspects of investigation, but adequate methods for analyzing complex motor control in mouse models have not been fully established. Here, we report the development of a novel running-wheel system that can be used to evoke simple and complex stepping patterns in mice. The stepping patterns are controlled by spatially organized pegs, which serve as footholds that can be arranged in adjustable, ladder-like configurations. The mice run as they drink water from a spout, providing reward, while the wheel turns at a constant speed. The stepping patterns of the mice can thus be controlled not only spatially, but also temporally. A voltage sensor to detect paw touches is attached to each peg, allowing precise registration of footfalls. We show that this device can be used to analyze patterns of complex motor coordination in mice. We further demonstrate that it is possible to measure patterns of neural activity with chronically implanted tetrodes as the mice engage in vigorous running bouts. We suggest that this instrumented multipeg running wheel (which we name the Step-Wheel System) can serve as an important tool in analyzing motor control and motor learning in mice.

Long-Range GABAergic Connections Distributed throughout the Neocortex and their Possible Function.

Features and functions of long-range GABAergic projection neurons in the developing cerebral cortex have been reported previously, although until now their significance in the adult cerebral cortex has remained uncertain. The septo-hippocampal circuit is one exception - in this system, long-range mature GABAergic projection neurons have been well analyzed and their contribution to the generation of theta-oscillatory behavior in the hippocampus has been documented. To have a clue to the function of the GABAergic projection neurons in the neocortex, we view how the long-range GABAergic projections are integrated in the cortico-cortical, cortico-fugal, and afferent projections in the cerebral cortex. Then, we consider the possibility that the GABAergic projection neurons are involved in the generation, modification, and/or synchronization of oscillations in mature neocortical neuron activity. When markers that identify the GABAergic projection neurons are examined in anatomical and developmental studies, it is clear that neuronal NO synthetase (nNOS)-immunoreactivity can readily identify GABAergic projection neurons. GABAergic projection neurons account for 0.5% of the neocortical GABAergic neurons. To elucidate the role of the GABAergic projection neurons in the neocortex, it will be necessary to clarify the network constructed by nNOS-positive GABAergic projection neurons and their postsynaptic targets. Thus, our long-range goals will be to label and manipulate (including deleting) the GABAergic projection neurons using genetic tools driven by a nNOS promoter. We recognize that this may be a complex endeavor, as most excitatory neurons in the murine neocortex express nNOS transiently. Nevertheless, additional studies characterizing long-range GABAergic projection neurons will have great value to the overall understanding of mature cortical function.

Expression of COUP-TFII nuclear receptor in restricted GABAergic neuronal populations in the adult rat hippocampus.

The COUP-TFII nuclear receptor, also known as NR2F2, is expressed in the developing ventral telencephalon and modulates the tangential migration of a set of subpallial neuronal progenitors during forebrain development. Little information is available about its expression patterns in the adult brain. We have identified the cell populations expressing COUP-TFII and the contribution of some of them to network activity in vivo. Expression of COUP-TFII by hippocampal pyramidal and dentate granule cells, as well as neurons in the neocortex, formed a gradient increasing from undetectable in the dorsal to very strong in the ventral sectors. In the dorsal hippocampal CA1 area, COUP-TFII was restricted to GABAergic interneurons and expressed in several, largely nonoverlapping neuronal populations. Immunoreactivity was present in calretinin-, neuronal nitric oxide synthase-, and reelin-expressing cells, as well as in subsets of cholecystokinin- or calbindin-expressing or radiatum-retrohippocampally projecting GABAergic cells, but not in parvalbumin- and/or somatostatin-expressing interneurons. In vivo recording and juxtacellular labeling of COUP-TFII-expressing cells revealed neurogliaform cells, basket cells in stratum radiatum and tachykinin-expressing radiatum dentate innervating interneurons, identified by their axodendritic distributions. They showed cell type-selective phase-locked firing to the theta rhythm but no activation during sharp wave/ripple oscillations. These basket cells in stratum radiatum and neurogliaform cells fired at the peak of theta oscillations detected extracellularly in stratum pyramidale, unlike previously reported ivy cells, which fired at the trough. The characterization of COUP-TFII-expressing neurons suggests that this developmentally important transcription factor plays cell type-specific role(s) in the adult hippocampus.

Expression of EGFP by adenovirus-mediated gene transfer in the central nervous system.

A new method for investigating cortical microcircuitry uses adenovirus to introduce enhanced green fluorescent protein (EGFP) as a reporter gene to small groups of neurons. Adenovirus in solution containing 600 mM NaCl was injected into the cerebral cortex in anesthetized rats and monkeys, resulting in many EGFP-positive neurons in the interconnected distant brain regions as well as at the injection site. This result suggests that adenovirus with high NaCl concentration will be a kind of retrograde tracer. Thus, I have succeeded in finding a condition of adenovirus injection to retrogradely label cortical neurons to the full extent of their dendritic configurations. This system can be used to study the microcircuitry of central nervous system, and specific mammalian gene function within identified circuits in vivo using RNA interference and/or gene overexpression.

Parvalbumin neurons in the forebrain as revealed by parvalbumin-Cre transgenic mice.

Neurons expressing the calcium-binding protein parvalbumin (PV) constitute an abundant subpopulation of GABAergic neurons in the cerebral cortex. However, PV is not unique to the GABAergic neurons of the forebrain, but is also expressed in a small number of pyramidal neurons and in a large number of thalamic neurons. In order to summarize the PV neurons in the forebrain, we employed the PV-Cre transgenic mice in the present study. In the progeny of crossbreed between PV-Cre mice and GFP-Cre reporter mice, we found that the GFP-positive neurons include many excitatory neurons in the neocortex and the thalamus as well as GABAergic neurons in the cerebral cortex and basal ganglia. All the reported PV-positive GABAergic neurons in the cerebral cortex and the basal ganglia seemed to be included in the GFP-positive cells. We found GFP-positive layer V pyramidal neurons inhabit a broader neocortical area than was previously reported. They were located in the primary somatosensory, motor, and visual areas. The somatosensory area of the neocortex contained the greatest number of PV-positive pyramidal neurons. A large number of thalamic relay neurons and virtually all the reticular thalamic neurons appeared as GFP-positive. Thalamic relay nucleus and a neocortical area for the same modality corresponded and seemed to contain a characteristic amount of PV-positive excitatory neurons.

Rhythmically active enkephalin-expressing GABAergic cells in the CA1 area of the hippocampus project to the subiculum and preferentially innervate interneurons.

Enkephalins (ENKs) are endogenous opioids that regulate synaptic excitability of GABAergic networks in the cerebral cortex. Using retrograde tracer injections in the subiculum, we identified a hippocampal population of ENK-expressing projection neurons. In situ hybridization for GAD shows that ENK-expressing cells are a small GABAergic subpopulation. Furthermore, by extracellular recording and juxtacellular labeling in vivo, we identified an ENK-expressing cell in stratum radiatum of the CA1 area by its complete axodendritic arborization and characteristic spike timing during network oscillations. The somatodendritic membrane was immunopositive for mGluR1alpha, and there was both a rich local axon in CA1 and subicular-projecting branches. The boutons showed cell-type- and layer-specific innervation, i.e., interneurons were the main targets in the alveus, both interneurons and pyramidal cell dendrites were innervated in the other layers, and interneurons were exclusive targets in the subiculum. Parvalbumin-, but not somatostatin-, calbindin-, or cholecystokinin-expressing interneurons were preferred synaptic targets. During network activity, the juxtacellularly labeled ENK-expressing cell was phase modulated throughout theta oscillations, but silenced during sharp-wave/ripple episodes. After these episodes the interneuron exhibited rebound activity of high-frequency spike bursts, presumably causing peptide release. The ENK-expressing interneurons innervating parvalbumin-positive interneurons might contribute to the organization of the sharp-wave/ripple episodes by decreased firing during and rebound activity after the ripple episodes, as well as to the coordination of activity between the CA1 and subicular areas during network oscillations.

Novel strategy to selectively label excitatory and inhibitory neurons in the cerebral cortex of mice.

Revealing the connections of neuronal systems is critical for understanding how they function. The vast majority of neurons in all cortical areas consist of excitatory cells whose activity is controlled by inhibitory cells. Distribution and projection patterns of inhibitory and excitatory cells are key information to understand the organization of the nervous system. To investigate axonal projections, we developed a method to uniquely distinguish excitatory axons from inhibitory ones in the cortex using transgenic mice expressing Cre recombinase in the Ca2+/calmodulin-dependent protein kinase IIalpha-containing neurons. These animals were injected by an adenoviral vector engineered so that it directs red fluorescent protein expression in non-Cre-expressing cells, and green fluorescent protein in Cre-positive neurons. We demonstrated in vitro and in vivo that GFP-expressing neurons are GABA-immunonegative (excitatory), while the RFP-expressing cells are either GABAergic neurons or glial cells. One week after the viral vector injection RFP and GFP signals overlapped in a subset of cells but after 1 month, the two signals showed total segregation. Six months post-inoculation, GFP-labelling was clearly visible in axons but RFP remained only in somata and proximal dendrites. This technique can thus be used to differentiate excitatory axonal projections from inhibitory ones, and represent a unique tool in neuronal circuit analysis.

Long-distance corticocortical GABAergic neurons in the adult monkey white and gray matter.

A subgroup of GABAergic neurons has been reported to project over long distances in several species. Here we demonstrate that long-distance cortically projecting nonpyramidal neurons occur in monkeys in both white and gray matter. Nonpyramidal neurons were first identified morphologically. Visualization of Golgi-like details was achieved by retrograde infection from injections of an adenovirus vector, producing enhanced green fluorescent protein (EGFP) under control of a neuron-specific promoter. Injections in areas V1, V4, TEO, and posterior TE resulted in EGFP-expressing nonpyramidal neurons up to 1.5 cm distant from the injections, mainly in the white matter. Some neurons occurred in the gray matter, mainly in layer 3, but also in layers 5 and 6, and, very occasionally, layer 1. As control, we injected cholera toxin subunit B, a standard retrograde tracer, in V4, and observed a similarly wide distribution of neurons in the white matter. Second, the GABAergic identity of EGFP-expressing nonpyramidal neurons was established by colabeling for EGFP and GAD67 in selected tissue sections. Most neurons positive for EGFP and GAD67 were positive for somatostatin (SS; 90%). Of those neurons positive for EGFP and SS, almost all were also positive for neuronal nitric oxide synthase or m2 muscarinic receptor, but only 23% were also positive for calretinin. None were positive for parvalbumin. We conclude that long-distance projecting GABAergic neurons 1) are phylogenetically conserved, although in monkeys most gray matter neurons are in the upper layers, and 2) are heterogeneous in terms of their neurochemistry, location, and potentially function.

Improved Golgi-like visualization in retrogradely projecting neurons after EGFP-adenovirus infection in adult rat and monkey.

An adenovirus vector was generated using a neuron-specific promoter synapsin I and enhanced green fluorescent protein (EGFP) reporter (AdSynEGFP). In addition, two modifications were identified that resulted in robust and reliable retrograde transport and EGFP expression after injection of the virus into three different brain regions in adult rats (medial prefrontal cortex, posterior thalamic nuclear group, and CA1). These are postinjection survival times of 14 days and addition of high concentrations of NaCl (>or=600 mM) to the injection buffer. These modifications resulted in obvious improvement in the intensity of the EGFP signal and in the number of labeled cells. Use of anti-EGFP in immunofluorescence or immunoperoxidase processing further enhanced the signal so that Golgi-like filling of dendritic spines and axon collaterals was routinely achieved. Effectiveness of the AdSynEGFP for Golgi-like filling was confirmed in one rhesus monkey with injections in visual area V4. Because of the long-term viability of the infected neurons (at least up to 28 days in rats and 22 days in monkey), this AdSynEGFP is suitable for use in microcircuitry studies in combination with other fluorescently tagged elements, including anterogradely labeled extrinsic projections. The native EGFP signal (without antibody enhancement) may be sufficient for studies involving cultured cells or slices.

Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex.

Gamma-aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67-green fluorescent protein (GFP) knock-in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP-positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long-range projections by tracing GFP-labeled GABAergic neurons with axon branches traveled rostro-caudally and medio-laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network.

Efferent and afferent connections of GABAergic neurons in the supratrigeminal and the intertrigeminal regions. An immunohistochemical tract-tracing study in the GAD67-GFP knock-in mouse.

It has been reported in the cat and rat that inhibitory premotor neurons, which send their axons to motoneurons of the trigeminal motor nucleus (Vm) are distributed in the reticular regions around the Vm, especially in the supratrigeminal region (Vsup) and the intertrigeminal region (Vint). In the present study, we examined neuronal connections of GABAergic neurons in the Vsup and Vint in the mouse by utilizing the adult heterozygous GAD67-GFP knock-in mouse, in which green fluorescence protein (GFP) is expressed in GABAergic neurons under the control of the endogenous GAD (GAD67) gene promoter [Yanagawa, Y., Kaneko, K., Kanbara, N., Totsuka, M., Yagi, T., Obata, K., 2001. Development of mouse expressing GFP in GABAergic neurons. Neurosci. Res. Suppl. 25, S77; Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J.-I., Obata, K., Kaneko, T., 2003. Green fluorescent protein expression and colocalization with calretinin, parvalbumin and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60-79]. The connections were examined light- and electron-microscopically by combining the anterograde or the retrograde tract-tracing method with the immunohistochemical method for GFP. The data indicated that the Vsup and Vint of the mouse contained GABAergic neurons, which received projection fibers from the marginal layer of the nucleus of the spinal tract of the trigeminal nerve (Vc) on the ipsilateral side and sent their axons to the Vm on the contralateral side. Some of these GABAergic neurons may represent Vm-premotor neurons that receive nociceptive input from the Vc to elicit jaw-opening reflex by inhibiting jaw-closing Vm-motoneurons.

GABAergic basal forebrain neurons that express receptor for neurokinin B and send axons to the cerebral cortex.

Neurons expressing neurokinin B (NK3) receptor in the basal forebrain region of rats were characterized histochemically by combining immunocytochemistry, in situ hybridization and retrograde labeling, and electrophysiologically by whole-cell clamp recording. NK3 receptor-immunoreactive neurons were found in the basal forebrain region including the substantia innominata, where axon terminals immunoreactive for preprotachykinin B, the precursor peptide of neurokinin B (NKB), were densely distributed. More than 90% of NK3 receptor-expressing neurons in the basal forebrain region showed signals for glutamate decarboxylase mRNA, indicating that almost all NK3 receptor-expressing neurons were gamma-aminobutyric acid (GABA)ergic neurons. On the other hand, only a few NK3 receptor-immunoreactive neurons showed immunoreactivity for choline acetyltransferase or parvalbumin in the substantia innominata, ventral pallidum, and globus pallidus, although the distribution of NK3 receptor-expressing neurons overlapped with those of cholinergic neurons and parvalbumin-positive neurons. After injection of wheat germ agglutinin into the cerebral cortex, NK3 receptor immunoreactivity was detected in about 25% of retrogradely labeled basal forebrain neurons, indicating that NK3 receptor-expressing neurons send projection fibers to the cerebral cortex. In the whole-cell clamp recording study, a selective NK3 receptor agonist evoked membrane depolarization or inward currents with decrease of input impedance in 10 of 100 cortically projecting neurons recorded in the basal forebrain region. Because NKB-producing striatal neurons send axons selectively to the basal forebrain region, the present results suggest that the release of NKB by those striatal neurons induces an inhibitory effect on cortical neurons via facilitation of GABAergic basal forebrain neurons expressing NK3 receptor.

Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse.

Gamma-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many medium-sized spherical somata emitting intense GFP fluorescence were observed in layer I.

Changes of immunocytochemical localization of vesicular glutamate transporters in the rat visual system after the retinofugal denervation.

To clarify which vesicular glutamate transporter (VGluT) is used by excitatory axon terminals of the retinofugal system, we examined immunoreactivities and mRNA signals for VGluT1 and VGluT2 in the rat retina and compared immunoreactivities for VGluT1 and VGluT2 in the retinorecipient regions using double immunofluorescence method, anterograde tracing, and immunoelectron microscopy. Furthermore, the changes of VGluT1 and VGluT2 immunoreactivities were studied after eyeball enucleation. Intense immunoreactivity and mRNA signal for VGluT2, but not for VGluT1 immunoreactivity, were observed in most perikarya of ganglion cells in the retina. Immunoelectron microscopy revealed that VGluT1- and VGluT2-immunolabeled terminals made asymmetrical synapses, suggesting that they were excitatory synapses, and that VGluT1-immunolabeled terminals were smaller than VGluT2-labeled ones in many retinorecipient regions, such as the dorsal lateral geniculate nucleus (LGd) and superior colliculus (SC). Double immunofluorescence study further revealed that almost no VGluT2 immunoreactivity was colocalized with VGluT1 in the retinorecipient regions. After wheat germ agglutinin (WGA) injection into the eyeballs, WGA immunoreactivity was colocalized in the single axon terminals of LGd and SC with VGluT2 but not VGluT1 immunoreactivity. After unilateral enucleation, VGluT2 immunoreactivity in the LGd, SC, nucleus of the optic tract, and nuclei of the accessory optic tract in the contralateral side of the enucleated eye was clearly decreased. Although only a small change of VGluT2 immunoreactivity was observed in the contra- and ipsilateral suprachiasmatic nuclei, olivary pretectal nucleus, anterior pretectal nucleus, and posterior pretectal nucleus, moderate reduction of VGluT2 was found in these regions after bilateral enucleation. On the other hand, almost no change in VGluT1 immunoreactivity was found in the structures examined in the present enucleation study. Thus, the present results support the notion that the retinofugal pathways are glutamatergic, and indicate that VGluT2, but not VGluT1, is employed for accumulating glutamate into synaptic vesicles of retinofugal axons.