Involvement of presynaptic N-methyl-D-aspartate receptors in cerebellar long-term depression.

At the cerebellar synapses between parallel fibers (PFs) and Purkinje cells (PCs), long-term depression (LTD) of the excitatory synaptic current has been assumed to be independent of the N-methyl-D-aspartate (NMDA) receptor activation because PCs lack NMDA receptors. However, we now report that LTD is suppressed by NMDA receptor antagonists that act on presynaptic NMDA receptors of the PFs. This effect is still observed when the input is restricted to a single fiber. Therefore, LTD does not require the spatial integration of multiple inputs. In contrast, it involves a temporal integration, since reliable LTD induction requires the PFs to fire two action potentials in close succession. This implies that LTD will selectively depress the response to a burst of presynaptic action potentials.     

Evidence for a multiple innervation of purkinje cells by climbing fibers in the immature rat cerebellum

Climbing fiber (CF)–-Purkinje cell (PC) relationships were studied electrophysiologically on the cerebellum of 8 to 15 day old rats. Some animals were rendered agranular by x-irradiation from birth; some others were treated with 3-acetyl pyridine 3 days before study to selectively destroy the CF. Unitary extracellular recordings in 8–9 day old normal rats revealed that more than 50μ of the PC units each exhibited either two types of all-or-none climbing fiber responses (CFR) or stepwise graded CFRs. The other PC units only presented one type of all-or-none CFR. These activities were entirely mediated via CF since they persisted at the same age in x-irradiated rats, but were absent in animals treated with 3-acetyl pyridine. Interaction experiments were performed between juxtafastigial and Inferior Olive stimulations on 49 PC units in 8–9 day old normal rats. Collisions between impulses set up in CFs were disclosed in 21 out of the 24 PCs which exhibited only one type of CFR. In the three others and in each of the 25 PCs that displayed two types of all-or-none CFRs, or CFRs graded by steps, no collision was detected. Moreover intracellular recordings of epsp's mediated via CFs in PCs of 8–9 day old normal rats revealed that they frequently fluctuated in stepwise fashion. These results demonstrate that in the immature rat more than 50μ of PCs are each innervated by at least two distinct CFs; later on, the disappearance of the supernumerary synapses between CF and PC leads, as early as day 15, to the one-to-one relationship between CF and PC.     

Enhancement of both long-term depression induction and optokinetic response adaptation in mice lacking delphilin

In the cerebellum, Delphilin is expressed selectively in Purkinje cells (PCs) and is localized exclusively at parallel fiber (PF) synapses, where it interacts with glutamate receptor (GluR) delta2 that is essential for long-term depression (LTD), motor learning and cerebellar wiring. Delphilin ablation exerted little effect on the synaptic localization of GluRdelta2. There were no detectable abnormalities in cerebellar histology, PC cytology and PC synapse formation in contrast to GluRdelta2 mutant mice. However, LTD induction was facilitated at PF-PC synapses in Delphilin mutant mice. Intracellular Ca(2+) required for the induction of LTD appeared to be reduced in the mutant mice, while Ca(2+) influx through voltage-gated Ca(2+) channels and metabotropic GluR1-mediated slow synaptic response were similar between wild-type and mutant mice. We further showed that the gain-increase adaptation of the optokinetic response (OKR) was enhanced in the mutant mice. These findings are compatible with the idea that LTD induction at PF-PC synapses is a crucial rate-limiting step in OKR gain-increase adaptation, a simple form of motor learning. As exemplified in this study, enhancing synaptic plasticity at a specific synaptic site of a neural network is a useful approach to understanding the roles of multiple plasticity mechanisms at various cerebellar synapses in motor control and learning.     

Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the adult staggerer mutant mouse

Intracellular recordings from Purkinje cells (PC) in the cerebellum of adult staggerer mutant mice revealed that the orthodromic response of PCs to juxtafastigial (JF) stimulation closely resembled a climbing fiber response (CFR). However, for most of the PCs studied, these responses were graded in a stepwise manner when the stimulus strength was increased. The underlying excitatory synaptic potentials (EPSPs) had the typical shape of EPSPs mediated through climbing fibers (CFs), but their size fluctuated in discrete steps, the highest one reaching the firing level. In the same PCs, the size of the spontaneous EPSPs fluctuated in a similar fashion and the frequency of each step was in the range of CF-mediated EPSPs. These results strongly suggest that in staggerer mice several CFs synapse with each PC instead of a single CF as in normal adults. Furthermore, the activation through some of these CFs does not reach the firing level of the corresponding PC.     

Junctophilin-mediated channel crosstalk essential for cerebellar synaptic plasticity

Functional crosstalk between cell-surface and intracellular ion channels plays important roles in excitable cells and is structurally supported by junctophilins (JPs) in muscle cells. Here, we report a novel form of channel crosstalk in cerebellar Purkinje cells (PCs). The generation of slow afterhyperpolarization (sAHP) following complex spikes in PCs required ryanodine receptor (RyR)-mediated Ca(2+)-induced Ca(2+) release and the subsequent opening of small-conductance Ca(2+)-activated K(+) (SK) channels in somatodendritic regions. Despite the normal expression levels of these channels, sAHP was abolished in PCs from mutant mice lacking neural JP subtypes (JP-DKO), and this defect was restored by exogenously expressing JPs or enhancing SK channel activation. The stimulation paradigm for inducing long-term depression (LTD) at parallel fiber-PC synapses adversely established long-term potentiation in the JP-DKO cerebellum, primarily due to the sAHP deficiency. Furthermore, JP-DKO mice exhibited impairments of motor coordination and learning, although normal cerebellar histology was retained. Therefore, JPs support the Ca(2+)-mediated communication between voltage-gated Ca(2+) channels, RyRs and SK channels, which modulates the excitability of PCs and is fundamental to cerebellar LTD and motor functions.     

Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.     

Phospholipase Cbeta4 and protein kinase Calpha and/or protein kinase CbetaI are involved in the induction of long term depression in cerebellar Purkinje cells

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI.     

Extent of multiple innervation of purkinje cells by climbing fibers in the olivocerebellar system of weaver,reeler, and staggerer mutant mice

Multiple innervation of cerebellar Purkinje cells (PCs) by climbing fibers (CFs) has been described recently in adult weaver, reeler, and staggerer mutant mice, instead of the monoinnervation found in normal adults. In the present study, the extent of this multiple innervation was estimated by two methods, using both evoked and spontaneous activity of the olivocerebellar system. Concordant values were obtained: the mean number of CF collaterals per PC was between 3.5 and 4 in weaver and staggerer and close to 3.2 for the multiply innervated PCs of reeler mice. These values are of the same order of magnitude as those for the transient multiple innervation in developing rats (Mariani and Changeux, 1981a, b).     

Inhibition of associative long-term depression by activation of β-adrenergic receptors in rat hippocampal CA1 synapses

The aim of this study was to investigate the role of β-adrenergic receptors in modulating associative long-term depression (LTD) at CA1 synapses in rat hippocampal slices. Standard extracellular electrophysiological techniques were employed to record field excitatory post-synaptic potential (fEPSP) activity and to induce associative LTD. Two independent Schaffer collateral pathways were elicited in hippocampal CA1 areas. In one (weak) pathway, the stimulating intensity was adjusted to elicit small fEPSP activity (20–30% of the maximum response). In contrast, 80–90% of the maximum response was evoked in the other (strong) pathway. Associative LTD of weak pathway could be induced by paired stimulation of weak and the strong pathways, repeated 100 times at 0.167 Hz. The associative LTD of weak pathway was NMDA receptor- and phophatase 2B dependent, because bath application of 50 μM D, L-AP5 or 10 μM cypermethrin blocked its induction. Bath application of 1 μM isoproterenol inhibited associative LTD, and this effect was blocked by timolol, suggesting the involvement of β-adrenergic receptors. The inhibitory effect of β-adrenergic receptors on LTD induction was blocked in slices pretreated with inhibitors of protein kinase A and mitogen-activated protein kinase, suggesting that these signal cascades are downstream effectors following activation of β-adrenergic receptors. Nevertheless, bath application of timolol or cypermethrin alone did not have significant effect on associative LTD induction, suggesting neither endogenous function of β-adrenergic receptor nor endogenous PKA activity does have a role in associative LTD induction.     

Does synaptic elimination contribute to the organization of cerebellar microzones of climbing fiber projection?

In adult rats whose cerebellar Purkinje cells (PCs) remain polyinnervated by olivary climbing fibres (CFs) after postnatal irradiation, topographical maps of responsive PCs to mechanical stimulation of the third row of contralateral vibrissae show that these cells are more numerous and more diffusely distributed than in the normal rat. PCs responding with the "best responses" are distributed evenly from the midline to 400 microns lateral in the contralateral hemivermis of lobule VII, and not in a parasagittal microzone centred on the plane 200 microns as in the normal rat. Thus it seems likely that synaptic elimination should contribute to microzone formation during postnatal development of the normal cerebellum.     

Insulin modulates hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and phosphatidyl-inositol-3-kinase-dependent manner

Insulin and its receptor are both present in the central nervous system and are implicated in neuronal survival and hippocampal synaptic plasticity. Here we show that insulin activates phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), and results in an induction of long-term depression (LTD) in hippocampal CA1 neurones. Evaluation of the frequency-response curve of synaptic plasticity revealed that insulin induced LTD at 0.033 Hz and LTP at 10 Hz, whereas in the absence of insulin, 1 Hz induced LTD and 100 Hz induced LTP. LTD induction in the presence of insulin required low frequency synaptic stimulation (0.033 Hz) and blockade of GABAergic transmission. The LTD or LTP induced in the presence of insulin was N-methyl-d-aspartate (NMDA) receptor specific as it could be inhibited by alpha-amino-5-phosphonopentanoic acid (APV), a specific NMDA receptor antagonist. LTD induction was also facilitated by lowering the extracellular Mg(2+) concentration, indicating an involvement of NMDA receptors. Inhibition of PI3K signalling or discontinuing synaptic stimulation also prevented this LTD. These results show that insulin modulates activity-dependent synaptic plasticity, which requires activation of NMDA receptors and the PI3K pathway. The results obtained provide a mechanistic link between insulin and synaptic plasticity, and explain how insulin functions as a neuromodulator.     

Diminished climbing fiber innervation of Purkinje cells in the cerebellum of myosin Va mutant mice and rats

Myosin Va is an actin-based molecular motor that is involved in organelle transport and membrane trafficking. Here, we explored the role of myosin Va in the formation of synaptic circuitry by examining climbing fiber (CF) innervation of Purkinje cells (PCs) in the cerebella of dilute-neurological (d-n) mice and dilute-opisthotonus (dop) rats that have mutations in dilute-encoded myosin Va. Anterograde labeling of CFs with biotinylated dextran amine (BDA) revealed that they arborized poorly and that their tips extended only half way through the thickness of the molecular layer (ML) in adult d-n mice. Using immunohistochemistry specific for vesicular glutamate transporter 2 (VGluT2) to visualize CF synaptic terminals, we found that during development and in adulthood, these terminals did not ascend as far along the proximal shaft dendrites of PCs in d-n mice and dop rats as they did in normal animals. An irregular distribution of BDA-labeled bulbous varicosities and VGluT2 spots along CF branches were also noted in these animals. Finally, VGluT2-positive CF terminals were occasionally localized on the PC somata of adult d-n cerebella. These phenotypes are consistent with our electrophysiological findings that CF-mediated excitatory postsynaptic currents (EPSCs) were significantly smaller in amplitude and faster in decay in adult d-n mice, and that the regression of multiple CFs was slightly delayed in developing d-n mice. Taken together, our results suggest that myosin Va is essential for terminal CF extension and for the establishment of CF synapses within the proper dendritic territories of PCs.     

Involvement of synaptic NR2B-containing NMDA receptors in long-term depression induction in the young rat visual cortex in vitro

Activation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in various forms of synaptic plasticity depending on the receptor subtypes involved. However, the contribution of NR2A and NR2B subunits in the induction of long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) in layer II/III pyramidal neurons of the young rat visual cortex remains unclear. The present study used whole-cell patch-clamp recordings in vitro to investigate the role of NR2A- and NR2B-containing NMDARs in the induction of LTD in visual cortical slices from 12- to 15-day old rats. We found that LTD was readily induced in layer II/III pyramidal neurons of the rat visual cortex with 10-min 1-Hz stimulation paired with postsynaptic depolarization. D-APV, a selective NMDAR antagonist, blocked the induction of LTD. Moreover, the selective NR2B-containing NMDAR antagonists (Ro 25-6981 and ifenprodil) also prevented the induction of LTD. However, Zn2+, a voltage-independent NR2A-containing NMDAR antagonist, displayed no influence on the induction of LTD. These results suggest that the induction of LTD in layer II/III pyramidal neurons of the young rat visual cortex is NMDAR-dependent and requires NR2B-containing NMDARs, not NR2A-containing NMDARs.     

Long-term depression requires postsynaptic AMPA GluR2 receptor in adult mouse cingulate cortex

Synaptic long-term depression (LTD) is thought to be important for various brain functions such as learning, memory, and development. Although anterior cingulated cortex (ACC) has been demonstrated to contribute to learning and memory, no studies has been reported about the synaptic mechanisms for cingulate LTD. Here, we used integrative genetic, pharmacological and electrophysiological approaches to demonstrate that AMPA GluR2, but not GluR3, subunit is critical for cingulate LTD. We found that LTD was absent in adult cingulate slices of GluR2 knockout mice. Furthermore, postsynaptic injections of peptides that inhibit AMPA GluR2-PDZ interactions blocked the induction of LTD. To determine if the requirement for AMPA receptor-PDZ interaction is time-dependent, we injected the same inhibiting peptide into the postsynaptic cells 5 min after the induction of LTD. We found that LTD was not affected by the peptide, providing the first evidence that postsynaptic AMPA GluR2-mediated depression occurs rapidly (within t = 5 min). Genetic deletion of GluR3 did not affect cingulate LTD. Our results provide the first study of cingulate LTD mechanism using whole-cell patch-clamp recording in adult cortical slices and demonstrate that postsynaptic AMPA GluR2 subunit is crucial for synaptic depression in the ACC of adult mice.     

Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.     

Lgr4 Protein Deficiency Induces Ataxia-like Phenotype in Mice and Impairs Long Term Depression at Cerebellar Parallel Fiber-Purkinje Cell Synapses

Cerebellar dysfunction causes ataxia characterized by loss of balance and coordination. Until now, the molecular and neuronal mechanisms of several types of inherited cerebellar ataxia have not been completely clarified. Here, we report that leucine-rich G protein-coupled receptor 4 (Lgr4/Gpr48) is highly expressed in Purkinje cells (PCs) in the cerebellum. Deficiency of Lgr4 leads to an ataxia-like phenotype in mice. Histologically, no obvious morphological changes were observed in the cerebellum of Lgr4 mutant mice. However, the number of PCs was slightly but significantly reduced in Lgr4^−/− mice. In addition, in vitro electrophysiological analysis showed an impaired long term depression (LTD) at parallel fiber-PC (PF-PC) synapses in Lgr4^−/− mice. Consistently, immunostaining experiments showed that the level of phosphorylated cAMP-responsive element-binding protein (Creb) was significantly decreased in Lgr4^−/− PCs. Furthermore, treatment with forskolin, an adenylyl cyclase agonist, rescued phospho-Creb in PCs and reversed the impairment in PF-PC LTD in Lgr4^−/− cerebellar slices, indicating that Lgr4 is an upstream regulator of Creb signaling, which is underlying PF-PC LTD. Together, our findings demonstrate for first time an important role for Lgr4 in motor coordination and cerebellar synaptic plasticity and provide a potential therapeutic target for certain types of inherited cerebellar ataxia.     

Activation of receptors negatively coupled to adenylate cyclase is required for induction of long-term synaptic depression at Schaffer collateral-CA1 synapses

Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl) glycine (DCGIV; 5 microM), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 microM), resulted in a long-lasting depression of synaptic strength. When zaprinast (20 microM) was combined with a cell-permeant PKA inhibitor H-89 (10 microM), the need for mGluR IIs was bypassed. DCGIV, when combined with a "submaximal" low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 5 microM), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)-a-Cyclopropyl-[3- 3H]-4-phosphonophenylglycine (CPPG; 10 microM), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 microM), was sufficient to elicit CLTD. Inhibition of PKA with H-89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity.     

Persistent Synapse Loss Induced by Repetitive LTD in Developing Rat Hippocampal Neurons

Synaptic pruning is a physiological event that eliminates excessive or inappropriate synapses to form proper synaptic connections during development of neurons. Appropriate synaptic pruning is required for normal neural development. However, the mechanism of synaptic pruning is not fully understood. Strength of synaptic activity under competitive circumstances is thought to act as a selective force for synaptic pruning. Long-term depression (LTD) is a synaptic plasticity showing persistent decreased synaptic efficacy, which is accompanied by morphological changes of dendritic spines including transient retraction. Repetitive induction of LTD has been shown to cause persistent loss of synapses in mature neurons. Here, we show that multiple, but not single, induction of LTD caused a persistent reduction in the number of dendritic synapses in cultured rat developing hippocampal neurons. When LTD was induced in 14 days in vitro cultures by application of (RS)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR) agonist, and repeated three times with a one day interval, there was a significant decrease in the number of dendritic synapses. This effect continued up to at least two weeks after the triple LTD induction. The persistent reduction in synapse number occurred in the proximal dendrites, but not the distal dendrites, and was prevented by simultaneous application of the group I/II mGluR antagonist (S)-a-methyl-4-carboxyphenylglycine (MCPG). In conclusion, we found that repetitive LTD induction in developing neurons elicits synaptic pruning and contributes to activity-dependent regulation of synapse number in rat hippocampal neurons.     

Long-term depression of kainate receptor-mediated synaptic transmission

Kainate receptors (KARs) have been shown to be involved in hippocampal mossy fiber long-term potentiation (LTP); however, it is not known if KARs are involved in the induction or expression of long-term depression (LTD), the other major form of long-term synaptic plasticity. Here we describe LTD of KAR-mediated synaptic transmission (EPSC(KA) LTD) in perirhinal cortex layer II/III neurons that is distinct from LTD of AMPAR-mediated transmission, which also coexists at the same synapses. Induction of EPSC(KA) LTD requires a rise in postsynaptic Ca(2+) but is independent of NMDARs or T-type voltage-gated Ca(2+) channels; however, it requires synaptic activation of inwardly rectifying KARs and release of Ca(2+) from stores. The synaptic KARs are regulated by tonically activated mGluR5, and expression of EPSC(KA) LTD occurs via a mechanism involving mGluR5, PKC, and PICK1 PDZ domain interactions. Thus, we describe the induction and expression mechanism of a form of synaptic plasticity, EPSC(KA) LTD.     

Hypothalamic CRF immunoreactivity in genetically hypothyroid (hyt/hyt) mice

The induction of hypothyroidism in young rats by feeding thiouracil to their mothers during pregnancy has been shown to depress hypothalamic content of bioactive and immunoactive corticotropin-releasing factor (CRF). The present study was done to determine whether genetically hypothyroid young mice (hyt/hyt) born to euthyroid mothers (+/hyt) exhibited a similar depression in hypothalamic CRF immunoreactivity. Young euthyroid and hypothyroid littermate mice were examined by radioimmunoassay for hypothalamic CRF content at 15, 20, 25, or 30 days of age. Mean CRF content was depressed insignificantly (to about 80% of normal) by hypothyroidism, at 15-25 days of age. However, after weaning by the mother, 30-day-old hypothyroid pups demonstrated significantly depressed hypothalamic CRF levels (71%). It is suggested that maternal factors may be assisting in the maintenance of hypothalamic CRF until after weaning. Furthermore, genetic hypothyroidism does not appear to have nearly as marked an influence as thiouracil feeding on hypothalamic CRF levels.