Specific Interneuron Subpopulations Research Articles

Somatostatin interneurons recruit pre- and postsynaptic GABAB receptors in the adult mouse dentate gyrus.

The integration of spatial information in the mammalian dentate gyrus (DG) is critical to navigation. Indeed, DG granule cells (DGCs) rely upon finely balanced inhibitory neurotransmission in order to respond appropriately to specific spatial inputs. This inhibition arises from a heterogeneous population of local GABAergic interneurons (INs) that activate both fast, ionotropic GABAA receptors (GABAAR) and slow, metabotropic GABAB receptors (GABABR), respectively. GABABRs in turn inhibit pre- and post-synaptic neuronal compartments via temporally long-lasting G-protein dependent mechanisms.The relative contribution of each IN subtype to network level GABABR signal setting remains unknown. However, within the DG, the somatostatin (SSt) expressing IN subtype is considered crucial in coordinating appropriate feedback inhibition on to DGCs. Therefore, we virally delivered channelrhodopsin-2 to the DG in order to obtain control of this specific SSt IN subpopulation in male and female adult mice.Using a combination of optogenetic activation and pharmacology, we show that SSt INs strongly recruit postsynaptic GABABRs to drive greater inhibition in DGCs than GABAARs at physiological membrane potentials. Furthermore, we show that in the adult mouse DG, postsynaptic GABABR signalling is predominantly regulated by neuronal GABA uptake, and less so by astrocytic mechanisms. Finally, we confirm that activation of SSt INs can also recruit presynaptic GABABRs, as has been shown in neocortical circuits. Together, these data reveal that GABABR signalling allows SSt INs to control DG activity and may constitute a key mechanism for gating spatial information flow within hippocampal circuits.Significance statement GABAergic interneurons provide powerful inhibition to cortical circuits, by directly regulating the activity of other neurons through metabotropic GABAB receptors. While much is known about the basic properties of GABAB receptor signalling, current knowledge of the relative contribution made by specific interneuron subpopulations to this inhibitory neurotransmission mechanism is less well understood. Our results help address this knowledge gap by showing that the somatostatin expressing interneuron subpopulation provide powerful GABAB receptor-mediated feedback inhibition in the mouse dentate gyrus. Furthermore, GABAB receptor activation in dentate gyrus was found to be tightly regulated by neuronal GABA uptake, and not astrocytes, thus providing self-regulated feedback inhibition. Together, these data provide novel insights into cell-type specific GABAB receptor-mediated control of dentate gyrus circuitry.

Impaired GABAergic regulation and developmental immaturity in interneurons derived from the medial ganglionic eminence in the tuberous sclerosis complex

GABAergic interneurons play a critical role in maintaining neural circuit balance, excitation–inhibition regulation, and cognitive function modulation. In tuberous sclerosis complex (TSC), GABAergic neuron dysfunction contributes to disrupted network activity and associated neurological symptoms, assumingly in a cell type-specific manner. This GABAergic centric study focuses on identifying specific interneuron subpopulations within TSC, emphasizing the unique characteristics of medial ganglionic eminence (MGE)- and caudal ganglionic eminence (CGE)-derived interneurons. Using single-nuclei RNA sequencing in TSC patient material, we identify somatostatin-expressing (SST+) interneurons as a unique and immature subpopulation in TSC. The disrupted maturation of SST+ interneurons may undergo an incomplete switch from excitatory to inhibitory GABAergic signaling during development, resulting in reduced inhibitory properties. Notably, this study reveals markers of immaturity specifically in SST+ interneurons, including an abnormal NKCC1/KCC2 ratio, indicating an imbalance in chloride homeostasis crucial for the postsynaptic consequences of GABAergic signaling as well as the downregulation of GABAA receptor subunits, GABRA1, and upregulation of GABRA2. Further exploration of SST+ interneurons revealed altered localization patterns of SST+ interneurons in TSC brain tissue, concentrated in deeper cortical layers, possibly linked to cortical dyslamination. In the epilepsy context, our research underscores the diverse cell type-specific roles of GABAergic interneurons in shaping seizures, advocating for precise therapeutic considerations. Moreover, this study illuminates the potential contribution of SST+ interneurons to TSC pathophysiology, offering insights for targeted therapeutic interventions.

Impact of stress on inhibitory neuronal circuits, our tribute to Bruce McEwen

This manuscript is dedicated to the memory of Bruce S. McEwen, to commemorate the impact he had on how we understand stress and neuronal plasticity, and the profound influence he exerted on our scientific careers. The focus of this review is the impact of stressors on inhibitory circuits, particularly those of the limbic system, but we also consider other regions affected by these adverse experiences. We revise the effects of acute and chronic stress during different stages of development and lifespan, taking into account the influence of the sex of the animals. We review first the influence of stress on the physiology of inhibitory neurons and on the expression of molecules related directly to GABAergic neurotransmission, and then focus on specific interneuron subpopulations, particularly on parvalbumin and somatostatin expressing cells. Then we analyze the effects of stress on molecules and structures related to the plasticity of inhibitory neurons: the polysialylated form of the neural cell adhesion molecule and perineuronal nets. Finally, we review the potential of antidepressants or environmental manipulations to revert the effects of stress on inhibitory circuits.

A possible novel mechanism for cognitive resistance to neurodegeneration: modulation of interneuron activity in the hippocampus after exposure to enriched environments

AbstractBackgroundExposure to an enriched environment has been shown in many studies to be beneficial to brain structure and cognition by preserving neuronal integrity and strengthening the functioning and plasticity of neural circuits. These benefits derive from the added spatial, social, and sensory complexities in an enriched environment. It has been well known for a few decades that an enriched environment stimulates neurogenesis and cell proliferation in the dentate gyrus of the hippocampus, leading to an increased number of dentate granule cells (DGCs). However, the changes to other populations of cell types, particularly interneurons, has remained elusive. Interneurons are integral regulators of neurotransmission in the hippocampus. Damage to hippocampal interneurons have serious implications and lead to a decline in cognitive health. Our study sought to elucidate possible changes in interneurons affected by exposure to an enriched environment (EE) and enriched changing (EC) environment.MethodThe topographic distribution of nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPHd) positive neurons was studied in the dentate gyrus of the rat dorsal hippocampus.ResultEnvironmental enrichment increased the number of NADPHd positive neurons found in both enrichment groups. The elevated level of neurons was represented uniformly across almost all six layers of the dentate gyrus and was most significant in the expected granular cell layer (GRCL) and subgranular zone (SGZ).ConclusionThe overall preliminary results suggest that the brain has the ability to adapt to increased amounts of sensory stimulation. These changes highlight a possible new mechanism of physiological homeostasis and physical evidence of a cognitive reserve; with an increased demand for energy from new‐born DGCs and increased sensory input, the brain will regulate its energy expenditure via an increase in either the activity of existing interneurons, or the actual number of interneurons. Further research will involve running a restudy and observing the differences to the ventral hippocampus, CA1, and CA3 regions, and the effects on specific subpopulations of interneurons.

Opposed hemodynamic responses following increased excitation and parvalbumin-based inhibition

Understanding cellular contributions to hemodynamic activity is essential for interpreting blood-based brain mapping signals. Optogenetic studies examining cell-specific influences on local hemodynamics have reported that excitatory activity results in cerebral perfusion and blood volume increase, while inhibitory activity contributes to both vasodilation and vasoconstriction. How specific subpopulations of interneurons regulate the brain’s blood supply is less examined. Parvalbumin interneurons are the largest subpopulation of GABAergic neurons in the brain, critical for brain development, plasticity, and long-distance excitatory neurotransmission. Despite their essential role in brain function, the contribution of parvalbumin neurons to neurovascular coupling has been relatively unexamined. Using optical intrinsic signal imaging and laser speckle contrast imaging, we photostimulated awake and anesthetized transgenic mice expressing channelrhodopsin under a parvalbumin promoter. Increased parvalbumin activity reduced local oxygenation, cerebral blood volume, and cerebral blood flow. These “negative” hemodynamic responses were consistent within and across mice and reproducible across a broad range of photostimulus parameters. However, the sign and magnitude of the hemodynamic response resulting from increased parvalbumin activity depended on the type and level of anesthesia used. Opposed hemodynamic responses following increased excitation or parvalbumin-based inhibition suggest unique contributions from different cell populations to neurovascular coupling.

Region-specific involvement of interneuron subpopulations in trauma-related pathology and resilience

Only a minority of trauma-exposed individuals develops Posttraumatic stress disorder (PTSD) and active processes may support trauma resilience. Individual behavioral profiling allows investigating neurobiological alterations related to resilience or pathology in animal models of PTSD and is utilized here to examine the activation of different interneuron subpopulations of the dentate gyrus-amygdala system associated with trauma resilience or pathology.To model PTSD, rats were exposed to juvenile stress combined with underwater trauma (UWT) in adulthood. Four weeks later, individual anxiety levels were assessed in the elevated plus maze test for classifying rats as highly anxious ‘affected’ vs. ‘non-affected’, i.e. behaving as control animals. Analyzing the activation of specific interneuron subpopulations in the dorsal and ventral dentate gyrus (DG), the basolateral (BLA) and central amygdala by immunohistochemical double-labeling for cFos and different interneuron markers, revealed an increased activation of cholecystokinin (CCK)-positive interneurons in the ventral DG, together with increased activation of parvalbumin- and CCK-positive interneurons in the BLA of affected trauma-exposed rats. By contrast, increased activation of neuropeptide Y (NPY)-positive interneurons was observed in the dorsal DG of trauma-exposed, but non-affected rats. To test for a direct contribution of NPY in the dorsal DG to trauma resilience, a local shRNA-mediated knock down was performed after UWT. Such a treatment significantly reduced the prevalence of resilient animals.Our results suggest that distinct interneuron populations are associated with resilience or pathology in PTSD with high regional specificity. NPY within the dorsal DG was found to significantly contribute to trauma resilience.

M181. DEVELOPMENTAL PROGRESSION OF INTERNEURON NETWORK DEFICITS IN A 15Q13.3 MICRODELETION MOUSE MODEL – A GLIMPSE ON ADOLESCENT PRIMING FOR SCHIZOPHRENIA?

BackgroundSchizophrenia is a complex neurodevelopmental disorder. Patients typically start exhibiting symptoms during adolescence, coinciding with a critical period for the maturation of the prefrontal cortex. While previous studies have identified deficits in cortical interneuron integrity and network function in chronic patients, little is known about the maladaptive circuitry in the early prodromal phase of the disease. To assess pathophysiological changes during adolescence that might contribute to the disruption of cortical network function we have studied a 15q13.3 microdeletion mouse model Df[h15q13]−/+ resembling a human copy number variant (CNV) known to confer high risk for psychiatric disorders such as schizophrenia. Using a combination of histology, in vitro electrophysiology and electroencephalography (EEG) we explored the interneuronal connectivity and cortical network functionality in the Df[h15q13]−/+ mouse model from adolescence to early adulthoodMethodsImmunohistological analysis was performed on brain slices within the prefrontal cortex, dorsal hippocampus and amygdala region from Df[h15q13]−/+ and wild-type mice (N=8) at PND35 and PND70 (4 sections/brain). Sections were immunostained for markers of interneuron subtypes and respective synapses. Fluorescence images were recorded and processed with an Opera Phenix (PerkinElmer) using the 63x objective in confocal mode. EEG studies were performed on Df[h15q13]−/+ and wild-type mice within the age range of PND41 to PND70 (6). Mice were obtained from Taconic and housed within the experimental facility for at least one week prior to experimental procedures.ResultsWe initially confirmed that the adult Df[h15q13]−/+ microdeletion mouse model exhibits robust markers reminiscent of schizophrenia-linked pathology, such as the reduction of parvalbumin positive (PV+) interneurons, lower abundance of perineuronal net proteins (PNNs) and an impaired cortical processing of sensory information. We identified abnormalities in the number and distribution of interneuron synapses in the prefrontal cortex, hippocampus and amygdala, the phenotype in the adolescent brain, which were opposed to pathophysiological changes identified in adult Df[h15q13]−/+ microdeletion mice. We discovered an enhanced inhibitory drive from specific subpopulations of interneurons during adolescence that might contribute to deficits in the adult hippocampal and PFC network. Likewise, we found Df[h15q13]−/+ specific differences in cortical network processing between adolescent and adult mice revealed by EEG. To align the development of cortical network function to the progressive changes in network structure we performed longitudinal EEG recordings and uncovered particular abnormalities in basal and evoked oscillatory rhythms in adolescent and adult mice.DiscussionIn this study, we discovered abnormalities in the interneuron integration during a critical period for the maturation of the prefrontal cortex in a 15q13.3 microdeletion mouse model. Our findings provide novel insights into early deficits in the limbic and cortical neuronal networks that may drive circuit dysfunction in schizophrenia patients. Identification of adolescent pathophysiology in models for schizophrenia risk will provide the opportunity to explore new mechanisms for early intervention.

Optogenetic dissection of roles of specific cortical interneuron subtypes in GABAergic network synchronization.

An increase in the excitability of GABAergic cells has typically been assumed to decrease network activity, potentially producing overall anti-epileptic effects. Recent data suggest that inhibitory networks may actually play a role in initiating epileptiform activity. We show that activation of GABAergic interneurons can elicit synchronous long-lasting network activity. Specific interneuron subpopulations differentially contributed to GABA network synchrony, indicating cell type-specific contributions of interneurons to cortical network activity. Interneurons may critically contribute to the generation of aberrant network activity characteristic of epilepsy, warranting further investigation into the contribution of distinct cortical interneuron subpopulations to the propagation and rhythmicity of epileptiform activity. In the presence of the A-type K+ channel blocker 4-aminopyrdine, spontaneous synchronous network activity develops in the neocortex of mice of either sex. This aberrant synchrony persists in the presence of excitatory amino acid receptor antagonists (EAA blockers) and is considered to arise from synchronous firing of cortical interneurons (INs). Although much attention has been given to the mechanisms underlying this GABAergic synchrony, the contribution of specific IN subtypes to the generation of these long-lasting discharges (LLDs) is incompletely understood. We employed genetically-encoded channelrhodopsin and archaerhodopsin opsins to investigate the sufficiency and necessity, respectively, of activation of parvalbumin (PV), somatostatin (SST) and vasointestinal peptide (VIP)-expressing INs for the generation of synchronous neocortical GABAergic discharges. We found light-induced activation of PV or SST INs to be equally sufficient for the generation of LLDs, whereas activation of VIP INs was not. By contrast, light-induced inhibition of PV INs strongly reduced LLD initiation, whereas suppression of SST or VIP IN activity only partially attenuated LLD magnitude. These results suggest neocortical INs perform cell type-specific roles in the generation of aberrant GABAergic cortical network activity.

Early Development of Parvalbumin-, Somatostatin-, and Cholecystokinin-Expressing Neurons in Rat Brain following Prenatal Immune Activation and Maternal Iron Deficiency

Prenatal maternal infection and maternal iron deficiency during pregnancy are 2 early environmental insults associated with increased risk for schizophrenia in offspring. Substantial evidence suggests that abnormalities in inhibitory γ-aminobutyric acid (GABA) interneuron function, especially in the parvalbumin subtype of GABA interneuron, both developmentally and in adulthood, may contribute mechanistically to cognitive deficits and psychotic symptoms in schizophrenia. This study used a rat model to test whether prenatal immune activation with lipopolysaccharide (LPS; at gestation days, GD, 15 and 16) or maternal iron deficiency (from GD2 to postnatal day P7) or the combination of both insults alters major subtypes of GABAergic interneurons (parvalbumin, somatostatin, cholecystokinin) in brain regions relevant to schizophrenia (medial and dorsolateral prefrontal cortex [PFC], hippocampal CA1 and dentate gyrus, ventral subiculum) in offspring at P14 or P28. Prenatal LPS treatment significantly increased the density of parvalbumin-immunoreactive neurons at P14 in the medial PFC, dorsolateral PFC, and ventral subiculum of offspring born from iron-sufficient but not iron-deficient dams. Prenatal LPS also increased cholecystokinin neuron density in the medial PFC at P28, under both iron-sufficient and iron-deficient conditions. We observed a large increase in parvalbumin neuron density from P14 to P28 in the medial PFC and subiculum across all birth groups, that was not observed in other brain regions, and significant decreases in somatostatin neuron density from P14 to P28 in all brain regions examined across all birth groups, indicating differential developmental trajectories for parvalbumin and somatostatin neurons in various brain regions during this early postnatal period. Thus, it appears that the medial PFC and ventral subiculum, brain regions involved in circuitry modulating ventral tegmental dopamine and nucleus accumbens activities, may be regions vulnerable to effects of prenatal LPS on specific subpopulations of interneurons. It is known that the timing of maturation and expansion of parvalbumin neurons in early development provides threshold levels of inhibition that trigger critical periods for cortical plasticity, leading to long-term circuit consolidation. Thus, our finding of increased parvalbumin neuron density at early developmental times might suggest a mechanism by which an acute prenatal insult like LPS exposure could produce long-term changes in prefrontal cortical or subicular function.

ErbB4 in Laminated Brain Structures: A Neurodevelopmental Approach to Schizophrenia.

The susceptibility genes for schizophrenia Neuregulin-1 (NRG1) and ErbB4 have critical functions during brain development and in the adult. Alterations in the ErbB4 signaling pathway cause a variety of neurodevelopmental defects including deficiencies in neuronal migration, synaptic plasticity, and myelination. I have used the ErbB4-/- HER4heart KO mice to study the neurodevelopmental insults associated to deficiencies in the NRG1-ErbB4 signaling pathway and their potential implication with brain disorders such as schizophrenia, a chronic psychiatric disease affecting 1% of the population worldwide. ErbB4 deletion results in an array of neurodevelopmental deficits that are consistent with a schizophrenic model. First, similar defects appear in multiple brain structures, from the cortex to the cerebellum. Second, these defects affect multiple aspects of brain development, from deficits in neuronal migration to impairments in excitatory/inhibitory systems, including reductions in brain volume, cortical and cerebellar heterotopias, alterations in number and distribution of specific subpopulations of interneurons, deficiencies in the astrocytic and oligodendrocytic lineages, and additional insults in major brain structures. This suggests that alterations in specific neurodevelopmental genes that play similar functions in multiple neuroanatomical structures might account for some of the symptomatology observed in schizophrenic patients, such as defects in cognition. ErbB4 mutation uncovers flaws in brain development that are compatible with a neurodevelopmental model of schizophrenia, and it establishes a comprehensive model to study the basis of the disorder before symptoms are detected in the adult.

Expression of Interneuron Markers in the Dorsolateral Prefrontal Cortex of the Developing Human and in Schizophrenia

The onset of schizophrenia symptoms in late adolescence implies a neurodevelopmental trajectory for the disease. Indeed, the γ-aminobutyric acid (GABA) inhibitory system shows protracted development, and GABA-ergic deficits are widely replicated in postmortem schizophrenia studies. The authors examined expression of several interneuron markers across postnatal human development and in schizophrenia to assess whether protracted development of certain interneuron subpopulations may be associated with a particular vulnerability in schizophrenia. RNA was extracted postmortem from dorsolateral prefrontal cortex of individuals from age 6 weeks to 49 years (N=68) and from a cohort of normal comparison subjects and schizophrenia patients (N=74, 37 pairs). Expression levels of parvalbumin, cholecystokinin, somatostatin, neuropeptide Y, calretinin, calbindin, and vasoactive intestinal peptide were measured by quantitative reverse transcription-polymerase chain reaction. Changes in calretinin protein levels were examined by Western blot. Interneuron marker genes followed one of three general expression profiles: either increasing (parvalbumin, cholecystokinin) or decreasing (somatostatin, calretinin, neuropeptide Y) in expression over postnatal life, with the most dramatic changes seen in the first few years before reaching a plateau; or increasing to peak expression in the toddler years before decreasing (calbindin, vasoactive intestinal peptide). mRNA expression of all genes, with the exception of calbindin (which increased), showed a reduction (8%-31%) in schizophrenia. Somatostatin showed the most dramatic reduction (31%) in schizophrenia. It appears that a heterogeneous population of interneurons is implicated in schizophrenia. Further studies are needed to determine whether specific interneuron subpopulations are altered or whether common or distinct upstream pathways are responsible for interneuron deficits in schizophrenia.

Lamina-Specific Alterations in Cortical GABAA Receptor Subunit Expression in Schizophrenia

Dysfunction of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia is associated with lamina-specific alterations in particular subpopulations of interneurons. In pyramidal cells, postsynaptic γ-aminobutyric acid (GABA(A)) receptors containing different α subunits are inserted preferentially in distinct subcellular locations targeted by inputs from specific interneuron subpopulations. We used in situ hybridization to quantify the laminar expression of α1, α2, α3, and α5 subunit, and of β1-3 subunit, mRNAs in the DLFPC of schizophrenia, and matched normal comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 mRNA expression was 17% lower in layers 3 and 4, α2 expression was 14% higher in layer 2, α5 expression was 15% lower in layer 4, and α3 expression did not differ relative to comparison subjects. The mRNA expression of β2, which preferentially assembles with α1 subunits, was also 20% lower in layers 3 and 4, whereas β1 and β3 mRNA levels were not altered in schizophrenia. These expression differences were not attributable to medication effects or other potential confounds. These findings suggest that GABA neurotransmission in the DLPFC is altered at the postsynaptic level in a receptor subunit- and layer-specific manner in subjects with schizophrenia and support the hypothesis that GABA neurotransmission in this illness is predominantly impaired in certain cortical microcircuits.

ADVANCES IN NEUROIMAGING ALLOW PROSPECTIVE STUDY OF HUMAN BRAIN DEVELOPMENT

The onset of schizophrenia symptoms in late adolescence implies a neurodevelopmental trajectory for the disease. Indeed, the GABAergic inhibitory system shows protracted development and GABAergic deficits are widely replicated in post-mortem schizophrenia studies. We examined expression of several interneuron markers across postnatal human development and in schizophrenia to determine whether protracted development of certain interneuron subpopulations may make them particularly vulnerable in schizophrenia. Method: RNA was extracted from the dorsolateral prefrontal cortex of individuals (n=68) at developmental ages from 6 weeks to 49 years, and from a cohort consisting of controls and schizophrenia subjects (n=37 pairs). Expression levels of parvalbumin, cholecystokinin, somatostatin, neuropeptide Y, calretinin, calbindin, and vasoactive intestinal peptide were measured by quantitative reverse transcriptase-polymerase chain reaction. Western blots were used to examine changes in parvalbumin and calretinin protein levels. Results: Interneuron marker genes followed one of three general expressionprofiles, either increasing (parvalbumin, cholecystokinin) or decreasing (somatostatin, calretinin, neuropeptide Y) in expression over postnatal life with themost dramatic changes seenwithin the first decade of life before reaching a plateau; or a dynamic pattern increasing topeakexpressionat toddler age, thendecreasing (calbindin, vasoactive intestinal peptide). Expressionof all interneuronmarker genes,with the exception of calbindin (which increased), showed a significant reduction (between 8 31%) in schizophrenia. Somatostatin showed the most dramatic reduction (31%) in schizophrenia. Conclusions: Developmental changes in interneuron marker mRNAs extends well into school age suggesting that the inhibitory system takes years to mature in humans. It appears that a developmentally heterogeneous population of interneurons is affected in schizophrenia. Further studies will be required to determine if specific interneuron subpopulations are altered in schizophrenia or if common or distinct upstream pathways are responsible for the interneuron deficits in schizophrenia.

Kv4.3-mediated A-type K+ currents underlie rhythmic activity in hippocampal interneurons.

Hippocampal-dependent learning and memory processes are associated with theta frequency rhythmic activity. Interneuron and pyramidal cell network interactions underlie this activity, but contributions of interneuron voltage-gated membrane conductances remain unclear. We show that interneurons at the CA1 lacunosum-moleculare (LM) and radiatum (RAD) junction (LM/RAD) display voltage-dependent subthreshold membrane potential oscillations (MPOs) generated by voltage-gated tetrodotoxin-sensitive Na+ and 4-aminopyridine (4-AP)-sensitive K+ currents. They also exhibit prominent 4-AP-sensitive A-type K+ currents, with gating properties showing activation at subthreshold membrane potentials. We found that LM/RAD cells are part of specific interneuron subpopulations expressing the K+ channel subunit Kv4.3 and their transfection with Kv4.3 small interfering RNA selectively impaired A-type K+ currents and MPOs. Thus, our findings reveal a novel function of Kv4.3-mediated A-type K+ currents in the generation of intrinsic MPOs in specific subpopulations of interneurons that may participate in hippocampal theta-related rhythmic activity.

Characterization of the Circuits That Generate Spontaneous Episodes of Activity in the Early Embryonic Mouse Spinal Cord

In the developing nervous system, patterned spontaneous activity affects a variety of developmental processes. Thus, it is important to identify the earliest time that such activity occurs and to characterize the underlying circuitry. In isolated mouse spinal cord-limb preparations, highly rhythmic spontaneous activity occurred as early as embryonic day 11 (E11)-E12, when many lumbosacral motoneurons were still migrating and extending their peripheral projections. This activity required both electrical and chemical transmission, and acetylcholine, rather than glutamate, provided the main excitatory drive. Our data are consistent with motoneurons themselves playing a critical role in generating such activity by making excitatory connections on each other and on GABAergic interneurons via dihydro-beta-erythroidine hydrobromide (DHbetaE)-insensitive nicotinic receptors. This resulted in the generation of local bursts. Consistent with these observations, E12-E12.5 mouse motoneurons retrogradely labeled by HRP were observed to have extensive axon collaterals that projected locally within the lateral motor column and to interneuron-containing regions dorsal and medial of the lateral motor column. Cholinergic axons, presumably from motoneurons, were also observed in the ventral and lateral funiculi. However, for local bursts to propagate throughout the cord, a second DHbetaE-sensitive cholinergic pathway that also involved glycinergic interneurons was required. This circuit characterization should facilitate the use of genetic mutations that alter specific subpopulations of interneurons or cholinergic transmission to determine how modifying different aspects of this early activity affects subsequent development of the spinal motor circuit.

Firing Properties of Identified Interneuron Populations in the Mammalian Hindlimb Central Pattern Generator

Little is known about the network structure of the central pattern generator (CPG) controlling locomotor movements in mammals. The present experiments aim at providing such knowledge by focusing on commissural interneurons (CINs) involved in left-right coordination. During NMDA and 5-HT-initiated locomotor-like activity, we recorded intracellularly from caudally or descending projecting L2 and L3 CINs (dCINs) located in the ventromedial area of the lumbar spinal cord in newborn rats. This region is crucial for rhythmic motor output and left-right coordination. The overall sample of dCINs represented a heterogenous population with neurons that fired in all phases of the locomotor cycle and exhibited varying degrees of rhythmicity, from strongly rhythmic to nonrhythmic. Among the rhythmic, putative CPG dCINs were populations that fired in-phase with the ipsilateral or with the contralateral L2 locomotor-like activity. There was a high degree of organization in the dorsoventral location of rhythmic dCINs, with neurons in-phase with the ipsilateral L2 activity located more ventrally. Spikes of rhythmically active dCINs were superimposed on membrane oscillations that were generated predominantly by synaptic input, with little direct contribution from the intrinsic pacemaker hyperpolarization-activated inward current. For both ipsilaterally and contralaterally firing dCINs the dominant synaptic drive was in-phase with the ipsilateral L2 motor activity. This study provides the first characterization of putative CPG interneurons in the mammalian spinal cord. Our results suggest an anatomical and physiological separation of CPG commissural interneurons in the ventral horn and demonstrate that it is possible to target specific interneuron subpopulations in the mammalian locomotor network.

Hippocampal interneurons expressing glutamic acid decarboxylase and calcium‐binding proteins decrease with aging in Fischer 344 rats

Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4–5 months old) and aged (23–25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the hippocampus. In addition, such a profound alteration in interneuron number will likely alter inhibitory control of excitability and neuronal synchrony with behavioral states. J. Comp. Neurol. 394:252–269, 1998. © 1998 Wiley-Liss, Inc.

Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats

Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the hippocampus. In addition, such a profound alteration in interneuron number will likely alter inhibitory control of excitability and neuronal synchrony with behavioral states.

Embryonic cortical neurons differentiate into various types of interneurons when heterotopically transplanted into the adult rat brain

The adult cortex represents a heterogeneous mixture of different classes of pyramidal neurons and non-pyramidal interneurons. After grafting embryonic cortical anlage into the adult striatum, the present study investigated whether the development of different populations of interneurons in heterotopic cortical grafts is similar to the adult cortex. The presence of specific subpopulations of interneurons in grafts was assessed by immunocytochemistry using various antibodies against marker molecules for interneurons such as neuropeptides and calcium-binding proteins. These molecules are expressed to a different extent in specific subpopulations of cortical interneurons. Cortical primordia obtained on embryonic day 14 (1314) were stereotactically grafted into the center of the left striatum of adult recipient rats. After an 8-week differentiation period, host rats were perfusion fixed and immunocytochemistry was performed using antibodies against neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin, parvalbumin and calbindin D-28k. Within the grafts, the number of immunopositive interneurons as well as the intensity of immunostaining for different marker molecules corresponded well with those of the adult cortex. In contrast, the expression pattern in the graft demonstrated clear differences when compared with the surrounding host striatum. The present study demonstrates, that at E14 at least some cells of the cortical anlage are primed to develop into different classes of interneurons independent of their normal environment and their regular synaptic connections. Thus, different interneuron progenitor cells survive transplantation and develop cell-specific morphological and cytochemical characteristics. Differentiation into various subpopulations of neurons may be a prerequisite for potential therapeutic approaches in humans.

Differential effects of serotonin and raphe grafts in the hippocampus and hypothalamus: a combined behavioural and anatomical study in the rat.

Combined with a partial cholinergic deficiency, serotonergic lesions induce severe spatial learning deficits. Serotonergic lesions, however, have additional effects, such as reduced body weight and disruption of thermoregulation, which may be the cause of the observed learning deficits. Restoration of the serotonergic innervation of the hippocampus by raphe grafts reduces these learning deficits. The effects of the grafts may result from a direct support of spatial learning but may also be an indirect result of preventing some of the other effects of serotonergic lesions. In the present study we used raphe grafts to examine the selectivity and specificity of the effects of serotonergic lesions in the rat, and used the behavioural effects as an indication of successful transplantation in order to examine the fine details of such grafts. Raphe grafts in the hippocampus did not prevent the effects of the lesions on body weight, thermoregulation and exploratory behaviour but did minimize the effects of the lesions on spatial learning. In contrast, raphe grafts in the hypothalamus reduced the effects of the lesions on thermoregulation but failed to support learning. The grafted fibres showed termination specificity with the interneurons, which is typical of the serotonergic innervation of the normal hippocampus. The results indicate that the serotonergic innervation of the hippocampus functions locally to support spatial learning. This role of serotonin is independent of its involvement in modulation of body weight, thermoregulation or exploratory behaviour. The results confirm that the modes of serotonergic action in the hippocampus include the selective innervation of specific interneuron subpopulations.