Doublecortin expression levels in adult brain reflect neurogenesis

Progress in the field of neurogenesis is currently limited by the lack of tools enabling fast and quantitative analysis of neurogenesis in the adult brain. Doublecortin (DCX) has recently been used as a marker for neurogenesis. However, it was not clear whether DCX could be used to assess modulations occurring in the rate of neurogenesis in the adult mammalian central nervous system following lesioning or stimulatory factors. Using two paradigms increasing neurogenesis levels (physical activity and epileptic seizures), we demonstrate that quantification of DCX-expressing cells allows for an accurate measurement of modulations in the rate of adult neurogenesis. Importantly, we excluded induction of DCX expression during physiological or reactive gliogenesis and excluded also DCX re-expression during regenerative axonal growth. Our data validate DCX as a reliable and specific marker that reflects levels of adult neurogenesis and its modulation. We demonstrate that DCX is a valuable alternative to techniques currently used to measure the levels of neurogenesis. Importantly, in contrast to conventional techniques, analysis of neurogenesis through the detection of DCX does not require in vivo labelling of proliferating cells, thereby opening new avenues for the study of human neurogenesis under normal and pathological conditions.     

Atypical neuroleptics stimulate neurogenesis in adult rat brain

Schizophrenia has been treated effectively with atypical neuroleptics without serious side effects. We have shown previously that long-term treatment with atypical neuroleptics is correlated with an improvement of cognition in adult rats. We report here that atypical neuroleptics stimulate a 2- to 3-fold increase in newly divided cells in the subventricular zone in the rat and that some of these new cells in the subventricular zone and hippocampus also express a neuronal marker. We used bromodeoxyuridine (BrdU) to identify newly divided cells and confirmed the observation with antibody to a cell-cycle-specific, endogenous proliferating cell nuclear antigen (PCNA). Identification of BrdU-positive cells in the anterior subventricular zone (SVZa) particularly in rats treated with the atypical neuroleptics but not in those in the haloperidol-treated and control rats, suggests increased rostral migratory stream (RMS) cell traffic to replenish neurons in the olfactory bulb. Expression of a neuronal marker, NeuN, in BrdU-positive cells in rats treated with atypical neuroleptics, also suggests that these compounds may modulate in vivo differentiation of neuronal progenitor cells even within a day of BrdU injection. Our results indicate that atypical neuroleptics have a mechanism of action other than the previously proposed mechanisms, which might explain their role in improved cognition in animal and in schizophrenic patients. If substantiated by future studies, our findings may lead to an expanded use of atypical neuroleptics in other neurodegenerative diseases to stimulate neuronal replacement and repair.     

The dynamics of adult neurogenesis in human hippocampus

The phenomenon of adult neurogenesis is now an accepted occurrence in mammals and also in humans.At least two discrete places house stem cells for generation of neurons in adult brain. These are olfactory system and the hippocampus. In animals, newly generated neurons have been directly or indirectly demonstrated to generate a significant amount of new neurons to have a functional role. However, the data in humans on the extent of this process is still scanty and such as difficult to comprehend its functional role in humans. This paper explores the available data on as extent of adult hippocampal neurogenesis in humans and makes comparison to animal data.     

The role of neurogenesis during development and in the adult brain

Neural stem cells (NSCs) give rise to neurons during development. NSCs persist and neurogenesis continues in restricted regions of postnatal and adult brains. Adult‐born neurons integrate into existing neural circuits by synaptic connections and participate in the regulation of brain function. Thus, understanding NSCs and neurogenesis may be crucial in the development of new strategies for brain repair. Here, we introduce the lineage of NSCs from embryonic to adult stages and summarize recent studies on maturation and integration of adult‐born neurons. We also discuss the regulation and potential functions of adult neurogenesis in physiological and pathological conditions.     

Chemotherapy disrupts learning,neurogenesis and theta activity in the adult brain

Chemotherapy, especially if prolonged, disrupts attention, working memory and speed of processing in humans. Most cancer drugs that cross the blood–brain barrier also decrease adult neurogenesis. Because new neurons are generated in the hippocampus, this decrease may contribute to the deficits in working memory and related thought processes. The neurophysiological mechanisms that underlie these deficits are generally unknown. A possible mediator is hippocampal oscillatory activity within the theta range (3–12 Hz). Theta activity predicts and promotes efficient learning in healthy animals and humans. Here, we hypothesised that chemotherapy disrupts learning via decreases in hippocampal adult neurogenesis and theta activity. Temozolomide was administered to adult male Sprague–Dawley rats in a cyclic manner for several weeks. Treatment was followed by training with different types of eyeblink classical conditioning, a form of associative learning. Chemotherapy reduced both neurogenesis and endogenous theta activity, as well as disrupted learning and related theta‐band responses to the conditioned stimulus. The detrimental effects of temozolomide only occurred after several weeks of treatment, and only on a task that requires the association of events across a temporal gap and not during training with temporally overlapping stimuli. Chemotherapy did not disrupt the memory for previously learned associations, a memory independent of (new neurons in) the hippocampus. In conclusion, prolonged systemic chemotherapy is associated with a decrease in hippocampal adult neurogenesis and theta activity that may explain the selective deficits in processes of learning that describe the ‘chemobrain’.     

Modulation of adult hippocampal neurogenesis during myelin-directed autoimmune neuroinflammation

In chronic autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) clinical signs of cognitive dysfunction have been associated with structural changes in the hippocampus. Moreover, experimental studies indicate that inflammatory responses within the CNS modulate the homeostasis of newborn cells in the adult dentate gyrus (DG). However, it remained open whether such changes happen regardless of the primary immunological target or whether a CNS antigen-directed T lymphocyte-mediated autoimmune response may exert a specific impact. We therefore induced experimental autoimmune encephalomyelitis (EAE), a common model of MS serving as a paradigm for a CNS-specific immune response, by immunizing C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG) p35-55. In EAE animals, we found enhanced de novo generation and survival of doublecortin (DCX)-positive immature neurons when compared with controls immunized with CNS-irrelevant antigen (ovalbumine). However, despite activation of neurogenesis, we observed a reduced capacity of these cells to generate mature neurons. Moreover, the high number of newly born cells retained the expression of the glial marker GFAP. These effects were associated with downregulation of pro-neurogenic factors Neurogenin1 and Neurogenin2 and dysregulation of Notch, β-catenin, Sonic Hedgehog (Shh) signaling as suggested by altered gene expression of effector molecules. Thus, a CNS antigen-specific immune response leads to an aberrant differentiation of neural precursors associated with dysbalance of signaling pathways relevant for adult hippocampal neurogenesis. These results may further extend our understanding of disturbed regeneration in the course of chronic inflammatory CNS diseases such as MS.     

Cells expressing markers of immature neurons in the amygdala of adult humans

The polysialylated form of the neuronal cell adhesion molecule (PSA‐NCAM) is expressed by immature neurons in the amygdala of adult mammals, including non‐human primates. In a recent report we have also described the presence of PSA‐NCAM‐expressing cells in the amygdala of adult humans. Although many of these cells have been classified as mature interneurons, some of them lacked mature neuronal markers, suggesting the presence of immature neurons. We have studied, using immunohistochemistry, the existence and distribution of these immature neurons using post mortem material. We have also analysed the presence of proliferating cells and the association between immature neurons and specialised astrocytes. These parameters have also been studied for comparative purposes in the amygdalae of cats and squirrel monkeys. Our results demonstrate that cells coexpressing doublecortin and PSA‐NCAM, but lacking neuronal nuclear antigen expression, were present in the amygdala of adult humans. These cells were organised in elongated clusters, which were located between the white matter of the dorsal hippocampus and the basolateral amygdaloid nucleus. These clusters were not associated with astroglial specialised structures. No cells expressing the proliferative marker Ki67 were observed in the amygdaloid parenchyma, although some of them were found in the vicinity of the lateral ventricle. Immature neurons were also present in the amygdala of squirrel monkeys and cats. These cells also appeared clustered in monkeys, although not as organised as in humans. In cats these cells are scarce, appear isolated and most of the PSA‐NCAM‐expressing structures corresponded to processes apparently originating from the paleocortical layer II.     

Doublecortin expression in the adult rat telencephalon

Doublecortin (DCX) is a protein required for normal neuronal migration in the developing cerebral cortex, where it is widely expressed in both radially and tangentially migrating neuroblasts. Moreover, it has been observed in the adult rostral migratory stream, which contains the neuronal precursors traveling to the olfactory bulb. We have performed DCX immunocytochemistry in the adult rat brain to identify precisely the neuronal populations expressing this protein. Our observations confirm the presence of DCX immunoreactive cells with the characteristic morphology of migrating neuroblasts in the subventricular zone, rostral migratory stream and the main and accessory olfactory bulbs. We have also found putative migratory cells expressing DCX in regions were no adult neuronal migration has been described, as the corpus callosum, the piriform cortex layer III/endopiriform nucleus and the striatum. Surprisingly, many cells with the phenotype of differentiated neurons were DCX immunoreactive; e.g. certain granule neurons in the hilar border of the granular layer of the dentate gyrus, some neuronal types in the piriform cortex layer II, granule and periglomerular neurons in the main and accessory olfactory bulbs, and isolated cells in the striatum. Almost all DCX immunoreactive cells also express the polysialylated form of neural cell adhesion molecule and have a similar distribution to rat collapsin receptor-mediated protein-4, two molecules involved in neuronal structural plasticity. Given these results, we hypothesize that DCX expression in differentiated neurons could be related to its capacity for microtubule reorganization and that this fact could be linked to axonal outgrowth or synaptogenesis.     

AAV-mediated delivery of BDNF augments neurogenesis in the normal and quinolinic acid-lesioned adult rat brain

Brain-derived neurotrophic factor (BDNF) plays a major role in regulating the survival and fate of progenitor cells in the adult brain. In order to extend previous observations in the normal adult brain and advance our knowledge regarding the effect of BDNF on neurogenesis in the injured brain, this study directly compared the effect of BDNF on basal and injury-induced neurogenesis in relation to progenitor cell distribution and levels of neuronal differentiation and survival. BDNF was overexpressed in the subventricular zone (SVZ) via recombinant adeno-associated virus (AAV(1/2)) delivery, and newly generated cells were identified using bromodeoxyuridine (BrdU) labelling. Selective striatal cell loss was induced in a subgroup of rats by unilateral striatal injection of quinolinic acid (QA) 21 days after AAV(1/2) injection. In the normal brain, BDNF overexpression significantly increased BrdU-positive cell numbers in the rostral migratory stream, indicating enhanced progenitor cell migration. Following QA lesioning, we observed a reduction in BrdU immunoreactivity in the SVZ. Overexpression of BDNF restored BrdU-positive cell numbers in the QA-lesioned SVZ to that observed in the normal brain. Most significantly, BDNF enhanced the recruitment of progenitor cells to the QA-lesioned striatum and promoted neuronal differentiation in both the normal and QA-lesioned striatum. Our findings indicate that BDNF augments the recruitment, neuronal differentiation and survival of progenitor cells in both neurogenic and non-neurogenic regions of the normal or QA-lesioned brain. Enhanced expression of BDNF may therefore be a viable strategy for augmenting neurogenesis from endogenous progenitor cells.     

Cell population dynamics in the course of adult hippocampal neurogenesis: Remaining unknowns

Neural stem cells (NSCs) generate new neurons throughout life in the mammalian hippocampus. The distinct developmental steps in the course of adult neurogenesis, including NSC activation, expansion, and neuronal integration, are increasingly well characterized down to the molecular level. However, substantial gaps remain in our knowledge about regulators and mechanisms involved in this biological process. This review highlights three long-standing unknowns. First, we discuss potency and identity of NSCs and the quest for a unifying model of short- and long-term self-renewal dynamics. Next, we examine cell death, specifically focusing on the early demise of newborn cells. Then, we outline the current knowledge on cell integration dynamics, discussing which (if any) neurons are replaced by newly added neurons in the hippocampal circuits. For each of these unknowns, we summarize the trajectory of studies leading to the current state of knowledge. Finally, we offer suggestions on how to fill the remaining gaps by taking advantage of novel technology to reveal currently hidden secrets in the course of adult hippocampal neurogenesis.     

Endogenous adult neurogenesis and cognitive function recovery following traumatic brain injury in the rat hippocampus

BACKGROUND:Endogenous neural progenitor cells play a beneficial role for cognitive recovery following traumatic brain injury.However,there are few classification-control studies aimed at varying graded brain trauma.OBJECTIVE:To observe the effects of adult endogenous neurogenesis on cognitive function repair and regeneration of neural progenitor cells following varying graded traumatic hippocampal injury to determine the significance of endogenous neurogenesis in the repair of brain injury.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Key Laboratory of Injuries,Variations and Regeneration of Nervous System,Tianjin Medical University General Hospital,from February to October 2009.MATERIALS:Mouse anti-rat 5-bromodeoxyuridine (BrdU) and neuronal nuclei (NeuN) monoclonal antibodies were purchased from Millipore Corporation,USA.METHODS:A total of 45 Wistar rats were randomly assigned to three groups.Mild and severe injury groups were respectively subjected to (182 ± 2) kPa and (284 ± 4) kPa lateral fluid percussion to establish models of brain injury,and the control group was subjected to surgery with no lateral fluid percussion.MAIN OUTCOME MEASURES:Cognitive function was estimated using the Morris water maze.Proliferation,survival,and differentiation of newly generated cells in the injured hippocampus were observed through the use of immunofluorescent staining.RESULTS:At 7 days post-injury,the number of BrdU+ cells in the hippocampal dentate gyrus significantly increased in the mild and severe injury groups compared with the control group (P<0.01).At 61 days post-injury,the number of BrdU7NeuN+ cells in the hippocampal dentate gyrus was significantly greater in the mild injury group compared with the severe injury and control groups (P< 0.01).In addition,the control group exhibited the greatest proportion of surviving cells that differentiated into mature neurons compared with the injury groups (P< 0.01).Moreover,at 61 days post-injury,cognitive function in rats with mild injury recovered to normal levels,whereas the severe injury group exhibited cognitive deficits (P< 0.01).CONCLUSION:Traumatic brain injury may be a stimulation factor for proliferation of neural progenitor cells in the adult hippocampus but severe brain trauma does not lead to an increased number of newly generated cells.Endogenous adult neurogenesis repairs neurological functions to an extent.However,recovery of neurological function remains limited following severe traumatic brain injury.     

Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain

In the adult nervous system, multipotential stem cells of the subventricular zone of the lateral ventricles generate neuron precursors (type-A cells) that migrate via the rostral migratory stream to the olfactory bulb where they differentiate into neurons. The migrating neuroblasts are surrounded by a sheath of astrocytes (type-B cells). Using immunostaining, in situ hybridization and enzyme histochemistry, we demonstrate that the ecto-ATPase nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is expressed in the subventricular zone and the rostral migratory stream of the adult rat brain. This enzyme hydrolyses extracellular nucleoside triphosphates to the respective nucleoside diphosphates and is thought to directly modulate ATP receptor-mediated cell communication. Double labelling for the astrocyte intermediate filament protein GFAP and the glial glutamate transporter GLAST identifies the NTPDase2-positive cells as type-B cells. During development the enzyme protein is first detected at E18, long before expression of the astrocyte marker GFAP. It gradually becomes expressed along the ventricular and subventricular zone of the brain, followed by complete retraction to the adult expression pattern at P21. NTPDase2 is transiently expressed in the outer molecular layer of the dentate gyrus and within the cerebellar white matter and is associated with select microvessels, tanycytes of the third ventricle, and subpial astrocytes of the adult brain. Our results suggest that NTPDase2 can serve as a novel marker for specifying subsets of cells during in vivo and in vitro studies of neural development and raise the possibility that ATP-mediated signalling pathways play a role in neural development and differentiation.     

Expression of embryonic tau protein isoforms persist during adult neurogenesis in the hippocampus

Tau is a microtubule-associated protein with a developmentally regulated expression of multiple isoforms. The neonatal isoform is devoid of two amino terminal inserts and contains only three instead of four microtubule-binding repeats (0N/3R-tau). We investigated the temporal expression pattern of 0N-tau and 3R-tau in the rat hippocampus. After the decline of 0N- and 3R-tau immunoreactivity during the postnatal development both isoforms remain highly expressed in a few cells residing beneath the granule cell layer. Coexpression of the polysialylated neuronal cell adhesion molecule, doublecortin, and incorporated bromodeoxyuridine showed that these cells are proliferating progenitor cells. In contrast mature granule cells express the adult tau protein isoform containing one aminoterminal insert domain (1N-tau). Therefore a shift in tau isoform expression takes place during adult neurogenesis, which might be related to migration, differentiation, and integration in the granule cell layer. A model for studying shifts in tau isoform expression in a defined subset of neurons might help to understand the etiology of tauopathies, when isoform composition is crucial for neurodegeneration, as in Pick's disease or FTDP-17.     

Decline in adult neurogenesis during aging follows a topographic pattern in the mouse hippocampus

In the rodent brain, diverse functions are topographically distributed within the hippocampus. For instance, the dorsal (septal) hippocampus is involved in spatial memory, whereas the ventral (temporal) hippocampus is related to emotion and anxiety. Accumulating evidence shows that age-dependent decline in hippocampal neurogenesis is associated with impairments of these functions. However, little is known about whether the decline in dentate granule cell production during aging follows a topographic pattern. Here we quantitatively estimated specific populations of adult-born cells in young adult and middle-aged mice by using endogenous markers and determined whether age-dependent reductions in adult neurogenesis exhibited topographic differences. The numerical densities (NDs) of putative primary progenitors, intermediate neuronal progenitors, and neuronal lineages were higher in the dorsal dentate gyrus (DG) than in the ventral DG both in young adult and in middle-aged mice, but the ratios of the NDs in the dorsal DG to the NDs in the ventral DG noticeably increased with age. The age-related reductions in the numbers of these populations were larger in the ventral DG than in the dorsal DG. By contrast, the NDs of glial lineages were higher in the ventral DG than in the dorsal DG during life, and the numbers of glial lineages showed no significant age-related changes. Our findings suggest that neurogenesis, but not gliogenesis, wanes faster in the ventral hippocampus than in the dorsal hippocampus during aging. Such age-related topographic changes in hippocampal neurogenesis might be implicated in memory and affective impairments in older people.     

Proliferation, migration and differentiation of neuronal progenitor cells in the adult mouse subventricular zone surgically separated from its olfactory bulb

The subventricular zone of the adult mammalian forebrain contains progenitor cells that, by migrating along a restricted pathway called the ‘rostral migratory stream’ (RMS), add new neurons to the olfactory bulb throughout life. To determine the influence of the olfactory bulb on the development of these progenitor cells, we performed lesions that interrupt this pathway and separate the olfactory bulb from the rest of the forebrain. By labelling cells born at several survival times after the lesions with the thymidine analogue bromodeoxyuridine (BrdU), we found that disconnection from the bulb influences the rate of BrdU incorporation by the progenitor cells. The number of labelled cells in lesioned mice was almost half that found in control mice. In the disconnected migratory pathway, the number of neurons expressing calretinin was increased indicating that neuronal differentiation was enhanced: newly born neurons occurred within and around the RMS, most of them expressed calretinin and left the pathway starting about 2 weeks after the lesion. Thereafter, these neurons preserving their phenotype, spread for long distances, and accumulated ectopically in dorsal regions of the anterior olfactory nucleus and the frontal cortex. Finally, transplantation of adult subventricular cells into the lesioned pathway showed that the lesion neither prevents neuronal migration nor alters its direction. Thus, although the olfactory bulb appears to regulate the pace of the developmental processes, its disconnection does not prevent the proliferation, migration and phenotypic acquisition of newly generated bulbar interneurons that, since they cannot reach their terminal domains, populate some precise regions of the lesioned adult forebrain.     

Thyroid hormone regulation of N-methyl-D-aspartic acid receptor subunit mRNA expression in adult brain

Thyroid hormone is an essential modulator of brain development, but little is known about its actions in the adult brain. Hypothyroidism is associated with gene expression changes in both central and peripheral nervous tissue. Functional consequences of adult-onset hypothyroidism include an inability to produce long-term potentiation in rat hippocampus and impaired learning and memory in both rats and man. Long-term potentiation is a form of learning that is dependent on functional N-methyl-d-aspartic acid (NMDA)-preferring ionotropic glutamate receptors. This work examines the expression of ionotropic glutamate receptor subunit mRNA following surgical thyroidectomy with or without thyroid hormone replacement. In situ hybridization histochemistry was used to determine the mRNA levels of the NMDA receptor subunits NR1, NR2A, NR2B, the AMPA receptor subunit GluR1, and the kainate receptor subunit KA2. Reducing circulating concentrations of thyroid hormone by surgical removal of the thyroid gland 2 weeks before sacrifice decreased the expression of NR1 mRNA exclusively in the hippocampus. Conversely, hyperthyroidism selectively reduced NR2B mRNA expression in the dorsal hippocampus. Altering thyroid hormone status had no effect on the expression of KA2 or GluR1 subunit mRNA. The regulation of expression of NR1 and NR2B mRNA by thyroid hormone is a novel mechanism for explaining the relationship between thyroid hormone and cognitive function.     

经尾静脉注入大鼠骨髓间充质多能成体祖细胞向脑缺血灶微环境中的移行及效应

BACKGROUND： Multipotent adult progenitor cells （MAPCs） from the bone marrow have been shown to differentiate into neurons.
OBJECTIVE： To observe migration, survival, and neuronal-like differentiation of MAPCs by tail vein injection. DESIGN, TIME AND SETTING： Randomized, controlled experiment of neural tissue engineering was performed at the Laboratory for Cardio-Cerebrovascular Disease, Hospital of Integrated Traditional and Western Medicine, Tongji Medical College of Huazhong University of Science and Technology between September 2006 and August 2007. MATERIALS： Eighty Sprague Dawley rats, 3-6 months old, underwent cerebral ischemia/reperfusion by thread technique, and were randomly divided into model and MAPCs groups （n = 40）. METHODS： Mononuclear cells were harvested from bone marrow using the FicolI-Paque density gradient centrifugation method. After removing CD45 and glycophorin A-positive cells （GLYA＋） with immunomagnetic beads, CD45 GLYA adult progenitor cells were labeled with bromodeoxyuridine （5-bromo-2-deoxyuridine, BrdU）. A total of 1 mL cell suspension, containing 5 × 10^6 MAPCs, was injected into the MAPCs group through the tail vein. A total of 1 mL normal saline was injected into the model rats.
MAIN OUTCOME MEASURES： After 60 days, BrdU and neuron-specific enolase double-positive cells were observed using immunofluorescence. Cell morphology was observed under electron microscopy, and nerve growth factor mRNA was measured through RT-PCR. In addition, rat neurological functions were measured with behavioral tests.
RESULTS： Immunofluorescence revealed that MAPCs positive for BrdU and neuron specific enolase were found surrounding the ischemic focus in the MAPCs group. Microscopic observation suggested that MAPCs-derived neuronal-like cells connected with other nerve cells to form synapses. Compared with the model animals, the level of nerve growth factor mRNA was significantly upregulated in rats injected with MAPCs （P 〈 0.05）. In addition, rats in the MAPCs group performed better in behavioral tests than the model group on days 28 and 60 （P 〈 0.05）.
CONCLUSION： Transplanted MAPCs migrated to the ischemic region, survived, and differentiated into neuronal-like cells, resulting in stimulation of nerve growth factor mRNA and improved neurological function in ischemic rats.     

HVC interneurons are not renewed in adult male zebra finches

Adult neurogenesis is a widespread phenomenon in many species, from invertebrates to humans. In songbirds, the telencephalic region, high vocal center (HVC), continuously integrates new neurons in adulthood. This nucleus consists of a heterogenous population of inhibitory interneurons (HVC(IN)) and two populations of projection neurons that send axons towards either the robust nucleus of the arcopallium (HVC(RA)) or the striatal nucleus area X (HVC(X)). New HVC neurons were initially inferred to be interneurons, because they lacked retrograde labelling from the HVC's targets. Later studies using different tracers demonstrated that HVC(RA) are replaced but HVC(X) are not. Whether interneurons are also renewed became an open question. As the HVC's neuronal populations display different physiological properties and functions, we asked whether adult HVC indeed recruits two neuronal populations or whether only the HVC(RA) undergo renewal in adult male zebra finches. We show that one month after being born in the lateral ventricle, 42% of the newborn HVC neurons were retrogradely labelled by tracer injections into the RA. However, the remaining 58% were not immunoreactive for the neurotransmitter GABA, nor for the calcium-binding proteins, parvalbumin (PA), calbindin (CB) and calretinin (CR) that characterize different classes of HVC(IN). We further established that simultaneous application of parvalbumin, calbindin and calretinin antibodies to HVC revealed approximately the same fraction of HVC neurons, i.e. 10%, as could be detected by GABA immunoreactivity. This implies that the sum of HVC(IN) expressing the different calcium-binding proteins constitute all inhibitory HVC(IN). Together these results strongly suggest that only HVC(RA) are recruited into the adult HVC.